. .fi . A €913. mm..." Wmufwv ‘ My 5. .. . . L2: . 5.6mm. .. Hams... En. . y .. . tiabimn .hvl- -‘|.lt‘\' I 1 firm?! ii: i 21.1.... 7:». J..." ills“ 337...: . :5. $35.8 .mué {gagfifiém ‘ 3"... K. nrvi .....H v. 11:35:! L—riy . .. G3. Jr: 5.? .. ““3 LIBRARY Michigan State ZOOCI University This is to certify that the dissertation entitled RETROVIRAL VECTOR-BASED RNA INTERFERENCE AGAINST MAREK’S DISEASE VIRUS AND AVIAN LEUKOSIS VIRUS presented by M0 CHEN has been accepted towards fulfillment of the requirements for the Doctoral degree in Microbiology &Molecular Genetics rofessor’s Signature ' 7/7 Z/flf Date MSU is an Affirmative Action/Equal Opportunity Employer a.-.-—-—---—.-¢—-—---.--.-.-.--.---n-n-u-o-u-n-o-.-a-I-o-o--n-n-o-c-n-t-o-n-n-n—n-o-o-o-u----I-o-o-—u--u- PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K:IProj/Aco&Pres/CtRC/DateDue.indd RETROVIRAL VECTOR-BASED RNA INTERFERENCE AGAINST MAREK’S DISEASE VIRUS AND AVIAN LEUKOSIS VIRUS By Mo Chen A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Microbiology and Molecular Genetics 2008 ABSTRACT RETROVIRAL VECTOR-BASED RNA INTERFERENCE AGAINST MAREK’S DISEASE VIRUS AND AVIAN LEUKOSIS VIRUS By Mo Chen RNA interference (RNAi) is an evolutionarily conserved gene silencing process mediated by small RNAs including microRNAs (miRNAs) and small interfering RNAs (siRNAs), both of which are derived from larger precursor RNAs. RNAi based anti-viral systems include transient transfection of synthetic siRNAs and expression of short hairpin RNA (shRNA), shRNA-mirs and long hairpin shRNA (lhRNA), all of which are resemble of bioproducts of cellular miRNA biogenesis. The shRNA-mirs can be also expressed as larger transcripts containing several stern-loop structures resembling polycistronic miRNAs (termed multimir). The potential advantage of multimir is to generate multiple siRNAs from a single transcript and target multiple regions of viral RNA to prevent viral escape. Although most studies have used exogenous siRNAs to inhibit viral replication, vectors expressing shRNA-mirs in the context of a modified endogenous miRNA are more efficient and are practical for in viva delivery. The overall purpose of this project is to develop RNAi-based anti-viral strategies against two important chicken viruses: avian leukosis virus (ALV) and Marek’s disease virus (MDV), both of which are economically important and they have distinctive infectious cycles, which will provide different challenges for RNAi. First, I describe the constructions of replication competent retroviral vectors to deliver shRNA-mirs targeting subgroup B ALV, the most effective of which reduces expression of protein targets by as much as 90% in cultured avian cells. Cells expressing shRNA-mirs targeting the tvb receptor sequence or the viral env(B) sequence significantly inhibit ALV(B) replication. I demonstrate efficient antiviral RNAi in avian cells using shRNA-mirs expressed from RNA polymerase II promoters, including an inducible promoter, allowing for the regulation of the antiviral effect by doxycycline. Next, I use similar vectors to test RNAi against MDV and its close relative, herpesvirus of turkeys (HVT). Cells expressing shRNA-mirs targeting the MDV or HVT gB glycoprotein gene or the ICP4 transcriptional regulatory gene show significant inhibition of viral replication. Not only are viral titers reduced, but observed plaque sizes are significantly smaller when the virus is grown on cells in which RNAi is effective. I describe a modified retroviral delivery vector that expresses a shRNA-mir containing up to three RNAi target sequences (3mirs) and employ this vector with multiple targets within the MDV gB gene or within both the g3 and ICP4 genes. Finally, I test whether RNAi against MDV can protect chickens from lethal infections. Delivery of 3mirs co- targeting MDV g8 and ICP4 genes significantly reduces MDV viremia and MD incidence and increases overall survival rate by reducing MD-induced death. I discuss that the anti-viral effects I observe in chickens is unlikely due to RNAi induced interferon responses. DEDICATIONS To Casey Ausmus, Chen Daguang, Tang Yuhua iv ACKNOWLEDGMENTS This dissertation could not have been written without my mentor, Dr. Jerry Dodgson. Thanks for introducing me to a fascinating and challenging project and providing his supervision and expertise to guide me through my graduate program. I would also thank his guidance and encouragement for my academic career planning. I express my gratitude to my committee members Dr. Andrea Amalfitano, Dr. John Fyfe, Dr. Henry Hunt, and Dr. Yong-Hui Zheng for their guidance and time. I am very grateful to my lab-mate and friend, William Payne for his friendship, advice, and invaluable assistance. I want to thank Dr. Huan-min Zhang, Dr. John Dunn, Dr. Lucy Lee, Evelyn Young, Noah Koller, Barbara Riegle, Chang Shuang for their help, support, encouraging words, thoughtful criticism. I would like to thank my parents Chen Daguang and Tang Yuhua, for their love, support and understanding during the long years of my education. And finally, many thanks to Casey Ausmus, my husband and dearest fi‘iend. Thank you for always being there for me. TABLE OF CONTENTS LIST OF TABLES .................................................................................... ix LIST OF FIGURES .................................................................................. x LIST OF ABBREVLATIONS ..................................................................... xii CHAPTER 1 INTRODUCTION ................................................................................. 1 RNA interference ............................................................................ 3 RNAi as an antiviral strategy ............................................................... 7 Avian leukosis viruses (ALV) ............................................................ 11 Marek’s disease (MD) and Marek’s disease virus (MDV) ........................... 14 Retroviral vectors for RNAi ............................................................... 19 REFERENCES ................................................................................... 22 CHAPTER 2 INHIBITION OF AVIAN LEUKOSIS VIRUS REPLICATION BY VECTOR-BASED RNA INTERFERENCE ............................................................................ 30 ABSTRACT ....................................................................................... 31 INTRODUCTION ............................................................................... 32 RESULTS ......................................................................................... 35 GFP fluorescence in DF -1 cells is decreased by RCASBP(A) shRNA-mirs ...... 35 ALV(B) resistance through silencing of the tvb host receptor ....................... 39 DISCUSSION .................................................................................... 47 MATERIALS AND METHODS ............................................................... 51 Vector constructions ....................................................................... 51 Cell culture .................................................................................. 53 Virus propagation ........................................................................... 5 3 Virus infection .............................................................................. 54 ALV alkaline phosphatase (AP) challenge assay ...................................... 54 Statistical analysis .......................................................................... 55 Polyacrylamide gel electrophoresis—Northern analysis (PAGE—Northem) ........ 56 Western blot analysis ...................................................................... 56 AUTHORS’ CONTRIBUTIONS ............................................................... 57 ACKNOWLEDGEMENTS ..................................................................... 57 REFERENCES ................................................................................... 59 APPENDIX A. SUPPLEMENTARY DATA ............................................... 63 CHAPTER 3 INHIBITION OF MAREK'S DISEASE VIRUS REPLICATION BY RETROVIRAL VECTOR-BASED RNA INTERFERENCE .................................................... 66 ABSTRACT ....................................................................................... 67 INTRODUCTION ............................................................................... 68 vi RESULTS ......................................................................................... 72 Inhibition of HVT by retroviral delivery of shRNA-mirs targeted against the gB gene .......................................................................................... 72 Antiviral RNAi against MDV (GaHV-Z) using gB gene targets ..................... 75 Antiviral RNAi against MDV using a three target vector with ICP4 and/or gB target sequences ............................................................................ 79 DISCUSSION .................................................................................... 83 MATERIALS AND METHODS ............................................................... 86 Vector constructions ....................................................................... 86 Cells and viruses ........................................................................... 88 Delivery of RCAS and RCAN vectors into cell culture .............................. 88 Plaque assays ............................................................................... 89 Immunofluorescence assay ............................................................... 90 Plaque size measurement .................................................................. 90 Flow cytometry ............................................................................. 91 Reverse transcriptase PCR (RT-PCR) ................................................... 91 Quantitative real time PCR (qPCR) assay for viral genomes ........................ 92 Statistical analysis .......................................................................... 93 ACKNOWLEDGEMENTS ..................................................................... 94 AUTHORS’ CONTRIBUTIONS .............................................................. 94 REFERENCES ................................................................................... 95 APPENDIX A. SUPPLEMENTARY DATA ............................................. 102 CHAPTER 4 RETROVIRAL VECTOR-BASED RNA INTERFERENCE PROTECTS CHICKENS FROM MAREK’S DISEASE VIRUS INFECTION .......................................... 111 ABSTRACT ..................................................................................... 112 INTRODUCTION .............................................................................. 1 13 RESULTS ....................................................................................... 118 Infection of line 0 chickens with W Mdll strain of serotype 1 MDV ............ 118 RCASBP(A) delivering 3 siRNAs against the viral gB gene inhibits the vv+ 648A strain of serotype 1 MDV infection .................................................... 122 Effects of RCASBP(A)miRNA-1 874 against a W Md5 strain of serotype 1 MDV in 1515 x 71 chickens ..................................................................... 126 RCASBP(A)3mirs targeting both the MDV gB and ICP4 genes inhibits vv+ 648A serotype 1 MDV infection ........................................................ 128 DISCUSSION ................................................................................... 142 MATERIALS AND METHODS ............................................................. 148 Vector constructions ..................................................................... 148 Cells and viruses .......................................................................... 148 Delivery of RCAS vectors into DF -1 cells ............................................ 149 In vivo MDV challenge assay ........................................................... 149 Plaque assays to determine MDV load ................................................ 151 Stem loop RT-PCR for testing siRNA expression .................................... 152 Immunofluorescence assay .............................................................. 153 vii Statistical analysis ........................................................................ 154 REFERENCES ................................................................................. 1 55 FUTURE WORK .................................................................................. 159 REFERENCES ................................................................................. 1 62 viii LIST OF TABLES Supplemental Table 2-1. Target oligonucleotide sequences ................................... 65 Table 3-1.Effect of retroviral RNAi vector treatment using gB gene targets on FC126 HVT infection ....................................................................................... 73 Table 3-2. Reduction in MDV and HVT plaque sizes due to RNAi against gB ............ 74 Supplementary Table 3-1. Oligonucleotides used for clomng102 Table 4-1. MDV RNAi in vivo experimental design: number of chicks per group ...... 138 Table 4-3. Reduction of MDV viremia by RCASBP(A) delivered RNAi ................. 140 Table 4-4. Protection of RCASBP(A) delivered RNAi in chickens against challenge with MDV ................................................................................................ 141 ix LIST OF FIGURES Figure 1-1. Overview of microRNA (miRNA) and small interference RNA (siRNA) biogenesis .............................................................................................. 5 Figure 2-1. Diagram of avian shRNA-mir vectors ............................................. 37 Figure 2-2. RNAi vs. GFP using RCASBP(A)shRNA-mir .................................... 38 Figure 2-3. RCASBP(A)shRNA-mir directed against tvb reduces protein expression and inhibits ALV(B) entry .............................................................................. 40 Figure 2-4. Regulated promoter-driven inhibition of ALV(B) ................................ 43 Figure 2-5. RCASBP(A)shRNA-mir directed against env(B) reduces protein expression and limits ALV(B) infection ....................................................................... 45 Supplemental Figure 2-1. The chicken mir-30a cassette sequence ........................... 63 Figure 3-1. RCASBP(A)shRNA-mir directed against Mdll MDV gB reduces plaque numbers in both CEF (blue bars) and SOgE cells (purple bars) .............................. 76 Figure 3-2. Morphology of Mdll MDV plaques on CEF at 5 dpi ........................... 77 Figure 3-3. RCASBP(A)3mirs directed against subgroup MDV gB and/or ICP4 reduces titers in CEF .......................................................................................... 80 Figure 3-4. RT-PCR of gB mRN A expression fi'om CEF expressing RCASBP(A)3mirs .................................................................................. 82 Supplemental Figure 3-1. Diagram of avian shRNA-mir vectors ........................... 105 Supplemental Figure 3-2. The chicken mir-30a-sphngo cassette sequence ............... 107 Supplemental Figure 3-3. The chicken 3mir cassette sequence ............................ 108 Supplemental Figure 3-4. Quantitative PCR of gB copy numbers per chicken GAPDH gene from CEF expressing RCASBP(A)3mirs challenged with MDV .................... 110 Figure 4-1. General procedure for using RCASBP(A)miRNA/RCASBP(A)3mirs vectors in vivo .............................................................................................. 120 Figure 4—2. Immunohistochemical analysis of representative bursa at 5dpi .............. 121 Figure 4-7. RCASBP(A)3mirs directed against 648A MDV increased survival rates by reducing MD mortality ........................................................................... 125 Figure 4-3. Immunohistochemal analysis of representative MDV induced T cell lymphoma and RCAS induced B cell lymphoma in spleens ................................. 131 Figure 4-4. Histochemical analysis of representative MDV induced lesions in peripheral nerves ................................................................................................ 132 Figure 4-5. Schematic showing stem-loop RT-PCR miRNA assays ....................... 133 Figure 4-6. Birds present of RCASBP(A)3mirs showed various levels of siRNA expression .......................................................................................... 134 xi ABC ADOL ALV ALV(B) AP BAC BTA CEF CMV DMEM DNA Dox Dpi ECL EDTA EGFP ELISA Ery F ACS F BS H&E LIST OF ABBREVIATIONS Avidin—biotin—peroxidase complex Avian disease and oncology lab Avian leukosis virus Subgroup B Avian leukosis virus Alkaline phosphatase Bacterial artificial chromosome Bursal/thymic atrophy Chicken embryo fibroblasts Cytomegalovirus Dulbecco's modified Eagle's medium Deoxyribonucleic acid Doxycycline Day post inoculation Enzymatic chemiluminescence Ethylenediaminetetraacetic acid Enhanced green fluorescence protein Enzyme-Linked ImmunoSorbent Assay Erythroblastosis Fluorescence-activated cell sorting F eta] bovine serum Haematoxylin & eosin xii HIV hIRNA HRP HSV HVT IE LAT LL LTR MDV miRN A M-MLV mRN A PAGE-Northern PBL P body PBS PCR PFU(pfu) pH Pol 11 Human immunodeficiency virus Long-hairpin RNA Horseradish Peroxidase Herpes simplex virus Herpesvirus of turkeys Inna-abdominal Immediate early Latency-associated transcript Lymphoid leukosis Long terminal repeat Monoclonal antibody Marek’s disease virus microRNA Moloney murine leukemia virus Messenger RNA Polyacrylamide gel electrophoresis—Northern White peripheral blood cells Processing body Phosphate buffer solution Polymerase chain reaction Plaque-forming-units Potential of hydrogen Polymerase II xiii Pol HI pri-miRNA PVDF qPCR RCAN RCANBP(A) RCAS RCASBP(A) RISC RNA RNAi RSV RT-PCR SDS-PAGE shRNA siRNA SU SU(B)—rIgG Polymerase 111 Primary microRNA Polyvinylidene difluoride Quantitative real time PCR Replication competent ALV long terminal repeat [LTR] with no splice acceptor Subgroup A RCAS with pol gene from the Bryan strain of RSV Replication competent ALV long terminal repeat [LTR] with splice acceptor Subgroup A RCAS with pol gene from the Bryan strain of RSV RNA-induced silencing complex Ribonucleic acid RNA interference Rous sarcoma virus Reverse transcription polymerase chain reaction Sodium dodecyl sulfate polyacrylamide gel electrophoresis Short hairpin RNA Small interference RNA Surface region of ALV envelop protein Subgroup B ALV SU protein fused to a rabbit immunoglobulin tag xiv TRE UTR VARI VSV-G Vv wpi Tetracycline response element Untranslated region Van Andel Research Institute Vesicular stomatitis virus G protein Very virulent Very virulent plus Week post inoculation XV CHAPTER 1 INTRODUCTION RNA interference (RNAi) recently has been shown to provide an effective strategy to reduce the replication of several animal viruses, including retroviruses and herpesviruses. This has been done either by inducing the breakdown of viral-specific RNAs or host cell mRNAs required for the viral life cycle. The overall purpose of this project is to develop RNAi-based anti-viral strategies against two important chicken viruses: avian leukosis virus (ALV) and Marek’s disease virus (MDV). These two targets have been chosen since both are economically important and they have distinctive infectious cycles, which will provide different challenges for RNAi. In chapter 2 (Chen et al., 2007), I describe the construction of replication competent retroviral vectors to deliver short hairpin RNAs (shRNA-mirs) targeting the tvb host receptor gene for subgroup B ALV (ALV(B)). I observed that efficient RNAi based on single copy retroviral proviruses requires the RNAi target be presented in the form of an endogenous miRNA, preferably expressed from an RNA polymerase II (pol 11) promoter. I demonstrated significant reduction in ALV (B) replication as well as target gene expression in cell culture. The shRNA-mir vectors employ pol II promoters, including, in one case, an inducible promoter that allows for the regulation of the antiviral effect by doxycycline. In chapter 3 (Chen et al., 2008), I show significant reduction of replication of HVT (herpesvirus of turkeys) and pathogenic MDV in cell culture by targeting viral gB or ICP4 genes, which encode an essential membrane fusion protein and a transcriptional regulatory protein, respectively. I also constructed a modified retroviral delivery vector that expresses a shRNA-mir containing up to three RNAi target sequences and employed this vector against multiple targets within the MDV gB gene or within both the g8 and ICP4 genes. The use of targets within multiple genes potentially can provide a larger antiviral effect and/or make it more difficult for viral escape mutations to evolve. Finally (Chapter 4), I describe the delivery of retroviral vectors expressing RNAi targets against MDV into newly-laid chicken embryos. Chicks were hatched and challenged with virulent strains of MDV. We designed PCR-based assays to select chicks that retain RNAi expression. Preliminary results show that RNAi significantly reduced lytic phase MDV replication and disease incidence as well as increased survival rates. RNA interference RNA interference (RNAi) is an evolutionarily conserved gene silencing process mediated by small RNAs including microRNAs (miRNAs) and small interfering RNAs (siRNAs), both of which are derived from larger precursor RNAs (Figure 1-1.). miRNAs are mostly transcribed by RNA polymerase II as long dsRNAs termed primary miRNAs (pri-miRNAs;(Lee et al., 2004). Pri-miRNAs can be thousands of nucleotides (nt) long and contain multiple miRNAs in polycistronic transcripts. In the nucleus, pri-miRNAs are processed by a large microprocessor complex consisting of a RNase III enzyme Drosha (Lee et al., 2003) and a double-stranded (ds) RNA binding protein termed DGCR8/Pasha, which functions to bring Drosha closer to its pri-miRNAs substrates. Drosha cleaves pri-miRNAs and generates pre-miRNAs. A typical pre-miRNA is about ~60 nt long and composed of a loop region and an imperfect double-stranded stem that has a 5' monophosphate terminus and a 2-nt 3' hydroxyl overhang. Pre—miRNAs are exported fi'om the nucleus into the cytoplasm by Exportin 5 along with Ran in a GTP dependent manner (Bohnsack, Czaplinski, and Gorlich, 2004). Another function of Exportin 5 is to stabilize pre-miRNAs and protect them from degradation in the nucleus. In the cytoplasm, another RNase III enzyme called Dicer further processes pre-miRNAs 3 to produce the miRNA duplex. Dicer can also degrade long dsRNA into small RNA duplexes called siRNA duplexes (Bernstein et al., 2001). Dicer contains two functional domains that recognize precursors of miRNAs and siRNAs. The PAZ domain recognizes the 2-nt 3' overhang of pre-miRNAs, while the dsRBD domain binds long dsRNAs (Zhang et al., 2004). Dicer cuts pre-miRNAs about 22 nt away from the end of the stem to remove the loop and produce a miRNA duplex with 2-nt 3’-overhangs on both ends. Dicer processes long dsRNAs to siRNA duplexes in a similar manner. The miRNA duplex is then unwound by an unidentified helicase, and the strand whose 5' end is less stably double-stranded (termed the guide strand) is preferentially incorporated into the RNA-induced silencing complex, RISC (Schwarz et al., 2003). E3 Microprocessor if Drosha ‘..- ] DGCR8/Pasha £11333}? 1' dSRN A Pri-miFINA l mw _. ‘;"i?i--I?‘;w"‘"“ . . Dicer a» . "W ' q . ‘ pre—mIRNA 55;“; E, n. . Exportin 5 :13“ ' . 1:- Ago 1 _,____—+——--_~,~—-—-___ siRNA nucleus . cytoplasm TTITTI'I‘ITTTTTTTTTITTT miRNA Figure 1-1. Overview of microRNA (miRNA) and small interference RNA (siRNA) biogenesis. In the nucleus, primary miRNAs (pri-miRNAs) are processed by a large microprocessor complex consisting of a Drosha and DGCR8/Pasha. Drosha cleaves pri- miRNAs and generates pre-miRNAs. Pre-miRNAs are exported from the nucleus into the cytoplasm by Exportin 5. In the cytoplasm, Dicer cuts pre-miRNAs about 22 nt away from the end of the stem to remove the loop and produce a miRNA duplex with 2-nt 3’- overhangs on both ends. Dicer processes long dsRNAs to siRNA duplexes in a similar manner. The miRNA duplex is then unwound by an unidentified helicase, and the strand whose 5’ end is less stably double-stranded is preferentially incorporated into the RNA- induced silencing complex (RISC). Modified from Liu et al., 2008. How the miRNA-loaded RISC complex mediates gene silencing varies from one individual miRNA and its target to another, largely depending on the degree of complementarity of the miRNA to the target sequence as well specific cellular conditions. If a miRNA or siRNA is perfectly complementary to its target, the Ago protein in the RISC complex cleaves the mRNA 10 and 11 nt from the 5’ end of miRNA (or siRNA (Liu et al., 2004). The 5’ mRNA fiagrnent is then decapped and degraded by 3’exonucleases, whereas 3’ products can be degraded by 5’ exonucleases. Direct target mRNA cleavage by Ago is the major gene silencing mechanism by plant miRNAs (Dugas and Bartel, 2004) or artificial transfected siRNAs. Most animal miRNAs recognize their target mRNA using a “seed” region, extending 2 to 8 nt from the 5’ end of the miRNA. MiRNAs silence their target mRNAs by repressing translation as well as recruiting mRNA into special cellular organelles where translation can’t occur. Several models of translational repression have been suggested. In the “ribosome drop-off” model, miRNAs associated the RISC complex interfere with the translation elongation step (Petersen et al., 2006). In the cap-dependent model, Ago protein binds to the m7G cap of an mRNA target to disrupt the 5’cap recognition by eIF4F, thus inhibiting initiation of translation (Kiriakidou et al., 2007). A third model suggests that miRNA- induced gene silencing can be mediated by recruitment of mRNAs to a special cellular structure called the processing body (P body). The P body contains mRNA decapping and degradation enzymes but not ribosomes. It has been suggested that some mRNAs can be localized to the P body where translation is repressed and later re-enter polyribosomes for translation under stress conditions (Peters and Meister, 2007). RN Ai can regulate crucial cellular processes such as differentiation, proliferation, apoptosis, oncogenesis, and metabolis. RN Ai also plays important roles in viral infections (Grassmann and Jeang, 2008). Viral encoded miRNAs have been identified in retroviruses and herpesviruses. In herpes simplex virus (HSV), latency-associated transcripts (LATs) were predicted to encode pri-miRNA-like hairpins, which may play roles in regulating initiation of lytic infection and maintenance of latency. It has also been shown that LATs target host mRNAs involved in apoptosis (Gupta et al., 2006). (Burnside et al., 2006) identified MDV-encoded miRNAs (MDV-miR-6 to -8), which are located in the 5' end of LAT, antisense to the ICP4 gene, that are highly expressed in MDV transformed cells. Viruses can also produce RNAi suppressors which function either as RNA-decoys that saturate host RNAi machinery or as dsRNA binding proteins to sequester host anti-viral miRNA (Andersson et al., 2005; Bennasser, Yeung, and J eang, 2006). Finally, several RNA viruses contain nuclear capsid proteins that can protect their genomes from the cellular RNAi machinery (W esterhout, ter Brake, and Berkhout, 2006). RNAi as an antiviral strategy RNAi based anti-viral strategies have effectively inhibited several families of DNA and RNA viruses. Several RNAi-based antiviral drugs are currently being tested in clinical trials (Haasnoot, Westerhout, and Berkhout, 2007). We are particularly interested in studies with herpesviruses and retroviruses since MDV and ALV are members of these two virus families. In tests of RNAi against herpesviruses, viral transcriptional regulators often have been used as targets to inhibit the early stage of viral replication (Chang etal., 2004; Jia and Sun, 2003). Several groups have targeted viral glycoproteins which 7 mediate cell-to cell spread or viral DNA polymerase (Bhuyan et al., 2004; Palliser et al., 2006). Palliser et a1. (2006) demonstrated that siRNAs (including those targeting the g3 glycoprotein gene) could block lethal HSV-2 challenges in mice when administered exogenously either before or after the virus. Among studies of using retroviruses as RNAi targets, He et al., (2002) described RNAi against Rous sarcoma virus (RSV), a member of the avian leukosis and sarcomas virus family. They demonstrated that siRNA against viral gag gene can protect chicken embryos from lethal RSV infection. Several groups have reported effective RNAi strategies against human immunodeficiency virus 1 (HIV - 1) by targeting viral genes including gag, pol, vijf tat (essential for production of full length viral mRNA), rev, env and nef (essential for exporting unspliced viral mRNA and downregulating CD4+ after viral entry); the viral LTR region and the host receptor CD4+ gene (Kanzaki, Omelas, and Arganaraz, 2008). Recently studies show that HIV can rapidly mutate to escape RNAi either by point mutation in the target sequence 6r mutations outside the target that change the RNA structure so that viral RNA is inaccessible to the RISC complex. The strategies to counteract viral resistance include targeting higly conserved viral genes or host factors, as well as inhibiting several targets simultaneously using shRNA-mirs and long-hairpin RNAs (lhRNAs), which are capable of producing several siRN As from a single transcript. RN Ai based anti-viral systems include transient transfection of synthetic siRNAs and expression of short hairpin RNA (shRNA), shRNA-mirs and long hairpin shRNA (lhRNA). Synthetic siRNAs have successfuly reduced acute virus infection both in cell culture and animal models (Haasnoot, Westerhout, and Berkhout, 2007). Chemical modifications have significantly improved the half-life of siRNAs, increases the binding 8 affinity to siRNA targets and directed siRNAs to specific cell types, however, chemical modifications can also cause cell toxicity (de Fougerolles, 2008). The siRNA effect is transient in vertibrates due to the fact that there is no siRNA amplification process. Repetitive adminstration of low concentrations of siRNAs are needed to achieve long term anti-viral effects. ShRNAs are commonly used to inhibit chronic viral infection. shRNAs are structurely similar to pre-miRNAs. They contain a 19-22 nt stem region, a small apical loop and a 3'-terrninal UU overhang. ShRNAs are typically expressed in the nucleus from a polymerase III promoter, translocated to the cytoplasm by Exportin-5, and further processed by Dicer in the cytoplasm into functional siRNAs. The Pol III promoters (e.g., U6 and H1) naturally produce small stable RNAs and are well-suited to accommodate the design requirements for shRNA (Brumrnelkamp, Bernards, and Agami, 2002b; Sui et al., 2002). The shRNA cassette can be delivered into cells by plasmid or viral vectors. In some instances, overexpressed shRNAs have been shown to saturate miRNA biogenesis factors (e.g., Exportin 5), deplete endogenous miRNA, and induce acute liver toxicity (Grimm et al., 2006). lhRNAs differ fiom shRNA in that they contain a larger stem region (60 nt) so that multiple siRNAs can be produced by Dicer. Introduction of GU mismatch in the hIRNA stem region has been shown to reduce the likelihood of inducing the interferon response and limit off-target effects (Akashi et al., 2005; Konstantinova et al., 2006; Liu, Haasnoot, and Berkhout, 2007). Since shRNAs are expressed in the nucleus, exported into the cytoplasm and then processed by Dicer, a better strategy might be to generate the target siRNA sequence as part of a natural pri-miRNA stem-loop structure that could be recognized and processed by Drosha in the nucleus and more efficiently transported to the cytoplasm by Exportin 5. 9 A large portion of the miR-30a gene was cloned into a retroviral vector with minor changes in sequence to allow for insertion of target sequences without disrupting the native folding expected for the pri-miRNA (Silva et al., 2005). It has been showed that the U6 pol III promoter was no longer required in these vectors and that equal or better expression could be obtained fi'om pol II promoters, including inducible pol II promoters (Dickins et al., 2005). shRNA-mirs expressed fiom pol II promoters in retroviral and lentiviral vectors (in the form of integrated, single copy proviruses) have been shown to mediate gene silencing more potently than shRNA in both transient and stable transfection assays ((Das et al., 2006; Dickins etal., 2005; Silva etal., 2005; Stegrneier et al., 2005). The shRNA-mirs can be also expressed as larger transcripts containing several stem-loop structures resembling polycistronic miRNAs (termed multi-mir). The potential advantage of multimir is to generate multiple siRNAs from a single transcript and target multiple regions of viral RNA to prevent viral escape. In our studies, we initially chose the chicken miR-30a gene as a backbone to construct shRNA-mir constructs, because the homologous human miR-30a gene is identical in the stem region that gives rise to the miRNA. The human miR-30a gene also has been used to construct a large scale shRNA- mir library targeting nearly full complements of human and mouse genes. At least two putative endogenous chicken pri-miRNA-encoding genes generate transcripts predicted to contain 6 miRNA hairpins, one on chromosome 1 (Das et a1. 2006) and another on chromosome 4. We chose to use the latter and modify it to generate a cassette that contains three miRNA sites (miR-20b, miR-92-2 and miR-19b), each of which is flanked by a pair of unique restriction sites allowing for the insertion of up to three targets. Each of the three sites individually can generate an effective RNAi response (Chapter 3). 10 Avian leukosis viruses (ALV) Avian leukemia (leukosis) and sarcomas was the major cause of mortality in poultry at the beginning of the 20m centry. Rous first discovered tumors induced by Rous sarcoma virus (RSV). ALV was first known as Rous interfering factor because it can interfere with infection by RSV due to blockade of the cell receptor required for RSV entry. RSV, ALV and some defective endogenous retroviral elements comprise a large retrovirus family called the avian sarcoma and leukosis virus family (Payne, 1998). The ALV genome is composed of a dirneric single stranded RNA, which is about 7.5 kb in length. The genome contains three genes: gag encoding internal structural proteins; pol encoding reverse transcriptase; and env encoding envelope glycoproteins. Replication competent strains of RSV contain the above three genes and an additional viral oncogene, v-src, which originates from the cellular pro-oncogene, c-src, and encodes a tyrosine kinase dispensable for virus replication. Ten different ALV subgroups (designated A—J) have been identified based on properties determined by the surface region (SU) of envelope glycoprotein, host range, and viral neutralization (Barnard, Elleder, and Young, 2006). Subgroup A to E viruses can only infect cells with a specific kind of receptor encoded by a specific allele at one of three genetic loci designated as I‘m, tvb, and tvc. Five different alleles of the tvb gene have been identified. Three of these alleles, tvb”, tvb‘z", and tvbSZb, allow infection by ALV subgroup B (ALV(B)), subgroup D (ALV(D)), and subgroup E (ALV(B)). One allele, tvb“, permits infection by ALV(B) and ALV(D), but not ALV(E) and the tvb’ allele does not allow infection by ALV(B), ALV(D), or ALV(E). There is only one 11 single nucleotide difference between tvb” and tvb” (tvb‘l encodes a cysteine at residue 62 instead of a serine by tvb”, (Adkins, Brojatsch, and Young, 2000). The resistant allele tvb' was found to be identical to tvb" at residue 62 but to posses a premature stop codon that results in a protein product of only 57 N-terminal amino acids (Klucking, Adkins, and Young, 2002). Chickens that are homozygous for tvb' are viable, suggesting that the tvb receptor is not an essential protein for host survival. The protein encoded by tvb” allele (also called CARI for gytopathic ALSV receptor) is an avian member of the tumor necrosis factor receptor family and is most similar to the mammalian TRAIL receptors DR4 and DR5 (Brojatsch et al., 1996). ALV envelope glycoproteins are class I fusion proteins with an N-terminal surface subunit involved in receptor-binding and a C-terminal transmembrane subunit for directing the membrane fusion (Barnard, Elleder, and Young, 2006). The first step of viral entry involves the interaction of SU with the cellular receptOr, which induces a conformational change in the envelope protein under neutral pH that leads to the insertion of the fusion peptide into the cell membrane. The next step is to complete the fusion reaction in an acidic endosomal compartment. The receptor interference has been identified in ALV and RSV. Viruses requiring the same cellular receptors neither can superinfect infected cells sufficiently, nor can virus infect chronically infected cells or cells expressing endogenous env genes successfully. This is due to irreversible binding and/or down-regulated expression of the requisite subgroup-specific receptor, such that cells consequently become unavailable to the superinfection viruses of the same subgroup. 12 ALV can cause lymphoid leukosis (LL), a B-cell lymphoma of adult chickens, and erythroblastosis (Ery), a leukemia involving the proliferation of immature erythrocytes. ALV induces tumors by insertional activation of cellular proto-oncogenes due to altered regulation of expression as a result of proviral integration, as has been demonstrated for c-myc in the case of LL and c-erbB in the case of Ery (Payne, 1998). ALV family members can be classified as exogenous or endogenous. Exogenous virus can be transmitted vertically (from dam to progeny through the egg) or horizontally (from bird to bird). Horizontal transmission is commonly seen in subgroups A, B, and J. Infected birds rarely pass viruses to their progeny and are less likely to develop tumors. In contrast, vertical transmission mostly ends up with immunological tolerance, and no neutralizing antibodies being produced, and tumors. Several chicken lines contain endogenous ALV virus. The origin of endogenous viruses is not completely understood. Most endogenous viruses are replication defective and do not produce infectious virions, however, recombination can occur between endogenous loci and exogenous viruses that share sequence similarity (a major concern when choosing specific chicken lines for our in vivo experiments, see chapter 4). No vaccines or drugs are available for controlling ALV infection. The eradiation of infected chickens and selection of genetically resistant chicken lines have been successfully applied by the poultry industry. Genetic resistance to subgroup J ALV, a highly pathogenic virus, has not been recognized to date. We chose to employ RNAi against the tvb” gene encoding the ALV(B) receptor, as a model for antiviral RNAi in chickens for the following reasons. First, tvb, the product of tvb” allele, is required for infection by ALV(B) (Brojatsch et al., 1996). The interruption of an oncogenic strain of ALV(B) viral envelope protein with its receptor 13 significantly impairs viral replication in cell culture as well reduces viral induced sarcoma (Reinisova et al., 2008). Second, tvb appears to be non-essential since chickens homozygous for tvb', a severely truncated version of the protein, are viable (Klucking, Adkins, and Young, 2002). Third, several studies already showed that RNAi against viral receptors or co-receptors can efficiently reduce viral replication (Kanzaki, Omelas, and Arganaraz, 2008). RNAi targeting cellular receptors can potentially reduce viral entry by keeping viruses in the extracellular space for greater amounts of time and making them more susceptible to immune surveillance and clearance. Marek’s disease (MD) and Marek’s disease virus (MDV) MD is a lymphoproliferative disorder in which aggressive T cell lymphomas result from infection of susceptible chickens with MDV. MD is a major chronic infectious disease concern of the poultry industry, and it's also of increasing interest as a model for tumorgenesis (Osterrieder at al. 2006). There are four phases during MDV infection: early cytolytic infection, latency, the secondary cytolytic phase and transformation (Calnek, 2001). The first step of MD infection is inhalation of the virus through the respiratory tract, where phagocytic cells become the main target. Phagocytic cells carry the virus to lymphoid organs (thymus, spleen, and bursa of Fabricius) within a day. Here, activated CD4+ T cells and B cells become the primary targets for the cytolytic phase of the infection. Infected T cells then carry the virus to the skin, where fiee infectious virus assembles within the feather-follical epithelium and is shed to the environment, thereby infecting other chickens. The first cytolytic phase lasts about 3 to 5 days, and infected chickens start shedding infectious cell-free virus about 10 days after infection. One week post-infection, l\/fl3V enters the latent phase, which is characterized l4 by the presence of the viral genome mostly in CD4+ T cells without production of infectious progeny virus. Very few genes are being transcribed during this period. However, latency-associated transcripts (LATs) are transcribed during latency. LATs are antisense to the MDV immediate eary gene ICP4 mRNA. T-cell lymphomas (mainly CD4+ T cells and macrophages) deveIOp within two to six weeks of the infection. Latent infection is a prerequisite for oncogenic transformation and lymphomagenesis. Although MD-induced lymphomas require a sufficient number of latently infected T cells, only a small subset of T cells or a single transformed cell may proliferate to generate neoplasms. Several studies show that the viral oncoprotein, Meq, plays an important role in tumorgenesis. Meq interacts with cellular oncoproteins and binds to several host proteins that are involved in cell-cycle control (Jones et al., 1992; Liu et al., 1999). Through all the productive and latent phases, no extracellular virus can be detected within the host, suggesting an obligate cell-to-cell mode of virus spread. MDV is a member of the alphaherpesvirus family based on its 160-180 kb dsDNA genome organization. MDV encodes approximately 100 proteins. Many of the proteins are conserved between the three serotypes of MDV and, to a lesser extent, with those of other herpesviruses such as HSV 1. There are three different serotypes of MDV designated as MDV] (gallid herpesvirus type 2, GaHV-2), MDV2 (GaHV-3), and MDV3 (also known as turkey herpesvirus, or HVT). Lymphomas are only caused by MDVl. Different field isolates (strains) of MDV are characterized by different levels of virulence (the frequency and severity of MD they generate in vaccinated and/or unvaccinated birds). Passage of MDV in chicken embryo fibroblasts or duck embryo fibroblasts invariably reduces virulence, eventually to a completely non-pathogenic form. Since MDV spreads 15 by cell-to-cell contact, it is difficult, if not impossible, to isolate pure isogenic (or “clonal”) stocks of the virus, and each subsequent passage of a stock may select for new populations of genetic variants. The MDV genome contains long (UL) and short (Us) unique regions flanked by inverted repeats (IRL and IRS) and terminal repeats (TRs and TRL). The genes located in the UL and Us regions are largely homologous to those of HSV-1 (Kingham et al., 2001). Similar to HSV-l, MDV genes generally can be grouped as immediate early (IE), early and late genes, according to their timing of expression during the lytic cycle. IE genes typically encode transcription factors, some of which are required for subsequent expression of early and late genes. A small number of IE genes are activated immediately upon infection, in response to transcriptional activation, typically by the VP16 virion protein. The role of MDV VP16 (UL48), in contrast to that of HSVI VP16, is unclear, and null mutants of MDV VP16 are viable, although impaired for growth (Dorange et al., 2002). Early genes encode proteins for DNA replication while most late genes encode viral envelope glyproteins. MD has been successfully controlled by vaccination since the 19703. Initially, the most common vaccine was an antigenically related HVT strain. The emergence of more virulent strains of MDV has been a driving force in the recurring need for more effective vaccines. The current vaccine based, on an attenuated “serotype 1” strain (CVI988) is relatively effective against very virulent strains of MDV (WMDV). The major concern for vaccine based anti-MDV strategies is that vaccines do not induce a ‘sterile immunity’ in the vaccinated host, that is, vaccination generally blocks tumorigenesis but not viral l6 replication. Thus, MDV continues to multiply and evolve in vaccinated flocks. In addition to escaping vaccine control, MDV also appears to be evolving greater virulence and the ability to generate new and more acute disease symptoms (Nair, 2005; Witter, 1997). It is thought that vaccine development may be approaching its biological threshold (Witter and Kreager, 2004), although recent studies show improved vaccine protection by a meg-deleted MDV serotype 1 strain (Lee et al., 2008). We believe that new control measures not based on vaccination warrant further research. Recently, several MDV strains have been cloned as recombinant DNAs, either as bacterial artificial chromosomes (BACs) or cosrnids, which allow researchers to study viral and host factors that are critical for viral replication, disease development and lymphomagenesis. It’s been demonstrated that the gB, gE, g1, gM, UL49.5 and UL49- VP22 genes are required for MDV growth (Dorange et al., 2002; Schumacher et al., 2000; Schumacher et al., 2001; Tischer et al., 2002). Subsequent studies have shown that several other MDV genes are necessary either for full infectivity and/or for pathogenesis and oncogenesis in viva (U53, vLIP, vTR, vZL-8, RLORF4, meq, gC, LORFII and the pp38 gene; reviewed in Osterrieder et al. 2006). J arosinski et a1. (2007) have also shown that U52, UL13 and gC genes are required for horizontal transmission. We initially explored RNAi against the g3 gene (UL2 7), which encodes a major surface glycoprotein easy to detect by a monoclonal antibody. In herpesviruses, gB is the most conserved glycoprotein and essential for viral entry (Cai et al., 1988). Recent structural analysis of HSV-1 demonstrated that gB is the effector of fusion between the virus and host cell (Heldwein et al., 2006). In HSV-1, the interaction of g8 and its cellular receptor, paired immunoglobulin-like type 2 receptor alpha, is required for HSV-l infection (Satoh et al., 17 2008). Palliser et al (2006) showed that siRNA targeting HSV-2 gB inhibits viral replication and protects mice from lethal infection. Subrarnanian et al (2008) used a transient siRNA system to inhibit the Kaposi's sarcoma-associated herpesvirus gB gene and showed a significant reduction in viral infectivity in cell culture. Comparison of MDV glycoproteins with HSV-1 reveals that gB exhibits the greatest level of conservation (Afonso et al., 2001). Baigent et a1 (2006) generated a gB-deleted HVT BAC clone, and its inability to form plaques in cell culture indicates that gB is essential for HVT to spread fiom cell to cell. The construction of a random transposon-insertion mutant library of a MDV BAC clone further confirms that gB is required for viral replication (Chattoo, Stevens, and Nair, 2006). In addition to gB, we also have tested RNAi directed against an IE gene, ICP4. In HSV, ICP4 is a transcriptional regulator, proven to be essential for HSV replication (Compel and DeLuca, 2003; Grondin and DeLuca, 2000). The MDV ICP4 gene lies in the inverted repeat that flanks the unique short region of the genome, so there are two copies of ICP4 per MDV genome (Anderson, Francesconi, and Morgan, 1992). Several studies suggest that suppression of IE gene expression by latency-associated transcripts (LATs) antisense to the MDV ICP4 plays an important role in MDV latency (Cantello, Anderson, and Morgan, 1994; Cantello et al., 1997; Li et al., 1998). Burnside et al (2006) showed that MDV- encoded miRNA (MDV-miR-6 to -8) are located in the 5' end of the LAT region. Xie et a1 (1996) showed that expression of antisense RNA to ICP4 inhibited the proliferation of MDV-transformed MSBl cells by 77-fold. Random transposon mutagenesis study suggested that ICP4 is essential for MDV] growth in chicken embryo fibroblasts (Chattoo, Stevens, and Nair, 2006). We believed that by targeting ICP4, we 18 might potentially block or reduce the transcription of delayed early and late genes and thus inhibit viral replication. Retroviral vectors for RNAi Retroviral vectors are attractive for RNAi studies since they transfer genes efficiently, and, usually, a single proviral copy of the cassette is stably integrated into host cell DNA. Several retroviral vectors have been shown to effectively express shRNA in mammalian cells (Brummelkamp, Bemards, and Agarni, 2002a; Hemann et al., 2003; Qin et al., 2003; Rubinson et al., 2003; Stewart et al., 2003; Tiscornia et al., 2003). However, most of these vectors are replication-defective and require packaging cell lines or helper virus for growth. Drawbacks to this approach include relatively low titers, the potential for recombination with helper virus, and the fact that gene transfer in viva is usually confined to a lower percentage of cells in the region of injection. We chose to deliver shRNAs and/or shRNA-mirs using replication-competent retroviral vectors based on the Schmitt Ruppin A (SR-A) strain of Rous sarcoma virus (RSV), a member of the avian sarcoma leukosis virus family (Petropoulos and Hughes, 1991). RSV contains the full complement of viral genes (gag, pol, and env) as well as the src oncogene. In the RCAS vectors (replication competent ALV long terminal repeat [LTR] with splice acceptor), the region encoding src (dispensable for viral replication) has been replaced by a synthetic DNA linker. Foreign genes inserted into this linker are expressed from the viral LTR promoter via a sub-genomic splice site. The RCAN vectors (replication competent ALV long terminal repeat [LTR] with no splice acceptor) differ from the corresponding RCAS vectors in that they lack the splice site. In the RCAN 19 vectors, genes of interest are inserted along with the internal promoter of choice. Higher titer viruses subsequently have been generated by replacing the RSV SR-A pal gene with the pol gene of the Bryan strain of RSV. These vectors are termed RCASBP or RCANBP (Federspiel and Hughes, 1994). RCASBP and RCANBP vectors are well suited as delivery vectors for our RNAi system: (1) they routinely achieve high titers in avian cells (107). In fact, in avian cells, the RCAS vectors infect nearly all the replicating cells in a culture dish since they are competent to produce infectious viruses containing the gene of interest without the need for helper viruses or cells; (2) they are available in different subgroup types based on different env genes encoding envelope glycoproteins, which allow for delivery of a gene of interest using one subgroup, followed by later unrestricted challenge with another subgroup; (3) RCASBP/RCANBP vectors are available in different subgroup types (different env genes), and chicken lines exist that are resistant to infection by one or more subgroups (e.g., C/A birds are resistant to subgroup(A)). RCASBP/RCANBP vectors have been used in several gain- of-function studies (Holmen and Federspiel, 2000; Holmen, Melder, and Federspiel, 2001; Holmen et al., 1999). We and others have subsequently demonstrated several gene silencing strategies based on RNAi-RCAS vectors (Chen et al., 2007; Chen et al., 2008; Das etal., 2006; Harpavat and Cepko, 2006; Mayr et al., 2008). Harpavat and Cepko (2006) delivered RCAS expressing shRNA against the thyroid hormone processing enzyme deiodinase 2 using a mouse U6 promoter in the-chick retina and achieved about a 50% reduction in deiodinase 2 enzyme activities. Mayr et a1 (2008) transduced a TD-2 mouse pancreatic cancer cell line using RCAS vectors expressing luciferase reporter genes. They injected healthy mice with TD- 2 cells and then RCAS expressing shRNA against EGFR (via the human H1 promoter), 20 an oncoprotein for TD-2 cells. They showed a 37% reduction in luciferase expression up to 50 days post injection, indicating that RCAS was able to deliver RNAi inhibiting tumor progression in viva. 21 REFERENCES Adkins, H. B., Brojatsch, J ., and Young, J. A. T. (2000). 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Westerhout, E. M., ter Brake, O., and Berkhout, B. (2006). The virion-associated incoming HIV-1 RNA genome is not targeted by RNA interference. Retroviralagy 3, 57. 28 Witter, R. L. (1997). Increased virulence of Marek's disease virus field isolates. Avian Dis 41(1), 149-63. Witter, R. L., and Kreager, K. S. (2004). Serotype 1 viruses modified by backpassage or insertional mutagenesis: approaching the threshold of vaccine efficacy in Marek's disease. Avian Dis 48(4), 768-82. Zhang, H., Kalb, F. A., J askiewicz, L., Westhof, E., and Filipowicz, W. (2004). Single processing center models for human Dicer and bacterial RNase 111. Cell 118(1), 57-68. 29 CHAPTER 2 Chen, M., Granger, A. J., VanBrocklin, M. W., Payne, W. S., Hunt, H., Zhang, H., Dodgson, J. B., and Holmen, S. L. (2007). Inhibition of avian leukosis virus replication by vector-based RNA interference. Virology 365(2), 464-472. 30 ABSTRACT RNA interference (RNAi) has recently emerged as a promising antiviral technique in vertebrates. Although most studies have used exogenous short interfering RNAs (siRNAs) to inhibit viral replication, vectors expressing short hairpin RNAs (shRNA- mirs) in the context of a modified endogenous micro-RNA (miRNA) are more efficient and are practical for in viva delivery. In this study, replication competent retroviral vectors were designed to deliver shRNA-mirs targeting subgroup B avian leukosis virus (ALV), the most effective of which reduced expression of protein targets by as much as 90% in cultured avian cells. Cells expressing shRNA-mirs targeting the tvb receptor sequence or the viral env(B) sequence significantly inhibited ALV(B) replication. This study demonstrates efficient antiviral RNAi in avian cells using shRNA-mirs expressed fiom pol II promoters, including an inducible promoter, allowing for the regulation of the antiviral effect by doxycycline. 31 INTRODUCTION RNA interference (RNAi) is an evolutionarily conserved mechanism of gene silencing that has shown promise as an antiviral strategy in vertebrates (Cullen, 2006 and Hu et al., 2002). RNAi is mediated by short RNA “guide strand” Oligonucleotides that bind to, suppress translation of, and sometimes induce cleavage of complementary mRNAs. RNAi generally arises from two types of intermediary molecules: siRNAs and miRNAs (Tang, 2005 and Valencia-Sanchez et al., 2006). siRNAs are ~ 22-bp double- stranded (ds) Oligonucleotides that can be naturally processed from longer dsRNA but also can be chemically synthesized and introduced directly into cells or transcribed as short hairpin RNAs (shRNAs) that are then processed to siRNA (Nakahara and Carthew, 2004). Initially, shRNAs were most often transcribed fiom polymerase III (pol III) promoters delivered by exogenous DNA plasmids or retroviruses. More recently, endogenous miRNA genes, that are transcribed as larger RNA precursors from pol II promoters, have been found to more effectively generate an RNAi effect (Boden et al., 2004, Das et al., 2006 and Sun ct al., 2006). In this case, the stem region of a miRNA gene is replaced with the target sequence and its guide RNA complement (termed shRNA-mirs; Zeng et al., 2002, Silva et al., 2005 and Dickins et al., 2005). RNAi appears to play a large role in normal viral life cycles. Many viruses, including human immunodeficiency virus (HIV), herpesviruses and some adenoviruses, contain miRNAs as part of their genomes that may be used to suppress host genes or regulate their own gene expression (Cullen, 2006 and Sullivan and Ganem, 2005). Also, RNAi has been shown to be an innate antiviral defense mechanism in plants, insects and 32 nematodes, and recent evidence indicates that higher vertebrates, including humans, use miRNAs to protect against certain viral infections (Lecellier et al., 2005). Experimental RNAi strategies have been used successfully to inhibit viral replication. Hu et al. (2002) showed that siRNAs containing sequences of the gag gene of avian leukosis virus (ALV), when electroporated into chicken embryos, were effective at slowing viral propagation. RNAi inhibition of HIV has been studied more extensively, and siRNAs against the host cell CD4 co-receptor, the HIV long terminal repeat (LTR), and the viral p24 structural protein, among others, have all been successfirl in reducing the growth of HIV (Martinez et al., 2002 and Novina et al., 2002) . However, the transient nature of siRNAs and difficulties in delivery present major hurdles for in viva application of this approach. RNAi delivered by DNA plasmids can be longer lasting but faces similar obstacles. Retroviral vectors provide the opportunity to stably integrate the RNAi cassette into the recipient cell genome, usually as a single copy provirus, and such vectors recently have been used in large scale RNAi mutagenesis of endogenous genes (reviewed in Chang et al., 2006 and Root et al., 2006), but their use in inhibiting viruses has been limited. This study describes a vector-based RN Ai strategy against an important pathogen of chickens, ALV. ALV is an oncogenic retrovirus divided into different subgroups, designated A—J, based upon differences in the surface region (SU) of its envelope glycoprotein, encoded by the env gene (Coffin et al., 1997). Each viral subgroup varies in its prevalence and toxicity, with ALV(A), ALV(B), and ALV(J) cited as the most dangerous to agricultural chicken populations (Fadly and Smith, 1999). ALV enters a 33 host cell through interactions between the viral envelope glycoprotein and a host cell receptor protein, which may differ between viral subgroups (Coffin et al., 1997). ALV(B) infection, the subgroup targeted in this study, is mediated through the host-cell receptor protein CAR-1, encoded by tvb, a member of the tumor necrosis factor receptor (TNFR) superfamily (Brojatsch et al., 1996 and Brojatsch et al., 2000). Here, we explore reducing expression of both a host receptor gene, tvb, and a viral gene, env(B), to inhibit ALV replication. These two genes encode the proteins that mediate the virus—host cell interaction, the initial step in viral infection. We have modified a previously described replication-competent ALV(A) RN Ai vector (Bromberg-White et al., 2004) to deliver gene-specific shRNA-mirs in the context of the endogenous chicken miR-30a gene. We demonstrate that our vector system shows potential as an antiviral RNAi agent in avian cells and is amenable to inducible promoter-driven expression. 34 RESULTS GFP fluorescence in DE] cells is decreased by RCASBP(A) shRNA-mirs Retroviral entry vector plasmids were constructed containing 417 bp of the chicken miR-30a gene, slightly modified to provide useful restriction sites into which synthetic target sequence duplexes could be inserted (Materials and methods). Various entry vectors contained either no promoter or various pol III promoters (mouse U6, human H1, chicken U6-l, or chicken U6-2; Kudo and Sutou, 2005) with or without the 27 bp leader sequence normally present between U6 promoters and the start of U6 RNA (Paddison et al., 2004). Entry vectors with no promoter were recombined into a vector based on RCASBP(A) (replication competent, ALV LTR promoter, Splice acceptor, Bryan-strain pal gene, sub-group A; Bromberg-White et al., 2004; Figure 2-1). RCASBP vectors are designed such that the inserted sequence is expressed as a sub-genomic, spliced RNA transcribed from the viral LTR (Hughes et al., 1987). The shRNA-mir sequences downstream of pol III promoters were moved into the corresponding RCANBP(A) vector that lacks the relevant splice acceptor such that insert transcription depends on the internal promoter provided. Two target sequences within GF P were used (Materials and methods), along with a control sequence of the same length and similar base composition, but with a scrambled sequence. These viral vectors were propagated in DF-l avian fibroblasts stably expressing GF P. Since these viruses are replication competent in avian cells, they quickly spread throughout culture thereby delivering the shRNA to the majority of the cells. Analysis of GFP expression by fluorescence-activated cell sorting (FACS) revealed that none of the RNA polymerase III promoter constructs reduced GFP expression. In addition, shRNA expression fiom the pol III constructs was 35 not detectable by PAGE—Northern (data not shown). In contrast, both GFP target sequences achieved an 85—90% reduction in GFP fluorescence (Figure. 2-2) when transcribed from the viral LTR promoter in RCASBP(A), indicating a substantial and specific RNAi response. 36 LTR SD ‘1’ gag pol SA env SA LTR _. J MWhfiWflfil-fldiwga- Wéuré-zgé'alfirqufiw .-.-:- .-- ‘7 u- -'-u RCASBP(A)miRN A h RCANBP(A)U6-miRN A LTR saw RCANBP(A)TRE-miRN A LTR CMV Human Ubiquitin-C Promoter GFP WRE LTR FGlZ-CMV [If _, Figure 2-1. Diagram of avian shRNA-mir vectors. Top line: shRNA-mirs inserted into the RCASBP(A) vector are expressed from the viral LTR via a spliced RNA transcript. Within the mir-30a gene, a short-hairpin loop containing the target sequence and its complement flanking the native mir-30a loop sequence is synthesized and inserted between the Mlul and NcaI restriction sites. Second line: shRNA-mirs inserted into RCANBP(A) are meant to be transcribed from chicken U6 (or other pol III) promoters. Third line: shRNA-mirs inserted into RCANBP(A)TRE—miRNA are transcribed from a TRE (tetracycline-regulated) promoter (Holmen and Williams, 2005). Fourth line: shRNA-mirs inserted into the FGlZ-CMV defective lentiviral vector are transcribed from the CMV promoter. LTR — long terminal repeat; ‘1’ — packaging signal; SD — splice donor; SA — splice acceptor; attR — Gateway® recombination sites; WRE — Woodchuck Responsive Element. 37 160 200 n l n 120 a l n Counts 200 120 160 nlgnnl.‘ Counts 10‘ o 104 Figure 2-2. RNAi vs. GFP using RCASBP(A)shRNA-mir. DF-l cells stably expressing GFP were transfected with one of two RCASBP(A)shRNA-mir vectors containing sequences (Supplemental Table 2-1) directed against GFP and fluorescence was analyzed by FACS. The shaded area represents DF-l cells transfected by a shRNA-mir with a scrambled control sequence while the unfilled curve represents cells transfected with the (A) GFP-1 shRNA-mir or (B) GFP-2 shRNA-min 38 ALV(B) resistance through silencing of the tvb host receptor To reduce expression of the ALV(B) receptor, tvb, four different target sequences complementary to tvb” mRNA were tested. The DF-l cell line is homozygous for tvb”, an allele that allows infection by ALV(B) and (D), but not (B) (Klucking et al., 2002). The various tvb shRNA-mir cassettes were delivered to DF -1 cells using the RCASBP(A) retroviral vector (transcribed from the proviral LTR). After viral titer peaked, tvb expression was assessed by Western blot using supernatant fiom cells expressing the SU(B) protein fused to a rabbit immunoglobulin tag (SU(B)—rIgG) as a primary antibody. Lysates from mock-transfected cells, cells transfected with a shRNA-mir containing a scrambled sequence, and quail QT6 cells that do not express tvb were included as controls. The four shRNA-mir sequences reduced tvb expression to varying degrees, with two of the targets (vari2 and cshll) reducing expression significantly as compared with the scrambled shRNA-mir control (Figure 2-3A). RT—PCR measurements demonstrated that tvb mRNA levels are also reduced by these two constructs, although not to the same extent as tvb protein levels (data not shown), consistent with the expected inhibition of translation in addition to an associated reduction in mRN A levels. 39 Mock VARIl VARIZ CSHLl CSHLZ Scram QT6 or-Tvb I or-Tubulin w Relative ALV(B) Titer 0- Mock VARIl VAR12 CSHLICSHL2 Scrambled Figure 2-3. RCASBP(A)shRNA-mir directed against tvb reduces protein expression and inhibits ALV(B) entry. Four different shRNA-mir sequences against tvb” (VARIl, VARIZ, CSHLl, CSHL2; Supplemental Table 2-1), along with a scrambled control sequence, were delivered to DF-l cells via the RCASBP(A) vector. (A) Western blot of tvb expression fi'om cells infected with RCASBP(B)shRNA-mir vectors. Mock transfected cells and QT6 cells provide additional positive and negative controls, respectively. (B) RCASBP(B)AP titer on DF-l cells infected with RCASBP(A)shRNA- mir vectors, relative to the titer on mock-infected DF-l. The MOI of the undiluted RCASBP(B)AP challenge virus was 4.0. Histogram bars labeled with different letters are significantly different (P < 0.05). 40 To determine if the reduced tvb expression was sufficient to interfere with viral entry, a viral challenge assay was performed. DF-l cells expressing the tvb shRNA-mir sequences were infected with RCASBP(B)AP virus, a form of ALV(B) that expresses an alkaline phosphatase (AP) reporter gene. RCASBP(B)AP infectivity was then assayed using a short-term AP titer assay, which measures AP gene expression fi'om viruses that successfully gained entry into the cells. The level of interference to viral entry correlated with the level of reduction in tvb expression. Viral infectivity was significantly (P < 0.01) reduced, about 2.5-fold by the cshll shRNA-mir and 2-fold by vari2, when compared to mock-transfected DF-l cells (Figure 2-3B). The other two targets and a control scrambled sequence target did not significantly reduce ALV(B) infectivity. Expression of shRNA-mirs fiom pol II promoters allows for the use of various inducible promoters that can regulate the subsequent RNAi effect. Among other advantages, this provides a control that demonstrates that any resulting phenotype is dependent on the expression of the shRNA-mir and is not an unrelated response to the delivery vector. We transferred the shRNA-mir cassettes shown to be effective against tvb to the RCANBP(A)TRE—miRNA vector (Figure 2-1) in which the shRNA-mir cassette is expressed from a synthetic promoter regulated by a tetracycline response element (TRE) that is inhibited in the presence of a tetracycline tet-off transactivating protein and doxycycline (Holmen and Williams, 2005). As shown in Figure 2-4, a similar reduction in titer of the RCASBP(B)AP challenge virus was observed with these vectors as was seen when the shRNA-mirs were expressed fiom the viral LTR (Figure 2-3B). However, in the presence of doxycycline and the tet-off regulatory protein, the RNAi 41 effect was completely abolished (Figure 2-4). This demonstrates the utility of the RCANBP(A)TRE—miRNA vector system and confirms that the specificity of the antiviral effect is due to the expression of the shRNA-mir cassette. 42 0.8 0.6 0.4 Relative ALV(B) Titer 0.2 0 1 CSHLl Scram VARIZ MOCK CSHLl Scram VARIZ Tet-0ff + + + - + + + Dox + + + - - - _ Figure 2-4. Regulated promoter-driven inhibition of ALV(B). The miRNA sequences previously shown to be effective against tvb“ (VARIZ, CSHLI) were delivered to DF-l cells via the RCANBP(A)TRE—miRNA vector (Figure 2-1) that includes a regulated promoter whose activity is inhibited in the presence of doxycycline and the tet-off transactivating protein. These cells were challenged with ALV(B)-AP as in Figure 2-3 both in the presence (+) or absence (—) of doxycycline (Dox). Histogram bars labeled with different letters are significantly different (P < 0.05). 43 Pathogen-derived resistance through shRNA-mir silencing of env(B) The ALV(B) surface glycoprotein (SU), encoded by env(B), was targeted with five different shRNA-mir sequences (Materials and methods) predicted by two different algorithms. To determine which shRNA-mir reduced. SU(B) expression most effectively, RCASBP(A)—shRNA-mir vectors containing these target sequences were propagated in DF-l cells stably expressing SU(B)—rIgG. After the viral titers peaked, SU(B)—rIgG expression was assayed by Western blot (Figure 2-5A). The 510 shRNA-mir reduced SU(B)—rIgG expression very effectively, followed by the 112 shRNA-min Surprisingly, the 854 shRNA-mir that was chosen by both algorithms showed no decrease in expression as compared with the scrambled sequence control. Mock Scram 112 214 408 510 854 I'll _ .5 fl ., ._ _ (rt-SU(B) » ' ' a-Tubulin B 5 10 Scrambled Mock Figure 2-5. RCASBP(A)shRNA-mir directed against env(B) reduces protein expression and limits ALV(B) infection. (A) Five different shRNA-mir sequences directed against env(B) (112, 214, 408, 510, 854; Supplemental Table 2-1) were transfected into DF-l ~ cells stably expressing SU(B)—rIgG, the surface glycoprotein encoded by env(B) fused to a rabbit immunoglobulin tag. After viral titer peaked, SU(B)—rIgG expression was assayed by western blot. Mock-transfected and scrambled shRNA-mir transfected SU(B)—rIgG cells are included as controls. (B) The 510 shRNA-mir was incorporated into the FGlZ-CMV lentiviral vector (Figure 2-1) and delivered to DF-l cells via VSV-G pseudotyping (Materials and methods). After multiple rounds of infection to maximize the fraction of infected cells, cells were challenged with RCASBP(B) at an MOI of 0.01, and viral spread was measured by ELISA for the ALV p27 capsid protein 6 days post infection. 45 To assess the corresponding effects on ALV(B) replication, we employed a defective lentiviral delivery vector with a GFP reporter gene, F G] 2-cmv (Figure 2-1), such that ALV challenge virus replication could be specifically monitored by ELISA for the ALV p27 capsid protein. The 510 shRNA-mir was inserted into the vector, to be transcribed fiom the viral cytomegalovirus (CMV) promoter. The defective virus was transfected into 293FT cells along with a plasmid that provides the vesicular stomatitis virus G protein (V SV-G) to provide a fimctional envelope protein. The VSV—G- pseudotyped delivery virus was then used to infect DF-l cells. Multiple rounds of infection were performed to maximize the number of cells expressing the shRNA-mir. Analysis of GFP fluorescence indicated ~ 50% of the infected DF-l cells were GFP positive. These cells then were infected with RCASBP(B)AP at a multiplicity of infection (MOI) of 0.01, 0.1, and 1.0. The cells were incubated in the presence of the challenge virus for 2 days, after which the media was replaced. ALV(B) propagation was assayed by ELISA. No significant difference was observed in viral spread between the 510 and scrambled shRNA-mirs at MOI values of 0.1 and 1.0 (data not shown). However, at an MOI of 0.01, viral replication was reduced. After 6 days, the level of p27 capsid protein detected in the cells expressing the 510 shRNA-mir was approximately half that detected in cells expressing the scrambled shRNA-mir and a third that of the mock-transfected cells (Figure 2-SB). 46 DISCUSSION We have demonstrated the first use of retroviral vector-based RNAi against a viral pathogen in cultured chicken cells. Through the use of a shRNA-mir gene delivered via a replication competent retroviral vector, RCASBP(A), we successfully decreased expression of two genes necessary for ALV(B) replication, tvb and env(B). This decreased expression resulted in reduced ability of ALV(B) to infect and propagate in DF-l cells. In the case of the env(B) viral target, we were unable to use a replication- competent ALV(A) delivery vector because the vector alone reduced ALV(B) replication, presumably due to phenotypic mixing (Okazaki et al., 1975 and Choppin and Compans, 1970). Thus, we resorted to a defective lentiviral vector that infected ~50% of the DF-l cells. This probably contributes to the fact that we observed an antiviral effect only at low MOI, even though the 510 construct very substantially reduced env(B) expression when delivered by the RCASBP(A) vector. In addition, the shRNA-mir was expressed from the CMV promoter in the context of the lentiviral vector, and it is possible that this promoter is less efficient than the ALV LTR in DF-l cells. Moreover, Hu et al. (2002) found that siRNAs preferentially blocked late stages of viral replication (likely by being more effective against viral mRNAs as opposed to genomic RNA). This is consistent with our observation of no effect at high MOI, which mainly requires viral entry without additional spread, but a substantial effect at low M01 in which spread depends on multiple rounds of replication. Though increased shRNA-mir delivery or transcription efficiency may be required for increased resistance at higher MOIs, the low MOI experiment may be more representative of the viral concentrations that a live chicken would typically experience. In this regard, it is of note that even modest levels of 47 receptor interference-based resistance to ALV in vitro provide substantial resistance to in viva pathogenesis (Federspiel et al., 1991 and Salter et al., 1998). By demonstrating the use of replication-competent, vector-based RNAi in chicken cells against an important viral pathogen, this study has opened the door for in viva implementation. Anti-viral shRNA-mirs could be effectively delivered to chickens via transgenics (Salter et al., 1987, Mozdziak et al., 2003 and McGrew et al., 2004) or as a part of a vaccine potentially to create viral-resistant chicken populations (Hu et al., 2002). The use of shRNA-mirs over siRNAs ensures the highest level of success by maximizing the level of gene silencing. Also, multiple shRNA-mirs can be delivered on a single transcript, so several different target sequences could be included to reduce the chance that the target virus will mutate and evade shRNA-mir silencing (Sun et al., 2006). It should be noted that significantly greater levels of reduction in tvb and env(B) protein production (Figure 2-3 and Figure 2-4) were observed than the 2- to 3-fold reductions in viral titers that result. Given that ALV(A) requires nearly non-detectable amounts of the tvb receptor to initially infect the cell, receptor protein expression may be a particularly challenging target for RNAi. In the case of env(B), only ~ 50% of the cells appear to contain the defective lentiviral vector used to deliver the env(B) shRNA-mirs for ALV(B) challenge. Furthermore, as noted above, it's expected that the RNAi effect will be less at the stage of initial infection versus downstream viral replication. In these experiments, we chose env(B) as a target since our ALV(A) delivery vector shares homology in gag and pal with the ALV(B) challenge virus. Modifying the delivery virus and/or refining the targets, along with employing multiple targets, should allow one to achieve greater inhibition of viral titers. 48 We observed significant RNAi effects only when our shRNA-mirs were expressed fiom the viral LTR, TRE or CMV pol II promoters. Das et al. (2006) reported successful transcription of a chicken mir-30a-based shRNA-rnir in DF-l cells from a chicken U6 promoter, and Harpavat and Cepko (2006) describe an RCAS vector in which shRNAs are transcribed from a murine U6 promoter. In addition to the pol HI promoters in these vectors, they also have, at least, an upstream LTR that can transcribe the RNAi insert, potentially giving rise to one or more transcripts that could be processed to siRNA. However, if this explains why the other vectors worked, it is then unclear why this effect was not sufficient in the pol III promoter vectors we examined. In several experiments using other targets as either shRNA-mirs or shRNAs, we consistently failed to see an effect in chicken cells using any of a variety of both chicken and mammalian pol III promoters. The differences in vector performance likely relate to the sensitivity of the target systems under study and/or subtle differences in the vectors. Both Das et al. (2006) and Harpavat and Cepko (2006) used RCAS backbones for their pol III-driven cassettes, whereas we always used RCAN when employing a pal III promoter. In any case, transcription from the RNA polymerase 11 promoter opens the door for further regulation of shRNA-mir expression through tissue-specific or drug-inducible promoters. The use of a tet-regulated delivery vector allowed us to confirm that the antiviral effect we observed is specifically due to expression of the shRNA-mir cassettes employed (Figure 2-4). This study has demonstrated that retroviral RNAi may be a viable method for interfering with viral infection in chickens, while further developing an enhanced vector system for efficient gene silencing in avian cells. Further study is needed to demonstrate an antiviral effect in vivo, and the efficacy of implementing such an antiviral strategy, 49 either through inclusion of the shRNA-mir in a vaccine or through creation of transgenic chicken lines, remains to be explored. 50 . MATERIALS AND METHODS Vector constructions The chicken mir-30a gene is Accession No. M10001204 at rninase and is located at chr3:85,102,239-85,102,310 in build2 of the chicken genome sequence. This segment was amplified using an overlapping PCR technique (Ho et al., 1989) to insert Mlul and Noel restriction sites at the mir-30a target region. Primers used and the sequence of the mir-30a cassette are given in Supplemental Figure 2-1. PCR was done in 100 pl containing 50 pl 2X PCR master mix (Promega Corp), 100 ng of template, and primers at 1 uM each for 2 min at 95 °C followed by 30 cycles of 1 nrin at 94.5 °C, 1 min at 65 °C and 1 min at 72 °C. The modified chicken mir-30a cassette was inserted between BamHI and NotI sites in the pENTR3C vector (Invitrogen Corp), downstream of a pal IIl promoter (mU6, H1, cU6-l, cU6-2; Kudo and Sutou, 2005) with or without the 27 bp leader sequence normally present between U6 promoters and the start of U6 RNA (Paddison et al., 2004) or without any promoter. Two complementary (99 nt) Oligonucleotides containing the desired target sequence with 5' Mlul and 3' Ncol overhangs were synthesized (Invitrogen Corp), annealed and ligated into the target region of the chicken mir-30a-derived entry vector. Specific target sequences for each gene were obtained using an in-house algorithm developed at the Van Andel Research Institute (VARI) by Matt VanBrocklin and Kyle Furge or by an on-line program from RNAi Central at the Cold Spring Harbor Laboratory. Two shRNA-mirs predicted by each program for tvb (varil, 2 and cshll , 2, respectively) were tested. shRNA-mirs targeted against env(B) were named according to their first complementary nucleotide in the gene (Accession No. M14902). Three were obtained using the on-line Cold Spring 51 Harbor Lab algorithm (214, 510, and 854), and three using the in-house Van Andel Research Institute algorithm (112, 408, 854), with the 854 target sequence selected by both algorithms. In total, 12 synthetic duplexes were employed with target sequences for GFP, tvb, env(B), and a scrambled sequence was made as a negative control. Loop and flanking sequences are identical to the corresponding sequences of the chicken mir-30a gene. Sequences for all inserts are given in Supplemental Table 2-1. A Gateway® LR reaction was used to transfer the modified shRNA-mir gene into an appropriate Gateway- compatible destination vector (Holmen and Williams, 2005) according to the instructions fi'om the manufacturer (Invitrogen Corp.) The shRNA-rnir genes preceded by a pal 11] promoter were inserted into the RCANBP(A) destination vector and the mir-30a genes with no promoter were inserted into the RCASBP(A) destination vector. All entry vector inserts were confirmed by sequencing analysis, and retroviral vector constructs were verified by restriction enzyme mapping. The pEGFP-l construct (Clontech, Palo Alto, CA) was digested with BamHI and NatI, and the 0.7 kb GFP gene fragment was cloned into the BamHI and Natl sites of pcDNA3 (Invitrogen Corp.) to create pcDNA3—EGFP. A defective lentiviral vector, FG12-cmv, was kindly provided by Maria Soengas from the University of Michigan (Verhaegen et al., 2006). FG12-cmv was converted into a destination vector by digestion with Hpal and ligation with Gateway conversion cassette C.l (Invitrogen Corp). Positive clones were selected by digestion with EcoRI and XbaI, and an LR reaction performed to insert the appropriate shRNA-mir cassette. Vectors employed for the tet-off analysis have been described previously (Holmen and Williams, 2005). 52 Cell culture DF-l and QT-6 cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM, Invitrogen Corp.) with 50 U/ml each of penicillin and streptomycin (Invitrogen Corp), 10% fetal bovine serum (FBS, Hyclone), and 0.25 pg/ml of fungizone at 39 °C or in Leibowitz's L-15 and McCoy 5A media (1:1) supplemented with 10% FBS. 293FT cells were maintained in DMEM with 1% penicillin and streptomycin, 10 % FBS, and l>< MEM non-essential amino acid solution (Invitrogen Corp.) at 37 °C. DF-l cells stably expressing GFP were selected in 500 pg/ml of G418 (Invitrogen Corp.) after transfection of the pcDNA3—EGFP plasmid. Clones were isolated using cloning cylinders (Bellco Glass Inc.), expanded, and maintained in standard medium supplemented with 500 pg/ml of G418. GFP expression was confirmed using a Becton Dickinson FACSCalibur (Lewis et al., 2001). GFP expression was also detected by fluorescence microscopy. DF-l cells stably expressing SU(B) with a rabbit immunoglobulin tag, SU(B)—rIgG, were kindly provided by Mark Federspiel. SU(B)—rIgG expression was confirmed by Western blot analysis. Virus propagation Viral propagation was initiated by transfection of plasmid DNA that contained the retroviral vector in proviral form using calcium phosphate (F ederspiel and Hughes, 1997) or SuperFect Transfection Reagent (Qiagen, Inc.) according to the manufacturer's protocol. In standard transfections, DF-l cells were plated at 30% confluency, allowed to attach (2—3 h), and 5 pg of purified plasmid DNA was introduced by the calcium phosphate precipitation method previously described (Kingston et al., 1989), followed by a 5-min glycerol shock at 39 °C (15% glycerol in the medium). Viral spread was 53 monitored by assaying culture supernatants for ALV capsid protein by ELISA (Smith et al., 1979). Virus stocks were generated from cell supernatants by centrifugation at 2000>< TBE at 200mA for 30 min using a Western blotting apparatus (Hoefer). Blots were UV-crosslinked and subsequently probed with a 5' end-labeled oligonucleotide that corresponds to the sense sequence of the shRNA used in the viral vectors. Blots were probed with 100 pmol of sense sequence, which was end-labeled with T4 polynucleotide kinase (Invitrogen) and [732P]-ATP (125 pCi) (Amersham) and purified on a G-25 MicroSpin Column (Amersham). Probed membranes were washed and exposed to autoradiography film (Kodak). Western blot analysis Cell lysates were collected in 250 pl of 85 °C SDS loading buffer (50 mM Tris— HCI pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol). The cell lysates were boiled for 10 min, vortexed vigorously and centrifuged for 10 min at 13,200Xg. Alternatively, cells were lysed in 700 pl NP-40 lysis buffer (50 mM Tris—HCI pH 8.0, 150 mM NaCl and 1% NP-40) supplemented with 1 mM phenylmethylsulphonyl fluoride (PMSF), centrifuged for 6 min at 13,200 X g and boiled in SDS loading buffer 56 for 5 min. Proteins were electrophoresed on a 10% SDS—polyacrylamide gel for 2 h at 120 V and transferred to a nitrocellulose membrane for 1 h at 25 V. The membrane was blocked in 5% non-fat dry milk in TBS-T (25 mM Tris—HCl, pH 8.0, 125 mm NaCl, 0.1% Tween 80). To detect SU(B)—rIgG, the membrane was incubated with HRP- conjugated anti-rabbit IgG (Sigma) diluted 1:1000 in TBS-T for 1 h at room temperature. To detect tvb, membranes were incubated 1 h in 0.45-pm filtered media from SU(B)— rIgG expressing DF-l cells, washed with TBS-T and incubated with HRP-conjugated anti-rabbit IgG diluted 1:1000 in TBS-T for 1h at room temperature. Enzymatic chemiluminescence (ECL) substrate (Pierce Biotechnology) was added and the membrane developed on Kodak BioMax scientific imaging film (Kodak). Images in this dissertation are presented in color. AUTHORS' CONTRIBUTIONS MC carried out the Tvb studies and helped draft the manuscript. AG carried out the Ean studies and helped draft the manuscript. MV helped develop the RNAi algorithm and generated the defective viruses. HH and HZ participated in the design of the study and performed the statistical analysis. JD and SH conceived of the study, and participated in its design and coordination and helped to draft the manuscript. ACKNOWLEDGEMENTS We thank John A. T. Young for the SU(B)—rIgG construct and Mark Federspiel for the SU(B)—rIgG stable DF-l cells. We thank Maria Soengas for the FGlZ-cmv vector 57 and Kyle Furge (Laboratory of Computational Biology) for technical assistance. We thank Stephen Hughes and Andrea Ferris for the RCASBP(B)AP vector and helpful discussions. This work was supported in part by the USDA NR1 Competitive Grants Program (2004-35204-14780), and the Van Andel Research Institute. 58 REFERENCES Boden, D., Pusch, O., Silbermann, R., Lee, R, Tucker, L., Ramratnam, B., 2004. Enhanced gene silencing of HIV-1 specific siRNA using microRNA designed hairpins. Nucleic Acids Res 32(3), 1154-8. Brojatsch, J., Naughton, J., Adkins, H.B., Young, J.A., 2000. TVB receptors for cytopathic and noncytopathic subgroups of avian leukosis viruses are fimctional death receptors. 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Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mal. Cell 9(6), 1327-1333. 62 APPENDIX A. SUPPLEMENTARY DATA BamHI caaggatcCAAACTGCCCCATGCACCTCTGCATGTAACAACACAGCAACAG AAGTTGTAGCAATCTTAACTAACCTGCTTGGAGGCCTTTGCATGTTCATAG Ml uI NcoI TGTAAAGTAAACAGCTGAGGAACGCGT_fi*t_-.-jjfaf 3- gCCAtgG ACGTCAAG AAATGTTTGACACCTAAAGCATAGTTGTCTGTTTAAAACTTCT ATTCTATTTGGTTTATCACAGACATATGAGTTTAGGCCAAACCTTTCTACG TATTACATGTGATGCCTGTGTCTAGAAACCATCCAGTTACTCATAGGAGCT TGTGCTCATCAACATCATAACCCAAAAAACTGAATACAAAGTAAAACATGA AATAACTTAGGCTTTATTTTTAACTACAAACTTTCAATTGTGTTTCCAGAA NotI "Q1’3“}fTVfi;TqufjppHiqftt," PrimerA: 5’Cn4144-LtAAACTE”FC1\T’CACCTCT3' PrimerB: 5’7"? 4 ACGCGTTCCTC AGC TCTTTACT TTAC3' PrimerC: 5' " 3 1" ' ‘a 4 Cai,-4 ThGACGT’. A. CAT‘l lTTT GACA ‘CA3' PrimerD: S'ILLYW:.-“I‘ns“*' fl" :"535ff. 3' Supplemental Figure 2-1. The chicken mir-30a cassette sequence. Capital letters indicate nucleotides amplified from and identical to the sequenced chicken (Red Junglefowl) genome (chr3:85,102,102-85,102,595 of the May 2006 Assembly). PCR primers are individually colored except that the overlapping portion of primers B and C are in a common color (green). PCR originally used primers A with B and C with D in separate reactions followed by mixing the two resulting fragments and PCR with A and D primers (Ho et al., 1989). The complete sequence above was digested with BamHI and Natl and cloned into the BamHI and Natl sites of a plasmid that previously had the chicken U6-2 promoter/leader (Kudo and Sutou, 2005) inserted into the HindIII and BamHI sites of the pENTR3C vector (Invitrogen). The version of the entry plasmid that lacks a promoter and leader was made by excising the BamHI/HindIII promoter fragment and replacing it with an adapter. shRNA-mir vectors are made by inserting synthetic duplexes (Supplemental Table 2-1) with the requisite overhanging ends into the Mlul and NcaI sites shown above. 63 an adapter. shRNA-mir vectors are made by inserting synthetic duplexes (Supplemental Table 2-1) with the requisite overhanging ends into the Mlul and NcaI sites shown above. 64 Supplemental Table 2-1. Target oligonucleotide sequences OHUEUUDH kahhgowfiwwwfihhflohuwfi thflgfiuofluwgwhwflh Dwfiwhguoofiowhhgus OuwflwhmVflJflD « « D « JD « _ I _ . E . vnw OHUEUUUH HUUEHggUgUCcH ._. 4 1.— DD afiSDCJDW‘g 43:... I 4 «D a «D A a. JD 44*) a a. #53) DJDWIHU‘JCD a #5 H JD H .1 _ D as O— W JD « J< UGHUEJJD « JJ «D « «D « J «43D « «43 « JD « : «DD «cDmUmEUUgoeog o¢8 UGHUEOUEH Ugvflhgwwkho§0 (5 49$ {5 "a '5 °o d2» 93 $08 8 ‘D\ N“ '8’ 00; 59b? "3 ‘b e: F’ cs ¢ \ N 3 , fa \ b’ {1; (\Q b’ (1% Q! ‘3 l 9,2 lo" .5 l | Q b<\ I s \ V «5" V '1: x '1 ‘0‘" \°°:\ \q’ <9 >99 60‘ 94°F <9 {EN Constructs Figure 3-3. RCASBP(A)3mirs directed against subgroup MDV gB and/or ICP4 reduces titers in CEF. miRNAs against MDV gB and/or ICP4 (Supplemental Table 1), along with scrambled control sequences, were delivered to CEF via the RCASBP(A)3mirs vector (Supplemental Figure 3-1). Targets are identified by the number of their first nucleotide within the MDV gB or ICP4 coding sequence, respectively (Supplemental Table 1). In each case, the first sequence in a series is in the miR-20b site, the second in the miR-l 9b site and the third in the miR-92-2 site. Scrl, 2 and 3 indicate different scrambled control sequences with similar base compositions to the targets. gB targets are 1874, 596 and 1518, whereas ICP4 targets are 4902 or 6170. Cultures were infected with 100 pfu of Md5 MDV. Plaques were counted at 5 dpi and normalized to the mock-infected control as 1.0. Histogram bars labeled with different letters are significantly different (P < 0.01). 80 The multi-target RNAi vector then was used to explore the effectiveness of the MDV ICP4 gene as a target. We first tested individual target sequences within ICP4. Two of four ICP4 targets chosen were able to significantly reduce plaque numbers to about 30% of the control (Figure 3-3, only the two effective targets are shown). The effective gB and ICP4 targets were then combined within single multi-target vectors. Figure 3-3 shows that the addition of the best ICP4 target sequence (6170) to one or two gB targets enhances the antiviral effect observed, with the best construct (1874-gB, 6170- ICP4, 1518-gB) reducing the MDV titer to nearly 5% of the mock-infected control (Figure 3-3). Given that RNAi reduced MDV viral replication as a whole, we expected at least a concomitant reduction in viral target mRN A levels. Indeed, reverse transcriptase PCR (RT-PCR) measurements demonstrated that reduction of gB mRNA levels correlated well with the reduction in MDV plaque number (Figure 3-4). For one of the four experiments whose results are summarized in Figure 3-3, we also measured replication of MDV viral DNA via quantitative PCR comparing the level of MDV gB gene DNA to that of the host cell GAPDH gene (Supplemental Figure 3-4). These results correlated well (correlation coefficient = 0.70, similar to, or higher than, those observed by Bumstead et al., 1997 and Baigent et al., 2005) with the titers shown in Figure 3-3, although the inhibition was somewhat less pronounced, at least for vectors containing the 1874-gB target. PCR-based methods measure all forms of the viral genome (Baigent et al., 2005), including, presumably, genomes that fail to be infectious due to a deficit in, for example, gB protein. Thus, we view inhibition of viral titers as the most relevant quantitative measure of the RN Ai effect. 81 Figure 3-4. RT-PCR of gB mRNA expression fi'om CEF expressing RCASBP(A)3mirs. CEF were infected with 100 pfu of Md5 MDV, and RNA was isolated at 5 dpi. RT-PCR was performed as described in Materials and methods. GAPDH mRNA was assayed as a loading control. Mock no RT: RT-PCR was performed using RNA fiom Mock treatment without added reverse transcriptase. In each case, the first sequence in a series is in the miR-20b site, the second in the miR-19b site and the third in the miR-92-2 site. Scrl, 2 and 3 indicate different scrambled control sequences with similar base compositions to the targets. gB targets are 1874, 596 and 1518, whereas ICP4 targets are 4902 or 6170. Targets are identified by the number of their first nucleotide within the MDV gB or ICP4 coding sequence, respectively (Supplemental Table 1). 82 DISCUSSION We have demonstrated the use of retroviral vector-based RNAi against MDV, a major pathogenic threat in chickens. Three different shRNA-mir gene constructs, delivered via a replication-competent retroviral vector, successfully reduced viral replication using targets in two very different viral genes, g8 and ICP4. The gB glycoprotein gene is known to be essential for MDV replication (Schumacher et al., 2000) and is likely involved in viral spread from cell to cell, whereas the ICP4 gene, known to be essential in other herpesviruses, acts as a transcription factor. The ICP4 effect occurs in spite of the fact that this immediate early protein is transported within infectious virions in HSVl (Y a0 and Courtney, 1989), which could provide a source of the protein resistant to any RNAi effect. However, the complementation studies of Dargan and Subak-Sharpe (1997) suggest the need for additional de nova ICP4 synthesis during the viral life cycle after the initial infection. The results reported here confirm our earlier observation (Chen et al., 2007) and those of others (Baden et al., 2004, Silva et al., 2005 and Dickins et al., 2005) that embedding the RNAi target within a pri-miRNA gene transcribed by an RNA polymerase 11 promoter provides an effective delivery method. Since many pri-miRNA genes are polycistronic (Lagos-Quintana et al., 2001), the possibility exists to express multiple RNAi target sequences within a single transcript (Y u et al., 2003, Chung et al., 2006 and Das et al., 2006). Indeed, the endogenous chicken chromosome 4 miRNA cluster that we have modified to deliver three targets originally contained six potential miRNAs, although it has not been proven that all six are expressed. Our results demonstrate that all 83 three target sites in our vector can be functional. However, combining either three different gB gene targets or one ICP4 target and two gB targets, each of which was known to inhibit on its own, did not always increase the antiviral effect in an additive fashion. For example, although the 596-gB and 1518-gB targets were effective on their own, they did not significantly enhance the antiviral effect of 1874-gB when added to the multi-target vector (Figure 3-3). On the other hand, adding 1518-gB to 1874-gB plus 6170-ICP4 produced a greater reduction in MDV titer than did 1874-gB plus 6170-ICP4 alone [a direct comparison of the 1874—6170-scrambled control result with that of 1874— 6170—1518 gave a significant difference (P < 0.05 using Student's t test), but the difference did not reach significance in the pairwise comparison of all treatment groups using Duncan's test]. Clearly, many factors modulate the effectiveness of any given viral target gene and any specific target sequence within that gene. We are currently exploring the use of additional targets and additional combinations of targets within our multi-target vector to address some of these issues. Regardless of whether multiple targets provide a substantially greater antiviral effect, this approach may also have value in making it more difficult for the virus to evade the RNAi effect through a mutation in the target sequence(s). RNAi escape mutants have been documented for HIV, poliovirus, and hepatitis B and C viruses (reviewed in Leonard and Schaffer, 2006). However viral escape mutations appear to be less frequent in DNA viruses such as MDV, and Palliser et al. (2006) found no mutations in the UL29 target region in HSV-2 in the course of blocking lethal effects of HSV-2 using RNAi in viva. Retroviral-delivered RNAi reduced both the number and size of MDV plaques. These two properties are likely due to the same central effect, since reducing the size of 84 plaques to the point that they are no longer visible will inevitably reduce the titer. In any case, both observations suggest a potentially significant antiviral effect may be obtained via in viva delivery of RNAi against MDV. We are presently engaged in experiments to test this possibility. Although the reductions in titer that we have observed are generally modest, it should be noted that genetic resistance/ susceptibility to MDV is a highly multigenic trait (Vallejo et al., 1998 and Yonash et al., 1999) involving minor contributions from many polymorphic alleles to achieve, in some cases, high levels of total resistance. Thus, even a partial block in viral replication could significantly lower the frequency and/or the severity of Marek's disease in viva. RNAi inhibition of viral replication should also reduce the rate at which MDV. evolves in the field to evade vaccination or to increase virulence (Osterrieder et al., 2006). While replication- competent vectors are unlikely to be of practical value in the field, they provide a useful model system in which to test targets that might later be incorporated into replication- defective retroviruses or other viral vectors. Anti-viral shRNA-mirs could be effectively delivered to chickens via transgenics (Salter et al., 1987, Mozdziak et al., 2003 and McGrew et al., 2004) or as a part of a vaccine potentially to create viral-resistant chicken populations (Hu et al., 2002). Additional research will be required to demonstrate an antiviral effect in viva and to overcome the barriers to implementing such a strategy. 85 MATERIALS AND METHODS Vector constructions The construction of the RCASBP(A)miRNA vector and the corresponding pENTR3C-miR30a entry vector has been described previously (Chen et al., 2007). The pENTR3C-miR30a-sphngo entry vector (Supplementary Figure 3-2) was made in an analogous fashion using the overlapping PCR technique of Ho et al. (1989) to insert Sphl and NgoMIV restriction sites into the mir-30a target region. Primers used in the construction and the resultant insert sequence are shown in Supplemental Figure 3-2. The PCR product was cleaved with BamHI and Notl and inserted into the pENTR3C vector (Invitrogen Corp.) as described previously (Chen et al., 2007). The pENTR3C-3mir entry vector was constructed using similar methodology. We chose to use the chicken miRNA cluster at 3,968,600—3,970,600 on chromosome 4 of the v2.1 build of the chicken genome sequence. The genome sequence in this region includes a gap that can be filled using two overlapping chicken EST sequences (BU384584 and BU337076) that, together, likely correspond to most, but not all, of the pri-miRNA transcribed from this cluster. BU384584 includes miRNAs from the miR- l8b, miR-20b, miR-19b and miR-92-2 families and most of a member of the miR-363 family. In addition, there is (at least) a member of the miR-106a family just upstream of the BU3 84584 cluster. We chose to use the miR-20b, miR-19b and miR-92-2 segment of this cluster, deleting the miR-363 family member, but retaining part of the presumed 3' end of the pri-miRNA in our construct. The sequence of the resulting insert and the PCR primers used to construct it are shown in Supplementary Figure 3-3. Two PCR duplexes 86 were generated that were cleaved with, respectively, HindIII and AvrII and AvrlI and NotI, and then were cloned into HindIII—NotI-cut pENTR3C in a three-way ligation. pENTR3C-3mir contains the complete miR-l9b family member gene which can be removed and replaced using BspEI/EcoRI digestion and two small filler sequences at the sites of the miR-20b and miR-92-2 family members flanked by unique AvrII and BglII and BamHI and AgeI sites, respectively (Supplementary Figures 3-1 and 3-3). RNAi target sequences were chosen by computer analysis as described previously (Chen et al., 2007). Target sequences were embedded within oligonucleotides designed to mimic, as nearly as possible, the endogenous flanking and loop miRNA sequence(s) that were obtained in polyacrylamide gel-purified form from commercial sources (Invitrogen Corp. or Integrated DNA Technologies). Duplexes were annealed and ligated into appropriately-digested entry vectors. Oligonucleotide sequences used are listed in Supplementary Table 1. All entry vector inserts were confirmed by DNA sequence analysis, after which the relevant insert was transferred into the RCASBP(A) destination vector using a Gateway® LR reaction according to the manufacturer's recommendations (Invitrogen Corp.). Retroviral vector constructs were verified by restriction digestion using appropriate unique sites within insert cassettes. For the RCASBP(A)shRNA-mir 1874 construct, versions were tested that either contained or lacked an internal U6 promoter in addition to the viral LTR, with only the results of the former shown in Figure 3-1 and Figure 3-2. No significant difference in effect was observed between the two constructs. All other constructs lack the U6 internal promoter. 87 Cells and viruses DF-l cells were maintained in Leibowitz's L-15 and McCoy 5A media (1:1) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals), 50 pg/ml of gentarnicin (Invitrogen), and 0.25 pg/ml of fungizone at 39 °C. CEF cells were maintained in the same media supplemented with 4% calf serum (Invitrogen Corp). SOgE cells were maintained in Dulbecco's modified Eagle's medium with 7.5% FBS with selection in 1 mg/ml of G418 (Invitrogen Corp.) once every five passages (Schumacher et al., 2002). The serum content for MDV- or HVT-infected cells was reduced to 1% when cells reached confluence. Mdll (80 passages in CEF) and Md5 (42 passages in CEF) MDV strains and the FC126 strain of HVT were obtained from Richard Witter and Mohammad Heidari, USDA-ARS, Avian Disease and Oncology Lab (ADOL). The Mdll MDV used to infect SOgE cells was passed in these cells four times prior to use. Delivery of RCAS and RCAN vectors into cell culture Propagation of RCASBP(A) vectors in DF-l cells was initiated by transfection of plasmid DNA containing the retroviral provirus using SuperFect Transfection Reagent (Qiagen, Inc.) according to the manufacturer's protocol. Viral spread was monitored by assaying culture supernatants for ALV capsid protein by ELISA (Smith et al., 1979). RCASBP(A) stocks were generated fiom cell supernatants by centrifugation at 1500 Xg for 10 min at 4 °C and stored in aliquots at - 80 °C. RCASBP(A) vectors were delivered into CEF cells by infection of virus grown on DF-l cells. Briefly, 106 CEF were seeded on 10 cm dishes and allowed to attach overnight. Viral stocks were added to DF-l media at a MOI of 1.0. CEF were then incubated in virus-containing media with 4 pg/ml polybrene for 24 h before being replaced with fi'esh medium. Sample supernatants were 88 collected after one passage and infection was monitored by ELISA as described above. Prepagation of RCASBP(A) vectors in SOgE cells was initiated by transfection of plasmid DNA using a Nucleofactor Device with the manufacturer-supplied Basic Fibroblast Kit (Amaxa Biosystems). RCASBP(A) spread was monitored using a Becton- Dickinson FACSCalibur (BD Biosciences) machine using a subgroup(A) ALV-specific antibody (RAV-l, a gift fi'om Lucy Lee, ADOL). GFP expression from the RCASBP(A)- GFP vector was also detected by fluorescence microscopy as described previously (Chen et al., 2007). Plaque assays DF-l cells expressing shRNA-mir constructs were infected by FC126 HVT as follows: 5.0 X 105 cells were seeded on 6-well plates and allowed to attach overnight. Four hundred plaque forming units (pfu) were added in DF-l media followed by incubation for up to 4 dpi. Plaques were observed and counted under a LEICA DM IRB/E inverted microscope (Leica Wetzlar), and images were captured by a DEL-750CE digital system. Plaque assays were repeated four times (shRNA-mir30a) or three times (shRNA-mir30a-sphngo), each time in duplicate (Table 1). CEF and SOgE cells expressing shRNA-mir constructs were infected with 100 to 400 pfu of Mdll or Md5 strains of MDV in a similar fashion, except that plaques were counted at 5 dpi. Plaque assays employing RCASBP(A)miRNA (Figure 3-1) were repeated twice (CEF cells) or three times (SOgE cells). Plaque assays employing RCASBP(A)3mirs (Figure 3-3) were repeated four times. 89 Immunofluorescence assay DF-l cell monolayers (4 dpi with 400 pfu of FC126 strain HVT) were fixed with ice-cold 40% ethanol and 60% acetone and incubated with the primary L78 antibody (gift from Lucy Lee, ADOL) for 30 min as described (Lee et al., 1983). Cells were washed three times with phosphate-buffered saline (PBS) and incubated with the secondary antibody, FITC-labeled goat anti-mouse IgG (MP Biomedicals) for 30 min. After three washes with PBS, cells were examined under the fluorescent microscope. CEF cells were infected with Mdll strain MDV at 100 or 150 pfir and incubated up to 5 days. Plaques were stained in a similar manner except that the primary antibody was IAN86 (gift from Lucy Lee, ADOL). Plaque size measurement DF -1 cells previously treated with RCASBP(A)miRNA-sphngo were infected by 400 pfu of FC126 HVT. DF-l cells monolayers (4 dpi) in 6 well plates were then fixed with ice-cold 40% ethanol and 60% acetone. Plaque images were captured using a DEL- 750CE digital system. CEF previously treated with RCASBP(A)miRNA were infected by 100 to 400 pfu of Mdll strain MDV in a similar fashion, except that plaques images were captured at 5 dpi. For size comparisons, 8 to 15 randomly chosen plaques (a sufficient number to achieve statistical significance according to pilot experiments) for each treatment were analyzed using ImageJ (fi'eeware fi'om the National Institutes of Health). To ensure random sampling, all plaques in a 10 mm diameter circle in the center of the plate were measured; if fewer than 8 plaques were found in this area, the diameter of the circle was increased to 15 mm. Plaque images were first processed using Image] to increase the picture quality. Plaque size measurement was performed according to the 90 Measuring and Counting Objects Instruction. The unit and scale were set at 1.058 pixels/ pm based on a known micrometer ruler. Both HVT and MDV plaques were measured fi'om 3 independent experiments. Flow cytometry For flow cytometric analysis of RCASBP(A) propagation in SOgE cells, cells were incubated with the primary antibody, RAV-l , for 30 min at 4 °C. Cells were washed three times with DMEM containing 1% FBS followed by centrifugation (1000 xg, 5 min). Cells were then incubated with the secondary antibody, FITC-labeled goat anti- mouse IgG (MP Biomedicals), for 30min at 4 °C. After three washes, cells were analyzed using a Becton-Dickinson FACScaliber flow cytometer and CellQuest Pro software (BD Bioscience). Reverse transcriptase PCR (RT-PCR) CEF expressing various RCASBP(A)-3mirs were challenged with 100 pfu of Md5 strain MDV. RNA was collected at 5 dpi and treated with DNase I using the RNeasy kit according to manufacturer's instructions (Qiagen, Inc.). One micrograrn of RNA was reverse transcribed with Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen Corp,) in a 20- pl reaction volume. Amplifications of g8 and chicken GAPDH sequences were carried out in separate reactions using the following primers: GAPDH forward, TGACAAGTCCCTGAAAATTGTCA; GAPDH reverse, CAAGGGTGCCAGGCAGTT; gB forward, CCAGTGGGTTCAACCGTGA; and g8 reverse, CGGTGGCTI‘TTCTAGGTTCG (gifts from Robert Silva, ADOL). PCR was done in 50 pl containing 25 pl 2>< PCR master mix (Promega Corp.), 2 pL cDNA, and 91 primers at 2.5 pmol each for 2 min at 95 °C followed by 20 cycles (24 cycles for gB) of l min at 95 °C, 30 s at 52.5 °C (57 °C for gB) and 30 s at 72 °C. cDNA prepared from mock infection minus reverse transcriptase (no - RT Mack) and from CEF without Md5 (CEF) were used as negative controls. Quantitative real time PCR (qPCR) assay for viral genomes CEF expressing various RNAi constructs were challenged with 100 pfu of Md5 strain MDV. DNA was collected at 5 dpi using the DNeasy Blood & Tissue kit and treated with RNase 1 according to manufacturer's instructions (Qiagen, Inc.). DNA was diluted to a concentration of 20 ng/ pl for use in the qPCR assay. pCRBlunt (Invitrogen Corp.) plasmids (gifts from Dr. Robert Silva, ADOL) containing MDV gB and chicken GAPDH genes were used to construct standard curves. A series of lO-fold dilutions of plasmid DNA in 5 ng/ pl sheared salmon sperm DNA (Sigma), from 106 to 10 copies per pl, was used to generate standard curves. Amplifications of gB and chicken GAPDH sequences were carried out in separate reactions using the following primers and probes: gB forward, CGGTGGC'I'I'I'I‘CTAGGTTCG; gB reverse, CCAGTGGGTTCAACCGTGA; gB probe, FAM- CA'I'I'1'1'CGCGGCGGTTCTAGACGG-TAMRA; GAPDH forward, ACAGAGGTGCTGCCCAGAA; GAPDH reverse, ACTTTCCCCACAGCCTTAGCA; GAPDH probe, VIC-TCATCCCAGCGTCCACT-TAMRA (gifts fi'om Dr. Robert Silva, ADOL). Real-tirne qPCR assays were performed using an ABI 7900 HT machine (Applied Biosystems). Each reaction contained 0.5 pM of each primer and 0.2 pM of the corresponding probe, 7.5 pl TaqMan® Universal PCR Master Mix (Applied Biosystems) and 3 pl DNA template in a total reaction volume of 15 pl. The cycling parameters 92 consisted of 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for l min. Viral loads were expressed as the copy number of gB divided by that of GAPDH. Reactions were done in duplicate. The correlation coefficients (R2 values) of the standard curves were 0.999 (GAPDH) and 0.997 (gB). PCR amplification efficiency was > 0.96. Statistical analysis The reduction in HVT and MDV titer due to RNAi constructs was analyzed with a simple general linear model in which the RNAi treatment was treated as a fixed variable. The statistical analysis was accomplished with the SAS for Windows v9.1.3 (SAS Institute Inc., 2004). The differences in average viral titers among the RNAi treatments were compared with Duncan's multiple range tests. For convenient visual evaluation, however, the relative average virus titer for each of the RNAi treatments was calculated by dividing each by the average titer of the negative control, mock infection. The standard errors for the relative average HVT or NHDV titers were estimated from the variances and covariances corresponding to the average titer ratios between the RNAi treatments and the negative control (mock) following an approximate procedure as described by van Kempen and van Vliet (2000). The reduction in HVT and MDV plaque sizes by RNAi constructs was also analyzed using SAS with a simple general linear model in which the RNAi treatment was treated as a fixed variable. The differences in average plaque sizes among the RNAi treatments were compared with Tukey's Studentized Range tests. Images in this dissertation are presented in color. 93 AUTHORS' CONTRIBUTIONS MC carried out the experiments and helped draft the manuscript. WP assisted with cell culture experiments. HH and HZ participated in the design of the study and performed the statistical analysis. JD and SH conceived of the study, and participated in its design and coordination and helped to draft the manuscript. ACKNOWLEDGEMENTS This work was supported in part by the USDA NR1 Competitive Grants Program (2004-35204-14780). We thank Lucy Lee, Robert Silva, Taejoong Kim, Mohammad Heidari and Richard Witter of the USDA-ARS Avian Disease and Oncology Laboratory (East Lansing, M1) for gifts of antibodies, PCR primers and virus stocks and assistance in viral plaque assays. 94 REFERENCES Afonso, C.L., Tulman, E.R., Lu, 2., Zsak, L., Rock, D.L., Kutish, GE. (2001). The genome of turkey herpesvirus. J. Virol. 75 (2), 971—978. Baigent, S.J., Smith, L.P., Currie, R.J., Nair, V.K. (2005). Replication kinetics of Marek's disease vaccine virus in feathers and lymphoid tissues using PCR and virus isolation. J. Gen. Virol. 86 (Pt 11), 2989—2998. Berkhout, B., Haasnoot, J. (2006). Interplay between virus infection and the cellular RNA interference machinery. FEBS Lett. 580 (12), 2896—2902. Bhuyan, P.K., Kariko, K., Capodici, J ., Lubinski, J ., Hook, L.M., Friedman, H.M., Weissman, D. (2004). 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SUPPLEMENTARY DATA Supplementary Table 3-1. Oligonucleotides used for cloning Oligmucleotide Sequence (5’-3’) Scramble F CGCGTATTGCTGTTGACAGTGAGCGAGCTAACATAGTTGA CATAGAATAGTGAAGCAGCAGATGGTATTCTATGTCAACT ATGTTAGCGTGCCAACTGC Scramble R CATGGCAGTTGGCACGCTAACATAGTTGACATAGAATAC CATCTGCTGCTTCACTATTCTATGTCAACTATGTTAGCTCG CTCACTGTCAACAGCAATA FgB-l F CGCGTATTGCTGTTGACAGTGAGCGCGCTACGTCCATCGA AATI‘TAGTAGTGAAGCAGCAGATGGTACTAAAI I ICGATG GACGTAGCATGCCAACTGC FgB-l R CATGGCAGTTGGCATGCTACGTCCATCGAAATTI‘AGTACC ATCTGCTGCTTCACTACTAAAT'ITCGATGGACGTAGCGCG CTCACTGTCAACAGCAATA FgB-2 F CGCGTATTGCTGTI‘GACAGTGAGCGCTGTATTGAGATCGA TICAGATTAGTGAAGCAGCAGATGGTAATCTGAATCGATC TCAATACATTGCCAACTGC FgB-2 R CATGGCAGTTGGCAATGTATTGAGATCGATTCAGATTACC ATCTGCTGCTTCACTAATCTGAATCGATCTCAATACAGCG CTCACTGTCAACAGCAATA FgB-3 F CGCGTATTGCTGTTGACAGTGAGCGAGGGATCTCCATGGG TATATAGTAGTGAAGCAGCAGATGGTACTATATACCCATG GAGATCCCCTGCCAACTGC FgB-3 R CATGGCAGTTGGCAGGGGATCTCCATGGGTATATAGTACC ATCTGCTGCTTCACTACTATATACCCATGGAGATCCCTCG CTCACTGTCAACAGCAATA FgB-csh12 F CGCGTATTGCTGTTGACAGTGAGCGAACTAA'ITATGCCTT TATGAACTAGTGAAGCAGCAGATGGTAGTTCATAAAGGC ATAATTAGTGTGCCAACTGC FgB-csh12 R CATGGCAGTTGGCACACTAATTATGCCI I IATGAACTACC ATCTGCTGCTTCACTAGTTCATAAAGGCATAATTAGTTCG CTCACTGTCAACAGCAATA FgB-cshl3 F CGCGTATTGCTGTTGACAGTGAGCGAACAGAGGAACTTA AAGATCAGTAGTGAAGCAGCAGATGGTACTGATCTITAA GTTCCTCTGTGTGCCAACTGC FgB-cshl3 R CATGGCAG'ITGGCACACAGAGGAACTTAAAGATCAGTAC CATCTGCTGCTTCACTACTGATCTTTAAGTTCCTCTGTTCG CTCACTGTCAACAGCAATA Scramble-sph-ngo F CGAGCTAACATAGTTGACATAGAATAGTGAAGCAGCAGA TGGTATTCTATGTCAACTATGTTAGCGTG Scramble-sph-ngo R CCGGCACGCTAACATAGTTGACATAGAATACCATCTGCTG CTTCACTATTCTATGTCAACTATGTTAGCTCGCATG FgB-2-sph-ngo F CGCTGTATTGAGATCGATTCAGATTAGTGAAGCAGCAGAT GGTAATCTGAATCGATCTCAATACATTG 102 Supplementary Table 1 (continued) F gB-2-sph-ngo R CCGGCAATGTATTGAGATCGATTCAGATTACCATCTGCTG C'ITCACTAATCTGAATCGATCTCAATACAGCGCATG F gB-cshlZ-sph-ngo F CGAACTAATTATGCCI I IATGAACTAGTGAAGCAGCAGAT GGTAGTTCATAAAGGCATAATTAGTGTG F gB-cshlZ-sph-ngo R CCGGCACACTAATTATGCCI I IATGAACTACCATCTGCTG CTTCACTAGTICATAAAGGCATAATTAGTTCGCATG Md5-gB-254l F CGCGTAT'TGCTGTIGACAGTGAGCGCACCCTAAATATGAT AAGTTACTAGTGAAGCAGCAGATGGTAGTAACTTATCAT ATITAGGGT'ITGCCAACTGC Md5-gB-2541 R CATGGCAGTTGGCAAACCCTAAATATGATAAGTTACTACC ATCTGCTGC'ITCACTAGTAACTIATCATAT'ITAGGGTGCG CTCACTGTCAACAGCAATA Md5-gB-l874 F CGCGTATTGCTGTTGACAGTGAGCGCAGCACATTTGTCGA GCTTAATTAGTGAAGCAGCAGATGGTAATTAAGCTCGAC AAATGTGCTATGCCAACTGC Md5-gB-1874 R CATGGCAGTTGGCATAGCACATTTGTCGAGCTTAATTACC ATCTGCTGCTTCACTAATTAAGCTCGACAAATGTGCTGCG CTCACTGTCAACAGCAATA Md5-gB-1518 F CGCGTATTGCTGTTGACAGTGAGCGACTACAGCGAGTGC AACATTAGTAGTGAAGCAGCAGATGGTACTAATGTTGCA CTCGCTGTAGCTGCCAACTGC Md5-gB-1518 R CATGGCAGTTGGCAGCTACAGCGAGTGCAACATTAGTAC CATCTGCTGCTTCACTACTAATGTTGCACTCGCTGTAGTC GCTCACTGTCAACAGCAATA Md5-gB-596 F CGCGTATI‘GCTGTTGACAGTGAGCGAGGAACCTCCGTCAA TTGTATATAGTGAAGCAGCAGATGGTATATACAATTGACG GAGGTTCCCTGCCAACTGC Md5-gB-596 R CATGGCAGTTGGCAGGGAACCTCCGTCAATTGTATATACC ATCTGCTGCTTCACTATATACAATTGACGGAGGTTCCTCG CTCACTGTCAACAGCAATA Scramblel(Scrl)20b F CTAGGAGTATGCGATTGTATCAACTGTATCTTCTI‘GGCAT TGGACAAGATACAGTTGATACAATCGACAGTACTGTTA Scramblel(Scrl)20b R GATCTAACAGTACTGTCGATTGTATCAACTGTATCTTGTC CAATGCCAAGAAGATACAGTTGATACAATCGCATACTC Md5-gB-1874 20b F CTAGGAGTATAGATTAAGCTCGACAAATGTGCCTTGGCAT TGGACGCACAI I IGTCGAGCTTAATCCCAGTACTGTTA Md5-gB-1874 20b R GATCTAACAGTACTGGGATTAAGCTCGACAAATGTGCGTC CAATGCCAAGGCACAI I IGTCGAGCTTAATCTATACTC Md5-ICP4-4902 20b F CTAGGAGTATGAGAI I IATCAGACGCAGACTGCTTGGCAT TGGACCAGTCTGCGTCTGATAAATCTACAGTACTGTTA Md5-ICP4-4902 20b R GATCTAACAGTACTGTAGAI I IATCAGACGCAGACTGGTC CAATGCCAAGCAGTCTGCGTCTGATAAATCTCATACTC Md5-ICP4-4739 20b F CTAGGAGTATACCACTAGGACCGGAACAI I ICCTTGGCAT TGGACGAAATGTTCCGGTCCTAGTGGCCAGTACTGTTA Md5-ICP4-4739 20b R GATCTAACAGTACTGGCCACTAGGACCGGAACAI I ICGTC CAATGCCAAGGAAATGTTCCGGTCCTAGTGGTATACTC 103 Supplementary Table 1 (continued) Scramble2( Scr2) 1 9b F CCGGAGAGGTGCTGCTCACAGTAGCTAACACTGTAGACA TGGAAAI I IGCTAAAATTATTTCCATGTCTACAGTGTTAG CGACTGTGGTGGTGGTG Scramble2(Scr2)1 9b R AATTCACCACCACCACAGTCGCTAACACTGTAGACATGG AAATAAI I I IAGCAAAI I ICCATGTCTACAGTGTTAGCTA CTGTGAGCAGCACCTCT Md5-gB-596 19b F CCGGAGAGGTGCTGCTCACAGTAGGAACCTCCGTCAATT GTATAAI I IGCTAAAATTATTATACAATTGACGGAGGTIC CCACTGTGGTGGTGGTG Md5-gB-596 19b R AATTCACCACCACCACAGTGGGAACCTCCGTCAATTGTAT AATAAI I I IAGCAAATTATACAATTGACGGAGGTTCCTAC TGTGAGCAGCACCTCT Md5-ICP4-6l 70 CCGGAGAGGTGCTGCTCACAGTCCGTCAATGTATGAAAC 19b F GTAAAATTTGCTAAAATTAT'ITTACGTTTCATACATTGAC GTACTGTGGTGGTGGTG Md5 -ICP4-6 l 70 AATTCACCACCACCACAGTACGTCAATGTATGAAACGTA 19b R AAATAATTITAGCAAATITTACGTTI'CATACA'ITGACGGA CTGTGAGCAGCACCTCT Scramble3(Scr3)922 GATCCTCCAGCTAGCATAATTGACACAGAGTTTGTAGCTG F TGTGTAGAACTCTGTGTCAATTATGCTAGCGGGAGGAAA GGAGGA Scramble3(Scr3)922 CCGGTCCTCCTTTCCTCCCGCTAGCATAATTGACACAGAG R TTCTACACACAGCTACAAACTCTGTGTCAATTATGCTAGC TGGAG Md5-gB-1518 922 F GATCCTCCACTACAGCGAGTGCAACATTAGTTTGTAGCTG TGTGTAGAACTAATGTTGCACTCGCTGTAGCGGAGGAAA GGAGGA Md5-gB-1518 922 R CCGGTCCTCCI I ICCTCCGCTACAGCGAGTGCAACATTAG TTCTACACACAGCTACAAACTAATGTTGCACTCGCTGTAG TGGAG Md5-ICP4-6696 922 F GATCCTCCAI I I GTCGGTCTGTAATATTACI I IGTAGCTGT GTGTAGAAGTAATATTACAGACCGACAAAGGGAGGAAAG GAGGA Md5-ICP4-6696 922 R CCGGTCCTCCTITCCTCCCI I IGTCGGTCTGTAATATTACT TCTACACACAGCTACAAAGTAATATTACAGACCGACAAA TGGAG 104 attR] attR2 LTR SID 3 gag P01 SA env \ / LTR RCANBP(A)-U6-miRNA / Mir 30a \ U6 T—' > Mlul / \ NcaI Sense loop anti * attRl attR2 LTR SID D gag P01 SA env SA\ / LTR RCASBP(A)miRNA Mir 30a ‘/ \ ‘ > MluI / \ NCO] Sense loop anti * attRl attR2 LTR SP E 838 pol SA cm, SA\ / LTR T I RCASBP(A)miRNA-SphIIgo I——_r M 1' 3 0“ 4g Sphl . / \ NgoMV Sense loop anti * attRl attR2 LTR SID E gag P01 SA env SA\ LTR RCASBP(A)3mirs I l .i I I l————-l E I 3mirs / \ I i > \ :>——<:S>—<:; \Aer Bng BspEI EcaRI BamHI AgeI Supplemental Figure 3-1. Diagram of avian shRNA-mir vectors. Top line: shRNA-mirs inserted into RCANBP(A) are meant to be transcribed from chicken U6 (or other pol III) promoters. Such vectors have been ineffective in avian cells in our hands and were not 105 used after our initial HVT experiments. Second line: shRNA-mirs inserted into the RCASBP(A)miRNA vector (through use of pENTR3C-miR30a) are expressed from the viral LTR via a spliced RNA transcript. Within the mir-30a gene, a short-hairpin loop containing the target sequence and its complement flanking the native mir-30a loop sequence is synthesized and inserted between the Mlul and NcaI restriction sites. Third line: The RCASBP(A)miRNA-sphngo vector (generated using pENTR3C-miR30a- sphngo, Supplemental Figure 3-2) differs from RCASBP(A)miRNA only in that a smaller synthetic insert is required between engineered Sphl and NgoMIV sites more closely flanking the miRNA stem-loop structure. Fourth line: The RCASBP(A)3mirs vector is generated using pENTR3C-3mirs (Supplemental Figure 3-3) which allows for the insertion of three target site miRNAs between unique AvrII and BglII, BspEI and EcoRI, and BamHI and AgeI sites. These sites are within miR-20b, miR-l9b and miR92- 2 family member genes, respectively. 106 BamHI caaggatccAAACTGCCCCATGCACCTCTGCATGTAACAACACAGCAACAG AAGTTGTAGCAATCTTAACTAACCTGCTTGGAGGCCTTTGCATGTTCATAG Sphl TGTAAAGTAAACAGCTGAGGAATGTGTATTGCTGTTGACAGCatGCtgcct NgoMIV gactggcactgctgGCngCTGCCACAGACGTCAAGAAATGTTTGACACCT AAAGCATAGTTGTCTGTTTAAAACTTCTATTCTATTTGGTTTATCACAGAC ATATGAGTTTAGGCCAAACCTTTCTACGTATTACATGTGATGCCTGTGTCT AGAAACCATCCAGTTACTCATAGGAGCTTGTGCTCATCAACATCATAACCC AAAAAACTGAATACAAAGTAAAACATGAAATAACTTAGGCTTTATTTTTAA NOtI CTACAAACTTTCAATTGTGTTTCCAGAAGCAAGTTGCCGTGCAGCTgCggccgctCC PrimerA: 5’CAAGGATCCAAACTGCCCCATGCACCTCT3' PrimerB: 5’CAGCAGTGCCAGTCAGGCAGCATGCTGTCAACAGCAATACACATTCCTC3’ PrimerC: 5'TGCCTGACTGGCACTGCTGGCCGGCTGCCACAGACGTCAAGAAATG3’ PrimerD: 5’GAAGCGGCCGCAGCTGCACGGCAACTTGC3’ Supplemental Figure 3-2. The chicken mir-30a-sphngo cassette sequence. Capital letters indicate nucleotides identical to the sequenced chicken (Red Junglefowl) genome [v2.1 build of the chicken genome sequence]). PCR primers are individually colored except that the overlapping portion of primers B and C are in a common color (green). Nucleotides in black derive from genomic DNA used to template the PCR reactions. PCR originally used primers A with B and C with D in separate reactions followed by mixing the two resulting fragments and PCR with A and D primers (Ho et al., 1989). The complete sequence then was digested with BamHI and NotI and cloned into the BamHI and Natl sites of the previously described pENTR3C vector (lacking any additional promoter or leader sequences, Chen et al., 2007). This provides a mir30a cassette that relies on Sphl and NgaMIV digestion and insertion of a synthetic duplex consisting of a 68 nucleotide and a 76 nucleotide strand (both the 5’ and 3’ overhang are on the longer strand). 107 HindIII gagaagctTGCTCATTGACCACCGTGTCACAGCGTTGCATTTTGTTTCGTTGAAGGCAATTGCTC AvrII BglII AGGAAGCAGCTGTGTCCTAthcaTgAGATctAGTGCGTCCCGGCTCGGGGCAGCTTCTGGAGAA GATGCCTTCCGGAGAGGTGCTGCTCACAGTCAGTTTTGCAGGTTTGCATCCCAGCTTGCTAAAAT EcoRI TGCTGTGCAAATCCATGCAAAACTGACTGTGGTGGTGGTGAATtCCGGGGTTAGgAGCTTTCTTC BamHI AgeI AA CCCTCTTGCTGTGAAGATCCGAAGATGCC2CQCGCTGSaTCCatquVgGCTCIth9GflCCTTU ATCPG3T3ACLCAGCTTTGTCAGTGGGCAGCTCTCGTGGTGTCGTGGCCGACCCAAAGGGGTGGG thI AAGGAAGAAAGCTCCAGATCCTGGGGTTTGTCTGngccgcaga PrimerA: 5’GAGAAGCTTGCTCATTGACCACCGTG3’ PrimerB: 5’TGACCTAGGACACAGCTGCTTCCT3’ PrimerC: 5’GTGTCCTAGGTCATGAGATCTAGTGCGTCCCGGCTC3’ PrimerD: 5’GGCATCTTCGGATCTTCACAGCAAGAGGGGAAGAAAGCTCCTAACCCCGGAATTCACCACCA CCACAGTCB' P ' E. ' «‘11T“.-CVfT‘/‘T‘|T‘. 1T\I‘W“IV,'1"\ .‘ ""f .‘a'NrW‘“"' NTT‘TW-"‘ \ ‘3 V.“i,"~7,'llrar<7. 'w ‘v‘r «W’ a rlmer . 5 L L x.) 1 'J.“.H‘JH.I Gk, afar-127.“. F131- [1,. . . 1C. 1 . .1 1:1 - ‘.._.4...‘-4. . 47‘ 7:1 -..‘.;1a . e _ . 1 3T3A3A3’ PrimerF: 5' TCTGCGGCCGCAGACAAACCCCAGGATC3’ Supplemental Figure 3-3. The chicken 3mir cassette sequence. Capital letters indicate nucleotides identical to the sequenced chicken (Red Junglefowl) genome (~chr4:3,968,600-3,970,600 of the v2.1 build). That portion of the genome sequence shown as a gap was estimated using the overlapping BU384584 EST sequence and later confirmed by our own sequencing of the cloned PCR fragment. PCR primers are individually colored except that the overlapping portion of primers B and C and that of primers D and E are in a common color. Nucleotides in black derive from genomic DNA used to template the PCR reactions. PCR originally used primers C with D and E with F in separate reactions followed by mixing the two resulting fragments and PCR with C and F primers (Ho et al., 1989). The two nucleotides with a A above them flank a segment (including a miR-363 family sequence) that was deleted from this construct (corresponding to nucleotides 604-726 of the BU384584 EST). This product was digested with Aer and Natl, the PCR product from primers A and B was digested with HindIII and Aer, and both fragments were combined with HindIII and Natl-digested pENTR3C (lacking any additional promoter or leader sequences). The subsequent plasmid, pENTR3C-3mir, contains the complete miR-l9b family member gene which can be removed and replaced using BspEI/EcaRI digestion and two small filler sequences at the sites of the miR-20b and miR-92-2 family members flanked by unique .4er and Bng and BamHI and AgeI sites, respectively. The order of the three sites is miR-20b, miR-l9b, and miR-92-2 from 5’ to 3’. Any one, two or all three sites can be filled with synthetic duplexes with appropriate restriction site overhangs (Supplemental Table 1). Note that the miR-20b site expresses its primary miRNA from the 5' end of the hairpin as 108 opposed to the miR-19b and miR-92-2 sites, which express their primary miRNAs fiom the 3’ side of the hairpin or miR-30a, which expresses miRNAs fiom both strands of the stem. This requires that the orientation of the miR-20b target sequence and its complement within the stem-loop structure be reversed in comparison to the other sites. 109 g 16 2 13 o 10 - - E 8 _ or "6 6 ‘ 8‘ 33 U 0 J I T I l I I I I I I Ii I r WI I jl T ‘t- ’b ’5 ‘b “b '5 4°° .9" .9" 9° .‘3‘ to" .C“ :84" 9“” :34" eon 900 2950 {098 9g» ((6 «95° «6 (\o 9° ’3’ " '3’ 09° 9° 1" '3’ 183‘ Constructs Supplemental Figure 3-4. Quantitative PCR of gB copy numbers per chicken GAPDH gene from CEF expressing RCASBP(A)3mirs challenged with MDV. CEF transfected with the RNAi vector constructs listed were infected with 100 pfu of Md5 MDV and DNA was isolated at 5 dpi. Quantitative PCR was performed as described in Materials and methods. In each case, the first sequence in a series is in the miR-20b site, the second in the miR-l9b site and the third in the miR-92-2 site. Scrl, 2 and 3 indicate different scrambled control sequences with similar base compositions to the targets. gB targets are 1874, 596 and 1518, whereas ICP4 targets are 4902 or 6170. Targets are identified by the number of their first nucleotide within the MDV gB coding sequence. 110 CHAPTER 4 RETROVIRAL VECTOR-BASED RNA INTERFERENCE PROTECTS CHICKENS FROM MAREK’S DISEASE VIRUS INFECTION lll ABSTRACT RNA interference (RNAi) based anti-viral strategies have effectively inhibited several families of DNA and RNA viruses including retroviruses and herpesviruses. We and others have subsequently demonstrated several gene transfer studies in live chickens using replication competent retroviral vectors (RCAS/RCAN). We have previously shown that RCAS/RCAN retroviral vectors are capable of delivering effective shRNA- mirs/multi-mirs against Marek's disease virus (MDV, also known as gallid herpesvirus type 2) in cell culture. In this study, similar RNAi vectors are shown to protect chickens from MDV infection. Delivery of RCASBP(A)3mirs-l874-596-1518 targeting MDV gB glycoprotein gene reduced MDV-infected white peripheral blood cells (PBLs) titers and modestly reduced Marek’s disease (MD) incidence and levels of MD-induced death. RCASBP(A)3mirs-1874-6170-1518 co-targeting MDV gB glycoprotein gene and ICP4 transcriptional regulatory gene significantly reduces MDV viremia and MD incidence and increases overall survival rate by reducing MD-induced death. A stem-loop RT-PCR assay is developed that successfully measured siRNA expression levels in birds; however, no correlation was seen between siRN A expression level and the reduction in MDV replication. Embryonic injection of replication competent RCASBP(A) vectors results in rapid induction of B cell lymphoma in a low percentage of birds. We discuss that the anti-viral effects we observed in chicken is unlikely due to RNAi induced interferon responses. 112 INTRODUCTION RNA interference (RN Ai) based anti-viral strategies have effectively inhibited several families of DNA and RNA viruses including retroviruses and herpesviruses (Haasnoot, Westerhout, and Berkhout, 2007). RNAi can be delivered into cells and animals by transient transfection of synthetic small interfering RNAS (siRNAs) and expression of short hairpin RNA (shRNA), modified primary microRNAs (shRNA-mirs) and long hairpin shRNA (lhRNA), which mimic precursors of cellular microRNAs (miRNAs). Depending on the degree of complementarity of the siRNA/miRNA to the target sequence as well specific cellular conditions, RNAi can mediate gene silencing by means of direct degradation of mRNAs, repression of translation or recruitment of mRNAs to cellular processing bodies where translation can’t occur (Peters and Meister, 2007). Transfection of siRNAs has been used to efficiently Silence gene expression in chicken embryos (Hu et al., 2002). Hu et a1 (2002) described RNAi against Rous sarcoma virus, a member of the avian leukosis and sarcoma virus family. They demonstrated that siRNA against viral gag genes can protect chicken embryos fiom lethal RSV infection. Due to the fact that the siRNA effect is transient in vertebrates (there is no siRNA amplification process), chemical modifications and repetitive adminstration of low concentrations of siRNAs are commonly used to increase the stability of siRNA and achieve long term anti-viral effects (do Fougerolles, 2008). Plasmid or viral vector- delivered shRNAs/lhRNAs have been used to inhibit chronic viral infection (Akashi et al., 2005; Liu, Haasnoot, and Berkhout, 2007). More recently, shRNAs based on 113 naturally occurring miRNA primary transcripts (termed shRNA-mirs) have been developed and, in some cases, have proved to mediate gene silencing more potently than shRNA in both transient and stable transfection assays (Das et al., 2006; Dickins et al., 2005; Silva et al., 2005; Stegmeier et al., 2005). Typically, shRNA-mirs are constructed by replacing the stem region of a miRNA gene with the target sequence and its guide RNA complement, which can be recognized and processed by Drosha in the nucleus and transported to the cytoplasm by Exportin 5. The shRNA-mirs can be also expressed as larger transcripts containing several stem-loop structures resembling polycistronic miRNAs (termed multi-mirs). The potential advantage of using multi-mirs against viruses is to target multiple regions of a viral RNA or multiple viral RNAS to enhance efficacy and prevent viral escape. We previously developed retroviral vectors based on the replication-competent avian leukosis virus backbone (termed RCAS/RCAN) that express shRNA-mirs either as spliced sub-genomic RNAS transcribed from the retroviral long terminal repeat (LTR) promoter or as short RNAS transcribed from internal promoters (Chen et al., 2007). We showed the efficacy of such vectors in inhibiting replication of subgroup B avian leukosis virus in cultured chicken cells with RNAi targeted either against a viral gene (ean) or the host cell virus receptor gene (tvb). Marek's disease virus (MDV) is a member of the Alphaherpesvirinae. There are three different serotypes of MDV designated as MDVl (also called gallid herpesvirus type 2, GaHV-Z), MDV2 (GaHV-3), and MDV3 (also known as turkey herpesvirus, or HVT). MDVl is the only serotype that causes Marek’s disease, a lymphoproliferative disorder in which aggressive T-cell lymphomas result fi'om infection of susceptible chickens (Osterrieder et al., 2006). The most common current method for controlling MD 114 in the poultry industry is vaccination using an attenuated strain of MDVl , CVI988/Rispens. Recent vaccine studies aided by recombinant DNA technology have developed several new vaccines that show equal or better efficacy compared to CVI988/Rispens (Lee et al., 2008). However, since vaccines do not induce a ‘sterilizing immunity’ in the vaccinated host, MDV continues to evolve in vaccinated flocks and has shown the ability to generate new and more acute disease symptoms. We believe that new control measures not based on vaccination warrant further research. RNAi is a particularly attractive alternate strategy against MDV for several reasons. First, RNAi is sequence-specific, which minimizes deleterious side effects. Second, RNAi based on lhRNA and multi-mirs can deliver siRNAs targeting several viral genes or sequences simultaneously and thus minimize the chances that viruses will mutate to resistant forms. Third, RNAi has the potential to be delivered by infection with non-pathogenic viruses, much as vaccines are now administered. Finally, due to its sequence-specificity, RNAi potentially can be targeted against sequences of virulent viruses (e.g., w+ MDV) without impairing vaccine viruses themselves (W esterhout, ter Brake, and Berkhout, 2006). Thus, RNAi might be used in combination with existing vaccine-based strategies. Replication competent RCAS/RCAN vectors are attractive for RNAi studies in live chickens. Usually, a single proviral copy of the vector cassette is stably integrated into the chicken genome, thus minimizing the possibility of saturating cellular miRNA biogenesis factors. While for plasmid based vectors gene transfer in viva is usually confined to a certain percentage of cells in the region of injection, RCAS/RCAN vectors are replication competent and spread rapidly and efficiently in chickens (with appropriate subgroup specific receptors). Finally, RCAS/RCAN vectors 115 can infect and deliver RNAi to tissues that are not readily electroporated. We and others have subsequently demonstrated several gene transfer studies in viva based on RCAS/RCAN vectors (Das et al., 2006; Harpavat and Cepko, 2006; Holmen et al., 1999). In particular, Holmen et a1. (1999) used this system to express soluble (secreted) forms of the tva subgroup receptor protein in vivo and showed that this induced resistance to ALV(A) in these birds. Therefore, we chose to use similar vectors to deliver RNAi in vivo as a test for RNAi-based anti-viral effects. Typically, RCAS/RCAN-RNAi viruses are made by trarrsfecting DF-l cells (an immortalized chicken fibrobast cell line) with viral DNA. Fresh or early chicken embryos are then injected with a large amount of DF-l cells containing RCAS/RCAN to maximize the chances that foreign genes will be delivered and expressed in virtually every cell. If embryos free of endogenous proviruses related to RCAS/RCAN are used, viremic chicks can be easily identified at hatch using ELISA assays and continuously examined for acquired genotypes or phenotypes. In ova DNA electroporation can also be used to deliver RCAS/RCAN-RNAi, however, viral infection is more suited for systemic RNAi studies because it leads to more uniform infection and less chances of tissue damage from electroporation. We have previously shown that replication competent RCAS/RCAN retroviral vectors are capable of delivering shRNA-mirs/multi-mirs against MDV genes in cell culture (Chen et al., 2008). We demonstrated that cells expressing shRNA-mirs/multi- mirs targeting the MDV gB glycoprotein gene or the ICP4 transcriptional regulatory gene show significant inhibition of viral replication. In this study, we investigate whether RNAi based on replication competent RCAS vectors develivering shRNA-mirs/multimirs 116 against viral ICP4 and/or gB genes can measurably inhibit pathogenic MDV infection in live chickens. 117 RESULTS Infection of line 0 chickens with W Mdll strain of serotype 1 MDV We examined whether RCASBP(A) delivering siRNA against the MDV gB gene could protect chickens from W Mdll infection in a pilot trial. General procedures and set up for experimental groups are shown in Figure 4-1. and Table 4-1. Detailed procedures for individual trials are Shown in Table 4-2. Newly laid Line 0 embryos were injected with 106 DF-l cells producing RCASBP(A)miRNA-1874 or RCASBP(A)miRNA-scramble (negative control virus) and then sealed and hatched. ELISA assays showed that 100% of RCASBP(A)miRNA-1874 chickens and 98% of RCASBP(A)miRNA-scramble chickens exhibited widespread infection (presence of the provirus). Chicks were then challenged intraperitoneally with 1000 pfu of Mdll at hatch. At 5 dpi, five birds from Mdll challenged group were sacrificed and bursa tissues were sectioned and labeled for pp38 (MDV early gene) expression using the H19 monoclonal antibody. Two birds from the no vector group (no RCAS vector injection), two from RCASBP(A)miRNA-scrarnble group and one fiom the RCASBP(A)miRNA-1874 group showed relatively weak staining in less than 2 bursa follicles. On the contrary, positive control bursas (from other experiments using different chicken lines infected with MDV) showed strong pp38 expression in multiple follicles, suggesting that the immunohistochemal procedures were correctly executed (Figure 4-2). Four birds from each of the RCASBP(A)miRNA-scramble and RCASBP(A)miRNA-1874 and 5 birds from the no vector group were bled at 14 dpi for testing MDV viremia. There were no significant differences in MDV titer in peripheral blood lymphocytes (PBLs) among treatment groups (Table 4-3). MDV titers were relatively low among all treatment groups 118 (32 PFU/2 x 106 PBLs in both no vector and RCASBP(A)miRNA-1874 groups, and 72 PFU/2 X 106 PBLs in the RCASBP(A)mirRNA-scramble group). At the end of the experiment, the no vector group showed 2% MD-specific mortality and 25% MD incidence (combined MD mortality with MD lesion). RCASBP(A)miRNA-scramble showed 10% MD-Specific mortality and 24% MD incidence. RCASBP(A)miRNA-1874 showed 11% MD-specific mortality and 25% MD incidence (Table 4-4.). There were no significant differences in MD mortality or MD incidence among groups. A B cells lymphoma induced by ALV infection was observed in one bird from vector injected groups alone (data not shown). One chicken in the RCASBP(A)miRNA-1874 group developed B cell lymphoma and died 30 days after hatch, suggesting that rapid induction of a B cell lymphoma by RCASBP(A) vectors can occur in RCAS vector treated birds. This indicates the importance of histological examination of tumor sample in subsequent experiments. In summary, this pilot experiment allowed us to test our protocols, but line 0 proved surprisingly resistant to the Mdll strain of MDV, resulting in low expression of the MDV early antigen, pp38, low MDV titers in PBLS, and low MD mortality and MD incidence among all treatment groups. Given that low incidence, it was not surprising that RNAi treatment failed to induce a statistically significant change in viremia or MD. 119 Replication competent i RCASBP(A)mir 0r Transfection RCASBP(A)3mirs - . L of DF-l - 7K :1 g? & Test MDV pp38 cells L.“ “"33 , expression at 5dpi - w A“. - ; I‘Tr‘mf’ ’1'1 :f "1* ,' a" 4“ . ,. _ _w'1.'. l' . _ nu. W: Pass cells Virus spreads _ . Throughout". Test MDV vrrenua culture W at 14 and/or 28de Inject virus-infected 3 Cells into day 0 embryos , , . Test siRNA expression Somatic Infection at PBLs at 14 dpi ‘\I\ Identify infected chimeras A, ' \ Terminate birds at . . 8weeks post challenge MDV inoculation Exam MD symptoms Figure 4-1. General procedure for using RCASBP(A)miRNA/RCASBP(A)3mirs vectors in vivo. Propagation of RCASBP(A) vectors in DF-l cells was initiated by transfection of plasmid DNA containing the retroviral provirus. Embroys were injected with DF-l cells producing the RCASBP(A) vector virus. Viremic chicks were identified at hatch by ELISA for the ALV capsid protein p27. Chicks were challenged with pathogenic strains of MDV at hatch. At 5 days post inoculation (dpi), lymphoid tissues were sampled for immunohistochemical staining of MDV pp38 protein expression. Chickens were bled at 14 and/or 28 dpi for testing MDV viremia by number of infected peripheral blood leukocytes (PBL). In some trials, chickens were also bled at 21dpi for testing siRNA expression using the stem-loop RT-PCR assay. Chickens were kept up to 8 weeks post inoculation and examined daily for MD symptoms. 120 Figure 4-2. Imrnunohistochemical analysis of representative bursa at 5dpi. line 0 embryos were injected with RCASBP(A)miRNA-1874 targeting MDV gB or RCASBP(A)miRNA-scramble. Chicks were inoculated with 1000 pfu of passage 7 Mdll strain of MDV at hatch. Bursae were sampled and stained with pp38-specific mAb. Relatively low expression in few bursa follicles were seen in experimental groups compared with positive control samples collected from other research. 121 Various chicken lines differ substantially in their susceptibility to MD. Unfortunately, line 0 (chosen for its lack of ALV endogenous proviruses) had not been tested previously with Mdl 1, a W strain of MDV. Not only do MDV isolates vary in their initial levels of virulence as first isolated, the subsequent passage in culture of any field virus will gradually lower (attenuate) the virulence level. Thus, it clearly is critical to our experiments that we choose an isolate and a passage level of MDV that will produce a substantial (but not overwhelming) level of MD in the controls (no vector and scrambled insert vector) in hopes of observing a statistically significant level of reduction due to RNAi. Data became available from a separate experiment by one of our collaborators (Huanmin Zheng, ADOL, unpublished data ) that showed that line 0 is susceptible to the vv+ 648A strain of MDV and suggested an appropriate dose and passage number to use for firture tests in line 0. RCASBP(A) delivering 3 siRNAs against the viral gB gene inhibits the vv+ 648A strain of serotype 1 MDV infection. We examined whether RCASBP(A) delivering 3 siRNAs against the MDV gB gene could protect chickens from w+ 648A infection in trial 2. Detailed procedures for trial 2 are listed in Table 4-2. In trial 2 the RCASBP(A)3mirs-18746964518 vector (that had recently become available) was used. RCASBP(A)3mirs-l874-596-1518 contains three targets within the MDV gB gene. Although this multimir vector was not significantly more effective than the RCASBP(A)miRNA-1874 in cell culture (Chen et al., 2008; Chapter 3), it potentially might be more resistant to MDV escape mutants. A comparable scrambled 3 insert control vector (RCASBP(A)3mirs-scramble) was used in parallel. AS for trial 1, unincubated line 0 embryos were injected with DFl cells 122 expressing these vectors (or no vector controls), and then sealed and hatched. In trial 2, 15% of the RCASBP(A)3mirs-scramble-treated and 18% of RCASBP(A)3mirs-1874- 596-1518-treated embryos hatched, resulting in less than 20 chicks in each of these two groups available to be challenged with MDV. A higher hatching rate was observed in the untreated group (87% hatched, 24 chicks). Previous experience (e.g., Hohnen et a1. 1999; the pilot trial discussed above) had shown that injection with ALV vectors ofien reduces hatch rates. Both the injection process by itself and ALV replication appear to contribute to this. However, the extent of that reduction is highly variable and also depends on the age of the hens producing those embryos (relative to their laying cycle) and the storage time of embryos. In order to have sufficient embryos, it’s necessary to accumulate them over a certain period and store them at 4°C. Thus, the storage period will be inversely related to the productivity of the hens. As a result of these sources of variability, it often was difficult to estimate how best to divide the limited number of embryos available into our various experimental treatment groups. Newly hatched chicks in trial 2 were challenged with 500 pfu of vv+ 648A at passage 40. Due to the low hatching rate, only 3 birds from each of the no treatment and RCASBP(A)3mirs-1874-596-1518 groups and 1 from the RCASBP(A)3mirs—scramble control group were sacrificed at 5 dpi for testing MDV pp38 expression in lymphoid organs. None of birds showed pp38 expression in any lymphoid organs (data not shown). The RNAi treatment reduced MDV viremia at 14 dpi by about 70% (25 PFU/2 X 106 PBLs in RCASBP(A)3mirs-1874-596-1518, 84 PFU/2 X 106 PBLs in RCASBP(A)3mirs- scrarnble, and lOOPFU/Z X 106 PBLs in no vector, Table 4-3.) and 28 dpi (39PFU/2 X 106 PBLs in RCASBP(A)3mirs-l 874-596-1518, 60 PFU/2 X 106 PBLs in RCASBP(A)3mirs- 123 scramble, and 133PFU/2 X 106 PBLs in no vector). The antiviral RNAi effect is significant when comparing RCASBP(A)3mirs-1874-596-1518 vs. no vector (p<0.02, ANOVA, Fisher protected LSD) but not RCASBP(A)3mirs-1874-596-1518 vs. scramble, primarily due to the fact that we ended up with only 7 birds in the scramble group. The MDV meq protein has been shown to be consistently expressed in all MDV tumors but not in ALV induced lymphoid leukosis (Gimeno et al., 2005). ALV does not induce nerve enlargements, so birds with nerve enlargements are diagnosed as MD without further staining of tumors. Tumors from birds at risk of MD with no nerve enlargements were sectioned and tested for meq expression immunohistochemically (data not shown). The percentage of birds in the RCASPB(A)3mirs-1874-596-1518 group that exhibited MD (birds with nerve lesions and/or meq-positive tumors) was 57% vs. 67% and 74% in the RCASPB(A)3mirs-scramble and no treatment groups, respectively. However the reduction of MD incidence by RNAi did not achieve statistical significance (two- proportion z-tests). Figure 4-7a shows that RCASPB(A)3mirs-1874-596-1518 treatment very modestly increased the overall survival rate by reducing MD-induced death during the experiment period, however, the effect did not achieve statistical significance (log- rank test). 124 1.0 0.9— 1_‘ 0.8- 0.?- 0.54 l w 0.5— I 0.4- ‘ 0.3- 0.2- 0.1 - 0.0 l I I I I I 35 4o 45 so 55 Day post inoculation (Total-MD mortality)/Total .4 O r. 0.9% 0.8. 0.7- 0.6- 0.5— 0.4— 0.3- 0.24 0.14 0'010 ' 2'0 ' 3'0 ' 40 ' 5'0 ' 60 Day post inoculation (Total-MD mortality)/Total Figure 4-7. RCASBP(A)3mirs directed against 648A MDV increased survival rates by reducing MD mortality. Line 0 embryos were injected with RCASBP(A)3mirs delivering 3 siRNAs (1874, 596, 1518) targeting gB (a) or 3 siRNAs (1874, 6170, 1518) targeting g3 and ICP4 (b) along with scramble control sequences. Chicks were inoculated with 500 pfu of passage 40 648A strain of MDV at hatch and monitored through 8 weeks. Red, RCASBP(A)3mirs; green, scramble control; blue, no vector. 125 In summary, the results of trial 2 confirmed that line 0 was relatively susceptible to the vv+ 648A strain of MDV (passage 40), which resulted in about 74% total MD incidence in no treatment control group. No pp38 expression at 5 dpi was detected by immunohistochemitry in selected birds among all groups. Although we were limited in the number of birds we could sacrifice for this assay, this suggests that 5 dpi pp38 staining may not be a good indicator of 648A strain MDV replication in line 0 chickens. Delivery of RCASBP(A)3mirs-1874-596-1518 reduced MDV-infected PBL titers and modestly reduced MD incidence and levels of MD-induced death. Unfortunately, low numbers of birds within the treatment groups (especially in the scrambled control group) due to low hatching rates prevented us from attaining adequate statistical power. Effects of RCASBP(A)miRNA-1874 against a W Md5 strain of serotype 1 MDV in 1515 x 71 chickens. Because line 0 embryos were not available for trial 3, we tested the offspring of a 1515 x 71 cross, which are the standard birds for MDV susceptibility at ADOL and whose susceptibility to Md5 infection was well established (Witter, 1997). Due to its availability and because RCASBP(A)miRNA-1874 (single target in the g3 gene) was equally effective to RCASBP(A)3mirs-1874-596-1518 in cell culture (Chen et al., 2008; Chapter 3) but produced inconclusive results in the pilot trial, we chose to retest RCASBP(A)miRNA-1874 in trial 3. Vector treatment of unincubated embryos was done as in previous trials. Unfortunately, hatch rates again were low, this time even in the untreated group (35% in no treatment, 10% in scramble, and 18% in RCASBP(A)miRNA-1874), probably because the hens were old and at the end of their production cycles. The ELISA test could not be used to examine the presence of vector 126 proviruses because 1515 x 71 chickens naturally contain endogenous ALVs. Newly hatched chicks were challenged with 500 pfu of Md5 MDV (passage 7). Two birds from the Md5-challenged groups were sacrificed and lymphoid organs were tested for pp38 expression at 5 dpi. One bird fiom each of the scramble and no vector control groups showed relatively weak pp38 staining (bursa in scramble, thymus in no vector), whereas no pp38 expression was detected in the RCASBP(A)miRNA-1874 group (data not shown). There was only a modest reduction in MDV titer by RNAi treatment at 14 dpi (58 PFU/ 2 x 106 PBLs in RCASBP(A)miRNA-1874, 66 PFU/2 x 10" PBLs in RCASBP(A)miRNA-scramble, 77 PFU/2 X 106 PBLs in no vector, Table 4-3), and the reduction did not achieve statistical significance. No reduction in MD incidence or MD mortality was observed as a result RNAi treatment in this trial (Table 4-4) To summarize the results of trial 3, line 1515 x 71 birds, as expected, were relatively susceptible to W Md5 infection, which resulted in about 90% total MD incidence in the no vector treatment group. The RNAi vector, RCASBP(A)miRNA-- 1874, showed a slight but insignificant reduction in MDV-infected PBLs and had no effect on MD incidence or MD mortality. It was unclear whether chickens in the treatment group still contained the vector provirus and expressed siRN A against gB during the course of this experiment due to fact that we could not use ELISA assays in the 1515 x 71 chickens. Based on the very high infection levels achieved previously, it seems likely that the retroviral vectors successfully infected the birds, but stability of the provirus is uncertain, expecially since recombination with endogenous ALV proviruses in 1515 x 71 chickens is possible. Although we could have tested for the presence of the 127 vector using Southern blots or PCR, we chose not to pursue this firrther due to the poor embryo quality and insignificant results obtained in this trial. RCASBP(A)3mirs targeting both the MDV g8 and ICP4 genes inhibits vv+ 648A serotype 1 MDV infection. By the onset of trial 4, a RCASBP(A)3mirs vector containing twoRNAi targets against MDV g8 and one against the MDV ICP4 gene was available (termed RCASBP(A)3mirs-l874-6170-1518). This construct had shown the greatest reduction in MDV replication in vitro (6% of the no vector treatment or about 5-fold lower titers than were achieved with the two vectors used in earlier trials, Chen et al., 2008; chapter 3). This vector was utilized in trial 4 in line 0 chicks, again challenged with stain 648A MDV (SOOpfu, passage 40, Table 4-2). In this trial, we observed higher hatching rates in all groups, probably due to the fact that line 0 was near the beginning of its production cycle at this time. No lymphoid tissues were sampled for pp38 expression at 5 dpi because staining did not correlate with MD incidence even in control groups in previous trials. RNAi treatment using RCASBP(A)3mirs-1874-6170-1518 significantly reduced MDV-infected PBL titers at 14 dpi (20 PFU/2 X 106 PBLs in RCASBP(A)3mirs-1874- 6170-1518, 41 PFU/2 x 106 PBLs in RCASBP(A)3mirs-scramble, and 97 PFU/2 x 106 PBLs in no vector, Table 4-3). The reduction was about 50% relative to the scrambled control-treated birds and nearly 80% relative to the untreated birds. Although the scrambled control birds showed a reduction in viremia relative to the untreated in this trial, that was not observed generally over the course of four trials including trial 1, 2, 3 and 5 (Table 4-3). In order to differentiate MDV-induced tumors from RCASBP(A)- induced B cell lymphoma, tumors from birds at risk of MD with no nerve enlargements 128 were sectioned and tested for meq (MDV antigen) and En] (B cell marker) staining. Meq-specific staining was observed in a large percentage of cells in MD spleen tumors, whereas no or very few cells (the spleen naturally contains a small amount of B cells) expressed Bul. An example of this is shown in Figure 4-3. On the other hand, Spleens with B cell lymphoma expressed Bul but not meq. Only meq-positive tumors were viewed as evidence of MD in the absence of nerve enlargements. There are 7% of birds in scramble group and 8% in RNAi group showing RCAS induced B cell lymphoma without any MD symptoms. In this trial, the percentage of birds in the RNAi group that exhibited MD was 54%, which was significantly different from that in the no vector control group and scrambled insert control group (both 83%, Table 4-4.). Figure 4-6b shows that RCASPB(A)3mirs-l874-6170-1518 treatment significantly increased the survival rate by reducing MD-induced death compared with the scrambled control treatment but not no vector group (log-rank test: P < 0.01). ELISA assays showed that 100% of the birds injected with RCASBP(A)3mirs-1874-6170-1518 contained the retroviral vector (data not shown). Note that being p27-positive does NOT mean that all cells in the bird contain a provirus, since the birds are a mosaic of infected and uninfected cells. More important, even those cells (or birds) that contain the vector provirus might fail to produce siRNAs due to genetic (e.g., internal deletions) or epigenetic (e.g., DNA methylation) silencing of proviral expression. We therefore developed stem-loop RT- PCR assays to measure the levels of target-specific siRNAs in PBL from experimental birds (Figure 4-5). Figure 4-6 shows that the average siRNA level as measured by stem- loop PCR varied among birds. However, there was no significant correlation between the average of siRNA expression level and MDV-infected PBL titer at 14 dpi (data not 129 shown). It’s unclear what this lack of correlation may mean however, since there are other examples in which the measurable level of siRNA does not correlate with the RN Ai effect achieved (Dickins et al. 2005). Furthermore, the siRNA expression level is only an average across all harvested PBL, and we don’t know what the cell-to-cell variance may be and how this might affect cell-to-cell infectivity. In this regard, as a strictly cell- associated virus, the course of MDV replication may be less reflective of average cell properties than would a lytic virus that releases bursts of extracellular infectious viral particles. 130 MDV tumor T cell lymphoma RCAS tumor .8 cells lymphoma Figure 4-3. Immunohistochemal analysis of representative MDV induced T cell lymphoma and RCAS induced B cell lymphoma in spleens line 0 embryos were injected with RCASBP(A)3mirs-1874-6170-1518 targeting MDV g8 and ICP genes or RCASBP(A)3mir-scramble. Chicks were inoculated with 500 pfu of passage 40 648A strain of MDV at hatch. Spleen tumors were stained with meq-specific monoclonal antibody (mAb) and Bul-specific mAb. MDV induced T cell lymphoma shows many meq cells but no Bul cells. RCAS induced B cell lymphoma shows many Bul cells not but meq cells. 131 Vague ' sciatic Israelis! LIDV ind-cell lave Idols Nor-3| lam Figure 4—4. Histochemical analysis of representative MDV induced lesions in peripheral nerves. Chicks were inoculated with 500 pfu of passage 40 648A strain of MDV at hatch. Peripheral nerves (vagus, sciatic and brachial) were collected in selective chickens to identify inflammatory or neoplastic lesions using the haematoxylin & eosin (H&E) Staining. MDV induced nerve lesions in this figure are a mixture of type A (neoplastic in nature with massive infiltration of lymphoid cells that destroy the normal structure of the nerve) and type B (inflammatory in nature with edema, infiltration of plasma cells). 132 siRNA RT primer Ill” lllllll , mRNA \ / Step] I I l Stem loop RT-PCR llllllllllllllllllll cDNA Step2 Regular PCR Forward primer \ Figure 4-5. Schematic showing stem-loop RT-PCR miRNA assays. Stem—loop RT primers bind to the 3‘porti0n of siRNA but not mRNA, initiating reverse transcription of the siRNA. Then, the RT product is amplified using a siRNA specific forward primer and the universal reverse primer. Modified from Chen et al, 2005. 4— Reverse primer 133 to 00$; Birds presence of RCASBP(A)3mirs-187461 70-1 518 a 0'20 E . illil . w J? W ..u HHHIHI‘III Relative expression of siRNAs .l a: ll :l l. b A-W w ill; lEE : SI 1 5 Birds present of RCASBP(A)3mirs-18746170-1518 provirus Figure 4-6. Birds present of RCASBP(A)3mirs showed various levels of siRNA expression. Line 0 embryos were injected with RCASBP(A)3mirs-1874-6170-1518 targeting MDV g8 and ICP4 genes. Birds that showed presences of provirus by ELISA were selected for measuring siRNAs expression using stem-loop RT-PCR assays. (a). representative agarose gel picture with stem-loop RT-PCR product, DF-l cells, small RNAS for stem-loop RT-PCR assays were isolated from DF-l cells used for egg injection. noRT, no reverse transcriptase. (b) Relative siRNA expression from each bird was calculated as the average of 3 siRNAs level divided by the average of 3 siRNAs level in DF-l cells. Black, trial 4; grey, trial 5. 134 To summarize trial 4, the RNAi effect of RCASBP(A)3mirs-1874-6170-1518 significantly reduced MDV viremia and MD incidence and increased overall survival rate by reducing MD-induced death. A stem-loop RT-PCR assay was developed that successfully measured siRNA expression levels in birds; however, no correlation was seen between siRNA expression level and the reduction in MDV replication. Trial 5 was originally set up as a replica of trial 4, in which RCASBP(A)3mirs- 1874-6170-1518 again would be tested against challenge with the vv+648A strain of MDV in line 0 chickens. Unfortunately, the passage 40 648A strain MDV stock used in trial 4 was no longer available to us, so for trial 5 we obtained a sample of a passage 39 648A stock and then passed this once more in CEF prior to infecting chicks at hatch. One other change was that for the trial 5 infected PBL assays, an immunoperoxidase—based staining technique (Silva, Calvert, and Lee, 1997) was employed that allowed for more accurate detection of smaller MDV plaques than did direct microscopic examination. Otherwise, the same protocols used in trial 4 were repeated in trial 5, including the same challenge dose of 648A MDV (500 pfu). Again, we observed relatively high hatching rates even in vector-injected groups (25% in scrambled control and 23% in 3mirs RNAi treatment) because line 0 was at the peak of its production cycle. MDV titers in PBLs at 14 dpi were about 2 to 5 fold higher among all treatment groups compared to those in trial 4 (111 PFU/2 X 106 PBLs in RCASBP(A)3mirs-1874-6170-1518, 198 PFU/2 X 106 PBLs in RCASBP(A)3mirs-scramble, and 183 PFU/2 X 106 PBLs in no vector, Table 4- 3). This difference is probably due both to the different 648A stock used and to the new infected PBL assay. RNAi treatment again significantly reduced MDV titer compared to 135 either the scrambled control treatment or no vector treatment (p<0.01, ANOVA, Fisher protected LSD), in both cases by about 40% (Table 4-3). (The lower percentage reduction in trial 5 vs. trial 4 may relate to the improved ability of the new assay to detect small plaques. Our in vitro results previously demonstrated that retroviral-delivered RNAi reduced MDV plaque size as well as plaque number, Chen et al., 2008; chapter 3.) MD incidence was also slightly reduced by RNAi treatment; however, statistical significance was only achieved when comparing the RNAi treatment group with the scrambled control group (p<0.05, two-proportion z-tests, Table 4-4). Compared to trial 4, MD incidence increased in all treatment groups in trial 5 (Table 4-4), and RNAi treatment did not reduce the MD survival rate. This likely relates to the new viral stock that we had to use in the challenge. We again used stem-loop RT-PCR assays to examine the average siRNA expression levels in RNAi-treated birds. As in trial 4, there was no correlation between the average siRNA expression level and MDV titers in PBLs at 14 dpi. There was a modest correlation in siRNA expression and MD incidence, since eliminating birds with less than 10%, 20% and 50% siRNA expression (relative to the infected CEF control), average MD incidence was 74%, 70% and 61%, respectively. In trial 5 as in trial 4, the RCASBP(A)3mirs-1874-6170—1518 significantly reduced MDV viremia at 14 dpi. There was a reduction in MD incidence relative to both the scrambled control virus and no treatment control, but this was less dramatic. However, statistical significant was only achieved when comparing RCASBP(A)3mirs- 1874-6170-1518 with scramble but not no treatment. The new 648A virus stock used for trial 5 appears to have a higher virulence (relative to the 500 pfu dose used) than that used in trial 4 (higher MDV titer, higher MD incidence and mortality). Thus, this level of 1 36 virulence may have overcome the partial and modest effects of RNAi that we’ve been able to observe, especially in terms of disease progression. 137 Table 4-1. MDV RNAi in vivo experimental design: number of chicks per group Treatment Treatment CASBP(A) RNAi CASBP(A) scrambled s control Treatment V V V Bursa 1 5 ‘ 5 5 5 5 5 3 5 5 5 a numbers of birds in each experiment were targets we tried to reach without knowmg in advance what hatch rates might be or exactly how many embryos would become available to us. 138 Table 4-2. General procedures of evaluating RNAi induced anti-MDV effects in viva 2 Pilot Trial 5x2 c8630 madam £35 828 $23.33 38m «ones $55 2: mm<0m 22 6:0 -3er 32 -053 -vwwfi if m D m _ -oom Auk: VD: D2 DZ wmam wmqa wmaa a3 3mm 5 £88; 3mm 5 £68; 3mm 5 «was; 3mm 5 2885 3mm 5 2885 ac: commmoaxo <75? :ommmoaxo <73?w DZ CU DZ 5: DZ 3mm 5 $885 DZ _aev~ 8898‘? D2 889:? DE 889?? DE 899?? D2 8895? D2 86on z: 3ND, not done 139 Table 4-3. Reduction of MDV virerrria by RCASBP(A) delivered RNAi 8.5 Eur—r DmHDnE c3832 Dmhan—m 596:2 DmHDmm 52:32 DmfiDn—m :58: Z Dwflam 598: Z ©2441me mm on D “no Om meR. ON om—fiog 2 Dmflmm 882, oz ocmnmwi E. wmfiv av wwfioo M: ©2va cough oEEEom owfi_—D** mm mmflomi. mm omnwm ow VVflmN* ND vmfimm A o a m Ox <1- o o «— m m [\ Z sr N ._. t\ v— ON 00 oo 0\ oo .2 E H o o «q- rx \o ._ \o M O\ ..... m N N as \o .— 00 v 00 oo 0\ A < V On :0 (I) <1 <1— 0 N V') '— O\ to v r\ o oo .— * o oo Dd .— N —— tn <- oo —« ar- oo «- **Statistical difference of P<0.0001 from both no vector and scramble treatments within a column *Statistical difierence of P<0.05 fiom scramble treatment within a column 141 DISCUSSION We have described the use of retroviral vector-based RNAi against MDV in vivo and demonstrated that a RCASBP(A)3mirs vector targeting both the MDV g8 and ICP4 genes can significantly reduce MDV viremia and MD incidence as well as increase overall survival rate by reducing MD-induced death. Two critical components of MDV in vivo challenge experiments are the MDV strain, passage number and dose used and the line of birds chosen for infection. We used endogenous virus free line 0 chickens in all trials except trial 3 even though the susceptibility of line 0 chickens to various virulent strains of MDV in some cases had not been previously tested. Our pilot trial was unsuccessful because line 0 proved unexpectedly resistant to the W Mdll strain. Another important component in our in vivo experiments is the number of embryos hatched. We observed relatively low hatching rates in several trials due to the combination of egg injections, old hens, and reproduction cycles. Failures to achieve statistically significant reductions in MDV virerrria (trial 3) and MD incidence (tria12) result in part fi'om the fact that we were unable to test a sufficient number of birds. While Table 4-1 describes our initial targets for bird numbers, we were rarely able to fully populate each treatment group as desired (see Table 4-4 for the actual numbers under study). Nevertheless, in trials 4, we were able to demonstrate that RCASBP(A)3mirs vectors expressing three siRNAs, two of which targeted the MDV gB gene and one of which targeted the ICP4 gene could significantly reduce MDV viremia (up to 80% of no treatment, and 50% of scramble control) and MD incidence (30% less than no treatment and scramble control in general, 40% less than no treatment when eliminating birds with 142 low siRN A expression ) as well as increase overall survival rate by reducing MD induced death compared to scramble control. To our knowledge, this is the first time that an RNAi-based strategy has been successful in live chickens. Compared with the current MDV vaccine of choice, CVI988/Rispens, our RNAi vector is similar in the level to which it reduces MDV virerrria but less effective in terms of reducing MD disease (Lee et al., 2008). Several options exist to improve the level of the RNAi effect. The endogenous chicken chromosome 4 miRNA cluster that we have modified to deliver three targets originally contained six potential miRNAs, although it has not been proven that all six are expressed. We can incorporate more siRNAs into the miRNA cluster to target additional MDV genes. We showed that using multiple target genes may enhance the antiviral effect to a greater extent than using multiple targets within one gene (Chen et al., 2008). It would be interesting to co-target genes that are essential for viral replication, such as gB, and genes that are essential for horizontal transmission or required only for the pathogenic effect of MDV (Osterrieder et al., 2006). Foreign genes transferred by retroviral vectors can be silenced due to epigenetic chromatin modification processes resulting in DNA methylation and histone modification (Ellis and Yao, 2005). In order to address the vector silencing issue, we first confirmed the presence of vector viruses using an ELISA assay to detect the ALV p27 capsid protein. We initially tested PCR amplification and Southern blot assays to measure retention of the RNAi cassette. However, these two methods do not answer the more critical question, which is the extent of shRNA-mir expression in the birds. Therefore, in 143 trials 4 and 5, we used a stem-loop RT-PCR assay to directly test the expression levels of siRNAs. Our results indicate that the average siRNA level varied among birds. A large percentage of birds (about 76%) expressed relatively low levels of siRNA. Strikingly, there seems to be no correlation in siRNA level and MDV viremia. MDV infection in birds occurs in four phases (cytolytic, latency, secondary cytolytic, and transformation). We measured MDV viremia in PBLs at 14 dpi, at which time infected birds can be in either the latency or secondary cytolytic phases. This probably contributes to the high level of variability in MDV-infected PBL counts which makes observing such a correlation more difficult. Furthermore, the level of reduction in the target gB and ICP4 messages need not correlate exactly with MDV replication. In future experiments, it might help to measure MDV viremia at multiple time points, which could provide a more accurate picture of the RNAi effect. Although siRNA level might not be a good indicator of MDV viremia at 14 dpi, there was some modest indication that birds with relatively high siRNA levels were more likely to survive and be disease fi'ee. Thus, another option for future research may be to develop vectors with stronger promoters driving siRNA production. Birds infected with replication competent RCAS/RCAN vectors at hatch can develop ALV-induced lymphomas (lymphoid leukosis) due to proviral activation of the endogenous c-myc gene at about 4-6 months (Payne, 1998). Embryonic infection of chickens with ALV or RCAS/RCAN vectors (in our case) can also result in the rapid induction of B cell lymphomas involving integration into the c-myb locus. Most studies involve inoculations of 10-16 day chicken embryos fiom the SC line of White Leghorn chickens and observe development of B cell lymphomas at 4 to 5 weeks post egg 144 injection (Kanter, Smith, and Hayward, 1988; Pizer and Humphries, 1989; Pizer, Baba, and Humphries, 1992). It has been reported that the time of infection by RCAS affects tumor incidences and their oncogenic spectrum (Jiang et al., 1997). In our trials, we injected day 0 embryos from line 0 and observed rare B cell lymphomas as early as 9 days post injection, suggesting that the time of infection of RCAS also affects the time of development of B-cell lymphomas. Both gross tumor examination and H&E staining could not differentiate MDV-induced T cell lymphomas from RCAS-induced B cell lymphomas in our trials. So we used an irnmunohistochemical staining of the MDV oncoprotein meq as well as the B cell antigen Bul to differentiate MDV-induced lymphomas. Typically, less than 10% of birds in vector injected groups developed B cell lymphoma and died before the termination without any MD symptoms. We recognize that RCAS/RCAN vectors are not suitable for application in the field due to their oncogenic properties; however we believe them to be the optimal system to test for an antiviral RN Ai effect at this stage of our research. If this approach proves feasible, then a longer term attempt at germline or lentiviral vector-induced transgenics could be better designed based on what we learn. A major concern for the in viva application of RNAi is the possibility of stimulating interferon responses through the toll-like receptor (TLR) pathway. siRNAs can stimulate the type II interferon response through TLR3 in the TRIP—NF-KB cascade (Kleinman et al., 2008). 'siRNA containing irnmunostimulatory sequences such as UGUGU or GUCCUUCAA can trigger type I interferon responses through TLR7 (this effect can be eliminated by incorporating 2’-O-methyl (2’OMe) nucleotides in GU residues) (Homung et al., 2005; Judge et al., 2005). Robbins et a1 (2008) recently showed 145 that native siRNAs could significant inhibit influenza virus replication both in vitro and in viva, while the corresponding 2’OMe siRNAs were only effective in vitro but not in viva. The authors further showed that commonly used control GFP siRNAs were less potent in stimulating IFNa responses in vitro compared to 8 effective siRNAs against viral, angiogenic and oncogenic targets. This paper raised an alarm in interpreting results from RNAi-based anti-viral studies in viva. The chicken genome contains TLR genes that are capable of stimulating interferon responses (Schwarz et al., 2007). We have no direct proof that our RNAi constructs didn’t stimulate an interferon response, since we did not test interferon levels. However, we think it unlikely that the anti-viral effects we observed in viva are due to RNAi-induced interferon responses. First, shRNA-mirs based on the cellular miR-30a gene were shown not to stimulate interferon responses in a different study (Bauer et al., 2008). Second, we demonstrated that the vectors we used were able to significantly inhibit MDV replication BOTH in vitro and in viva. Our siRNAs lack immunostimulatory sequences that would be predicted to trigger type I interferon responses through TLR7/8. The TLR3 activation is based on a siRNA-class effect, which is independent of both siRNA sequence and chemistry. If our RNAi constructs were able to activate TLR3, then both effective siRNAs and scrambled control insert siRNAs should show anti-viral effects. Our results indicated that scrambled control insert siRNAs did not consistently reduce MDV viremia in viva. Finally, we delivered RNAi constructs through injecting day 0 chicken embryos which results in congenitally infected birds that are immunologically tolerant to the retroviral vectors (Payne, 1998). siRNAs can mediate off-target silencing because of the complementarity between the seed regions of the siRNAs and the 3' UTR sequences of unintended transcripts (Jackson et al., 2006). 146 Our design protocol seeks to eliminate significant complementarity other than to the desired target. We have verified that the target gene showed reductions in mRNA and/or protein that correlate with any anti-viral effect in vitro (Chen et al., 2008). 147 MATERIALS AND METHODS Vector constructions The construction of the RCASBP(A)miRNA and RCASBP(A)3mirs vectors as well as the corresponding pENTR3C-miR30a entry vector and pENTR3C-3mir entry vector has been described previously (chapters 2 and 3). The RCASBP(A)mirRNA vector delivering a single siRNA (1874) against the MDV gB gene (termed RCASBP(A)miRNA-1874) was used in the pilot trial and trial 3. RCASBP(A)3mirs delivering 3 siRNAs against the MDV gB gene was used in trial 2. RCASBP(A)3mirs delivering 2 siRNAs (1874 and 1518) against the MDV g8 and one against the ICP4 gene (6170) was used in trials 4 and 5 (termed RCASBP(A)3mirs-1874-6170-1518, Table 4-2). Cells and viruses DF-l cells were maintained in Leibowitz's L-lS and McCoy 5A media (1:1) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals), 50 rig/ml of gentamicin (Invitrogen), and 0.25 rig/ml of fimgizone at 39 °C. Chicken embryo fibroblasts (CEF) were maintained in the same media supplemented with 4% calf serum (Invitrogen Corp.). The serum content for MDV-infected CEF cells was reduced to 2% when cells reached confluence. Very virulent Mdll (7 passages in CEF), very virulent MDS (7 passages in CEF), and very virulent plus (vv+) 648A (40 passages in CEF) strains of serotype 1 MDV were obtained fi'om Richard Witter and Mohammad Heidari, USDA-ARS, Avian Disease and Oncology Lab (ADOL). Mdll was used as the 148 challenge virus in the pilot trial, Md5 in trial 3, and 648A in the remaining trials (Table 4- 2). Delivery of RCAS vectors into DF-l cells Propagation of RCASBP(A) vectors in DF -1 cells was initiated by transfection of plasmid DNA containing the retroviral provirus using SuperFect Transfection Reagent (Qiagen, Inc.) according to the manufacturer's protocol. Viral spread was monitored by assaying culture supernatants for ALV capsid protein by ELISA (Smith, Fadly, and Okazaki, 1979) Once the viral titer peaked, DF-l cells were counted and prepared for direct egg injection (see below). In viva MDV challenge assay Line 0 chickens were used in in viva trials except for trial 3 even though the susceptibility of line 0 chickens to various virulent strains of MDV in some cases had not been previously tested. Line 0 is a White Leghorn line that is genetically susceptible to all RCAS subgroups except subgroup E and is free of endogenous proviruses that are closely related to ALV (Astrin, Buss, and Haywards, 1979). Chickens from a 1515x71 cross were used in trial 3 because line 0 was at the end of its annual egg production cycle. General procedures and experimental set up are described in Figure 4-1, and Tables 4-1 and 4-2. This experimental design has a power of 0.9 or greater to detect 8% or greater mean differences between proportional characteristics, such as tumor incidence and number of chicks surviving for a certain period of time. Fresh or stored embryos (stored up to three weeks at 4°C) were somatically infected with RCASBP(A)miRNA or RCASBP(A)3mirs vectors by injecting unincubated 149 eggs near the blastoderrn with about one million DF -1 cells producing the RCAS vector virus. Viremic chicks were identified at hatch by ELISA for the ALV capsid protein p27 (this test can only be used in Line 0 or another line that does not contain an expressing endogenous provirus). Chicks with low (OD490