‘u o“ 1.0" .._ 7 \J O---oc*.o..O-f--1“N" five-9'9." ?-~"”.CQ"4O-‘~‘-Q~‘O(w‘~“-~.‘..‘..",’.v -w'-.. .. \‘fi‘. . . . E‘FECT 0F B-AZAURICIL RIBOSIDE AND PUROMYCIN ON THE TIME COURSE OF BIOCHEMICAL EVENTS IN THE REPLICATION CYCLE 0F AVIAN INFECTIOUS BRONCHITIS VIRUS ‘ Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY CHMLES W. MOORE 197 l .. . -. .. I .. .l ' ' v .1 ‘ 0-! v "' I . . " ,_. r e -. . ' _ - l , cl- ' . ‘ I. . ' .0 O .- ' a .v . I n , , . , I , ' . v". . ' ',. " , ‘. I . I "".’ . ‘ .'.'.ITw-A.I--II"—"""”.' " o a " - t'fi‘r' . .dfiu-U" . -.. . , . 1. on '1‘ , o .v.: - . . ’ ’ __‘,'.- f... , .0’1'1P’ .f,‘ ‘ .’ . 'f ‘ "”-. .' ’ :- '0' - . ~ ' - p '0 .’. ... " [M‘ . ' ." ““‘I ‘ . v’n- " '. , n!. r. y.' ' . <'l '. ." ' . . v o -.'9 . l0. ' —" —_———————— av 0 v - ..-c.‘q.~a. flan-v.‘ -p-.--...“.v.“4.~woomn m c o-“ 9-. 9.1"". ”CW L I B R A R Y Michigan State University l; VBINDI 7 BY mm; & s s' mun: Inc. ABSTRACT EFFECT OF 6-AZAURICIL RIBOSIDE AND PUROMYCIN ON THE TIME COURSE OF BIOCHEMICAL EVENTS IN THE REPLICATION CYCLE OF AVIAN INFECTIOUS BRONCHITIS VIRUS BY Charles W. Moore The production of avian infectious bronchitis virus (IBV) in chicken embryo kidney cells (CEKC) is in— hibited nearly 90% of normal by 50 ug/ml of 6-azauricil riboside (AUR) and 95% of normal by 5 ug/ml of puromycin. When AUR, an inhibitor of pyrimidine nucleotide biosyn- thesis, is added to IBV infected CEKC at various times after infection, there is a gradual increase in production of IBV until about 3 hours, after which infectious virus production ceases to increase further. Puromycin, a pro- tein synthesis inhibitor, added at various times after infection inhibits virus production through 4.5 hours, after which IBV production sharply increases. This information implies that synthesis of viral RNA begins shortly after infection and is completed by about 3 hours after infection. It also indicates that Charles W. Moore viral protein synthesis is completed between 4.5 and 5 hours, which, according to the results of another ex- periment, coincides very closely with the length of the replication cycle of IBV. EFFECT OF 6-AZAURICIL RIBOSIDE AND PUROMYCIN ON THE TIME COURSE OF BIOCHEMICAL EVENTS IN THE REPLICATION CYCLE OF AVIAN INFECTIOUS BRONCHITIS VIRUS BY I {0 00A Charles W. Moore A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1971 ACKNOWLEDGEMENTS I would like to express my sincere gratitude to Dr. Charles H. Cunningham, Professor of Microbiology and Public Health, for his guidance and encouragement through- out this investigation, and for his assistance in the preparation of this manuscript. I also wish to extend my sincere thanks to Mrs. Martha P. Spring, Department of Microbiology and Public Health, for her kind and invaluable assistance and in- struction in the proper virology and cell culture tech- niques. ii TABLE OF CONTENTS LIST OF FIGURES . . . . . . . . . INTRODUCTION . . . . . . . . . . LITERATURE REVIEW . . . . . . . . METHODS AND MATERIALS . . . . . . . Viruses . . . . . . . . . . Cell Cultures . . . . . . . Puromycin and 6 -Azauricil Riboside . Thymidine and Deoxycytidine . . . . Inoculation of CEKC tube Cultures . . Assay of Viruses . . . . . . . Replication Cycle . . . . . . Effect of Various Concentrations of the Inhibitors on the Replication of IBV Effect of Time of Addition of the In- hibitors on the Replication of IBV . RESULTS 0 O O O O O O O O O O O Replication Cycle . . Effect of Various Concentrations of the Inhibitors on the Replication of IBV Effect of Time of Addition of the In- hibitors on the Replication of IBV . DISCUSSION . . . . . . . . . . . LITERATURE CITED . . . . . . . . . iii Page iv 10 10 11 ll 11 12 12 14 14 14 14 26 3O LIST OF FIGURES Figure Page 1. Replication cycle of IBV . . . . . . 16 2. Effect of AUR on IBV replication . . . 18 3. Effect of puromycin on IBV replication . . . . . . . . . . 20 4. Effect of time of addition of AUR on IBV replication . . . . . . 22 5. Effect of time of addition of puromycin on IBV replication . . . . 25 iv INTRODUCTION Since the description of "an apparently new respir- atory disease of baby chicks" in 1939 (39), a great deal of information about the disease and the etiologic agent, avian infectious bronchitis virus (IBV), has been collected. Early work was concerned primarily with the disease it- self: the clinical symptoms and the treatment. Later work led to a description of the properties of the virus itself, as well as the nature of its component parts. This provided a basis for comparison with other viruses reveal- ing a relationship with the etiologic agents of other dis- eases, possibly including the human cold virus. Studies of the replication cycle of IBV have been directed primarily toward the physical and biological as- pects. Light and electron microscopy of IBV infected cells have contributed a significant amount of information, as has the use of fluorescent antibodies. However, rela— tively little has been done to study the replication cycle on a biochemical level. Therefore, a study of the time course of biochemical events in the replication cycle of IBV was initiated using metabolic inhibitors with known modes of action. LITERATURE REVIEW Avian infectious bronchitis virus (IBV) is a mem- ber of the Coronavirus group (2,15) which includes viruses causing the human common cold, mouse hepatitis virus, and others (3,24,30). These viruses seem to replicate in a similar manner and have several morphological characteris— tics in common (6,10). The protein coat of IBV surrounding the core of ribonucleic acid (RNA) (1) is covered by a lipid envelope (32). The specific gravity of the Beaudette strain is 1.24 as determined by isopycnic density gradient centri- fugation in cesium chloride. With a linear density grad- ient of sucrose, the specific gravity is 1.19. The sedi- mentation constant is 344 8 indicating that the particle is roughly a sphere between 80 and 100 mu in diameter (14). Infection by IBV occurs following attachment of the virion to specific receptor sites on the surface of the membrane of the host cell, which nearly always is a chicken cell. The earliest visible immunofluorescence is present 4 hours after infection, primarily in the peri- nuclear region, but eventually diffusing throughout the cytoplasm (11,29). Sections of IBV-infected cells fixed between 30 and 40 hours, have viral particles with an overall diameter of 67 to 110 mu (6,36). The virus buds from the cytoplasm into cisternae of the endoplasmic reticulum (15). During the budding process, the virus acquires the lipid envelope, which is composed, at least partially, of altered host cell membrane material (6,36). Extracellular virions possess club- or pear-shaped spikes, 20 mu in length, covering the surface of the envelope (7). Puromycin, an inhibitor of protein biosynthesis, consists of an aminonucleoside linked by an amide bond to the amino acid p-methoxyphenylalanine. A structural analogy to the amino acyl adenosine portion of amino acyl transfer RNA (tRNA) is responsible for its inhibitory activity. During normal protein synthesis (44), a complex composed of amino acyl tRNA, guanosine triphosphate (GTP), and Transfer Factor I is inserted into the amino acyl ("A") site on the ribosome where anticodon-codon hydrogen bonding holds the amino acyl tRNA in position. Peptidyl transferase, an integral part of the ribosome, removes the polypeptide from the peptidyl tRNA on the peptidyl ("P") site on the ribosome, and catalyzes the formation of a peptide bond between the carboxyl end of the polypeptide and the a-amino group of the amino acyl tRNA. The tRNA left in the "P" site is released and, with the help of GTP and Transfer Factor II, the ribosomal complex moves up one codon. This allows the translocation of the peptidyl tRNA from the ”A" site into the "P" site. Puromycin will move into the "A" site on the ribo- some instead of the amino acyl tRNA. Peptidyl transferase will then form a peptide bond between the carboxyl end of the polypeptide and the free amino group on the puromycin molecule. Since puromycin possesses neither an anticodon to hold it on the ribosome nor a carboxyl group with which to form a peptide bond, the polypeptidyl-puromycin chain is released from the ribosomal complex (19,34,35,40,43,45). By this mechanism, puromycin prevents synthesis of complete proteins. Puromycin has been used to study various aspects of in_yi£52_protein synthesis including the importance of N-formylmethionyl-tRNA (46) and GTP (21) in peptide init- iation, the required presence of ribosomes (33), and the roles of the ribosomal subunits (22). The effect of puromycin on viral infections in cell and tissue cultures has been used to study the cyto- pathic effects (CPE) produced by vaccinia virus and the relationship to the replication cycle of the virus (4). The CPE that normally occurred early in infection, as well as viral multiplication, were inhibited by treating in- fected cells with high concentrations (330 ug/ml) of puro- mycin. At lower concentrations (33 ug/ml), viral multiplication was still inhibited significantly, but the CPE was nearly the same as that in infected cells without puromycin. The CPE was interpreted as being caused by viral-induced proteins made early infection. Synthesis of poliovirus RNA did not occur when puromycin was added within 2 hours after infection of HeLa cells (26). When puromycin was added 2.5 hours after in- fection, synthesis of significant amounts of viral RNA occurred, but mature virions were not formed. This indi- cated that a protein required for production of viral RNA was synthesized during the first 2 hours after infection. Synthesis of pyrimidine nucleotides required for nucleic acid synthesis is inhibited by 6-azauricil ribo- side (AUR) (20,37). An important precursor in the bio- synthesis of pyrimidines is orotic acid. 0rotidine-5'- phosphate perphosphorylase catalyzes the reversible formation of orotidine-5'-phosphate (OSP) from orotic acid and S-phosphoribosylpyrophosphate (PPRP). Uridylic acid or uridine monophosphate (UMP), a precursor of all pyri- midine nucleotides required for the synthesis of both deoxyribonucleic acid (DNA) and RNA in most types of cells, is formed by the irreversible decarboxylation of orotidine- 5'-phosphate by 05P decarboxylase (25,27). Normal cell enzymes phosphorylate AUR to form 6-azauridine-5'-phosphate, or azauridylic acid, which is P the active form of the inhibitor. This form competitively inhibits OSP decarboxylase, thus preventing formation of UMP and resulting in the accumulation of orotidine. Hence, nucleic acid biosynthesis is inhibited due to a lack of pyrimidine nucleotides (20,37). The replication of type 5 adenovirus, a DNA virus, was studied by adding AUR to infected cell cultures at various intervals after infection (18). Thymidine and deoxycytidine were also added to allow synthesis of viral DNA. All samples were harvested after 30 hours of incu- bation and assayed for infectious virus, which was plotted against the time of addition of the inhibitor. The data, when interpreted explicitly, indicated that throughout the first 8 hours of infection the AUR sufficiently inhibited the accumulation of pyrimidine ribonucleotides to prevent synthesis of the RNA necessary for replication of the virus. When the inhibitor was added at any time after 8 hours, however, the pyrimidine nucleotide levels were suf- ficient to allow synthesis of increasing amounts of RNA which were directly related to the time after infection. By 16 hours, enough nucleotides were present to permit maximal synthesis of viral RNA and, subsequently, maximal production of infectious virus. The implicit interpretation is that synthesis of RNA essential for production of infectious adenovirus began about 8 hours after infection and, by 16 hours, enough viral RNA had been made to allow maximum virus production. At various intervals after infection, AUR.was added to cell cultures infected with an RNA containing virus, dengue-2 virus (41). After 21 hours of incubation, the samples were plaque assayed and the results plotted against the time of addition of AUR. A reversal type of experiment was also performed in which AUR was added to the cell cultures at the time of infection and then, at sequential time intervals, the inhibition by AUR was re- versed by the addition of uridine. Again, after 21 hours of incubation, the samples were plaque assayed and the results were plotted against the time of addition of uridine. The data inferred that production of infectious virus depended on a species of RNA whose synthesis began about 6 hours after infection. METHODS AND MATERIALS Viruses The 137th passage of the Beaudette strain of avian infectious bronchitis virus (IBV) adapted to chicken embryo kidney cells (CEKC) was assayed on primary CEKC and found to contain 8.6 x 106 plaque-forming units per milli— liter (pfu/ml). The stock extracellular virus from the CEKC was stored in screwcap vials at -90°C. Cell Cultures Primary CEKC were prepared from 17-19 day old embryos according to standard methods (13,16). The cells were suspended in Medium 199, Grand Island Biological Company (GIBCO), containing 2 mM glutamine, and supple- mented with vitamins and amino acids (GIBCO), 100 units/ml penicillin, 100 ug/ml dihydrostreptomycin, 50 units/m1 Mycostatin (GIBCO), and 0.1% sodium bicarbonate. Newborn calf serum (nbcs) was added to a final concentration of 5%. Cell cultures in 16 x 125 mm tubes (Rochester Scientific Co., Inc.) were inoculated with 1 ml of a 1:200 dilution of packed cells with a final concentration of 10 5 x 106 cells/m1. For plaque assays, 4 m1 of a 1:100 dilution of packed cells, final concentration of 107 cells/ ml, was dispensed into 15 x 60 mm plastic petri dishes (Falcon Plastics). All cell cultures were incubated at 37° in an atmosphere of 6-8% C02 and 80-85% relative humidity. Monolayers of cells formed in the tubes in about 48 hours, whereas 3 to 4 days were required for monolayers to form in petri dishes. Puromycin and 6-Azauricil Riboside Puromycin dihydrochloride and 6-azauricil ribo- side (AUR) (Nutritional Biochemical Co.) were prepared as 1 mg/ml stock solutions in Hanks' balanced salt solutions (HBSS) made with double glass distilled water. The solu- tions were sterilized by filtration (Falcon Plastics, 7103 Filter, 0.22 p average pore size) and stored at 4°C. Thymidine and Deoxycytidine Thymidine and deoxycytidine (Nutritional Bio- 4 M and 10-3 M solutions, chemical Co.) were prepared as 10- respectively, in HBSS made with double glass distilled water. The solutions were sterilized by filtration and stored at 4°C. Thymidine and deoxycytidine were always used in conjunction with AUR, at a final concentration of 10"5 M and 10-4 M, respectively, thereby providing the pyrimidine deoxyribonucleotides needed for DNA synthesis. 11 Inoculation of CEKC Tube Cultures Extracellular fluid was decanted from the tubes and the cells were washed twice with HBSS. Each tube was inoculated with 0.2 ml of stock IBV containing 1.7 x 106 pfu. This represented a multiplicity of infection of about 0.3. The inoculated cultures were incubated for 1 hour to allow adsorption of the virus to the cells. Fol- lowing the adsorption period, the inoculum was decanted and the monolayers were then washed 4 times with cold HBSS. Fresh medium was added to each tube and they were then placed in the incubator. This was considered 0 time for all experiments. Assayyof Virus Extracellular fluid from the infected cultures, containing released virus, was decanted into screwcap vials and stored at -90°C. At the time of use the virus was thawed at room temperature. Monolayers of CEKC in petri dishes were inoculated with 0.5 m1 of 10-fold di- lutions of each sample, with 1 culture per dilution and 3 plates per sample. The cultures were then treated as described in the plaque assay method (13,16). Replication Cycle At various intervals after infection of CEKC tube cultures with IBV, the extracellular fluid from 2 cultures 12 was pooled in a screwcap vial and stored at -90°C until the assay materials were prepared. Effect of Various Concentrations of the Inhibitors on the Repli- cation of IBV At 0 time, various concentrations of the inhibitors were added to tube cultures of IBV-infected CEKC. Two tube cultures per inhibitor concentration were used. The extracellular fluid from each sample was harvested and pooled 12 hours after infection and then assayed for in- fectivity. The virus yield for each concentration of inhibitor was calculated as per cent of normal yield, which was the yield of infectious virus from cultures not treated with the inhibitor. The per cent yield was then plotted against the concentrations of the inhibitors. The concentration of the inhibitor that was used in subsequent experiments was considered to be the concentration that inhibited IBV replication by about 90% but was not so toxic as to prevent the host cells from surviving during the lZ-hour incubation. Effect of Time of Addition of the Inhibitors on the Replication of IBV At each of several sequential intervals following infection of CEKC tube cultures with IBV, an inhibitor was added to 2 tubes. The final concentrations of the 13 inhibitors were 50 ug/ml for AUR and 5 ug/ml for puromycin. The extracellular fluid was harvested from all tubes 12 hours after infection, pooling each sample, and then assayed for infectivity. The relative concentrations of infectious virus were expressed as pfu/ml and then plotted against the time of addition of the inhibitor. RESULTS Replication Cycle A sharp increase in released infectious virus, indicating the end of a replication cycle, occurred between 4.5 and 5 hours after infection (Figure 1). These results agree with previous studies (28,29). Effect of Various Concentrations of the Inhibitors on the Repli- cation of IBV The optimum concentrations of the inhibitors were 50 ug/ml of AUR and 5 ug/ml of puromycin (Figures 2 and 3). These were the concentrations used in subsequent experi- ments. Effect of Time of Addition of the InhibiEors on the Repli- cation of IBV There was an increase in the production of in— fectious virus when AUR was added at anytime during the first 3 hours after infection, and a relatively constant production when AUR was added at anytime after 3 hours and through the last sample at 10 hours (Figure 4). This implies that synthesis of viral RNA began soon after 14 15 Figure l. Replication cycle of IBV. Each point is the average of 3 experiments and represents the concentration of infectious virus in the pooled extracellular fluid from 2 tubes taken at the indi— cated intervals after infection. 16 4 o :E\:mn= notches-.0600 2... .> 103 9 10 II 12 Time (How: After Infection) 17 Figure 2. Effect of AUR on IBV replication. Tube cultures of IBV infected CEKC were treated with various concentrations of AUR for 12 hours. The extracellular fluid was then harvested, pooling each sample, and assayed for released virus. Each point is the average of 3 experiments. Percent Normol Yield 90 70 60 50 3O 18 ‘IO 20 3O 40 50 Al"! Concentration (cg/inn 70 19 Figure 3. Effect of puromycin on IBV replication. Tube cultures of IBV infected CEKC were treated with various concentrations of puro- mycin for 12 hours. The extracellular fluid was then harvested, pooling each sample, and assayed for released virus. Each point is the average of 4 experiments. Percent Normol Yield 90 70 60 4O 20 I 2 3 4 5 6 Puromycin Concentrotion («g/ml) 21 Figure 4. Effect of time of addition of AUR on IBV replication. At each of several intervals after infection, AUR was added to 2 tube cultures of IBV infected CEKC to a final concentration of 50 ug/ml. The extracellular fluid was harvested from all cultures 12 hours after infection, pooling each sample, and assayed for infectious virus. Each point is the average of 2 experiments, each of which were assayed twice with an average variation of :0.05 log units. Virus Concentration (pfu/ml) 10’ 10‘ 22 l 2 3 4 5 e 7 8 9 Time of Addition of AUR (Hours After Infection) 23 infection and was completed between 2 and 3 hours after infection. Synthesis of viral proteins was completed between 4.5 and 5 hours after infection as indicated by the marked increase in virus production when puromycin was added at anytime after 4.5 hours (Figure 5). The time of completion of viral protein synthesis coincides very closely with the end of the IBV replication cycle and the release of infectious virus (Figures 1 and 5). The lowest yield of virus produced when inhibitors were added early in infection and analyzed 12 hours after infection (Figures 4 and 5) was about 10 times higher than the background level of extracellular virus normally present just prior to the release of new virus in the absence of inhibitors (Figure 1). This difference is due to the production of between 5% and 10% of the normal virus yield with the inhibitor concentrations used, since higher concentrations did not decrease the yield (Figures 2 and 3) and were toxic to the cells. 24 Figure 5. Effect of time of addition of puromycin on IBV replication. At each of several intervals after infection, puromycin was added to 2 tube cultures of IBV infected CEKC to a final concentration of 5 ug/ml. The extracellular fluid was harvested from all cultures 12 hours after infection, pooling each sample, and assayed for infectious virus. Each point is the average of 2 experiments with an average variation of i 0.03 log units. Virus Concentration (pfu/ml) 25 l 2 3 4 5 6 7 8 9 10 Time of Addition of Puromycin (Hours After Infection) DISCUSSION The results from the experiments were based on the assay of extracellular virus which had been released from infected cells. Adding an inhibitor to virus-infected cells at various intervals after infection caused varia- tions in the amounts of virus that were produced. The variation was dependent on two factors: first, the time of addition of the inhibitor and, second, the mode of action of the inhibitor which indicates the viral compon- ent whose synthesis was inhibited. The results were then interpreted as to the possible time course of biochemical events in the intracellular replication cycle of the virus. When AUR was added at the time of infection and at sequential intervals thereafter, the production of infectious IBV increased throughout the first 3 hours after infection and then remained fairly constant. This suggested that synthesis of viral RNA had been completed by approximately the third hour of the 4.5 to 5 hour IBV replication cycle. However, inhibition of infectious virus production was only an indirect effect of the AUR, 26 27 since AUR inhibits only the synthesis of pyrimidine nucleo- tides. Therefore, the most rigorous interpretation of the results is that by 3 hours after infection the nucleotide levels in the infected cells were high enough to permit synthesis of IBV RNA in sufficient amounts for maximum production of infectious virus. Extrapolating somewhat beyond the rather limited interpretations above, the fact that there was an gradual increase in infectious virus whenever AUR was added during the first 3 hours suggests that much of the RNA that accumulated during that interval eventually became incor- porated into the progeny virions and that it began accumu- 1ating very shortly after infection. The data do not show what proportion of the RNA made was released in progeny virus, nor how much was used only as messenger RNA (mRNA). Also, it did not indicate how much of the RNA was comple- mentary (-) to the parental or virion (+) RNA, although the RNA that was first made was almost certainly comple- mentary. It is not known whether the (+) strand or the (-) strand acts as mRNA for translation into proteins of IBV, or whether each is translated into different proteins. The use of (-) strands as mRNA has been described for Newcastle disease virus (NDV) and Sendai virus (38), both paramyxoviruses, in which there was a preferential syn- thesis of (-) strand RNA which then became associated with the polyribosomes of the infected cells. 28 A prerequisite for early synthesis of IBV RNA would be early synthesis of an RNA polymerase or trans- criptase. Synthesis of such an enzyme must, in fact, occur very shortly after the virion enters the cell, since, as the data suggest, production of IBV RNA occurred within the first hour after infection. A speculative, al- though plausible, explanation would be the existence of an RNA polymerase within the virion. Although an RNA poly— merase has not been determined for IBV or other Corona- viruses, such an enzyme has been reported for several other lipid enveloped, single stranded RNA viruses (17,31), namely: Newcastle disease virus (NDV) (23) and Sendai virus (38,42), both paramyxoviruses, influenza A (8,9,12), a myxovirus, and vesicular stomatitis virus (5), a rhab- dovirus. The possible presence of this enzyme in the virions of IBV is, then, worth considering in future studies. Puromycin inhibits the translation of viral pro- teins, which is a more direct effect on the replication of IBV than is the effect of AUR, and, therefore, the re- sults of the experiments involving puromycin are more con— clusive. Although a number of different species of pro- teins are coded for by the viral genome, the data indicate only the time of completion of the last protein species required for viral replication. 29 The experimental results indicated that puromycin inhibited virus replication to about the same degree through 4.5 hours of infection. However, production of infectious virus increased sharply at 5 hours and main— tained that level thereafter, indicating that puromycin ceased to be effective when added anytime after 4.5 hours. Therefore, synthesis of IBV protein was completed between 4.5 and 5 hours after infection. The time of completion of the synthesis of the last viral protein coincided very closely with the time of release of mature IBV virions which indicates that re- lease occurred very shortly after the synthesis of viral proteins was completed. This suggests a possible sequence for the last few events in the intracellular replication of IBV. Although it is not possible to identify specific proteins with the techniques employed, the last proteins to be synthesized could be virus specific membrane pro- teins or, perhaps, structural proteins which are assembled with the viral RNA to form the nucleocapsid. Maturation proceeds as the nucleocapsid buds through the membrane of the endoplasmic reticulum, adquiring its lipid envelope and surface projections, and is released from the cisternae and cytoplasmic vesicles into the fluid surrounding the cell as an infectious virion. LITERATURE CITED LITERATURE CITED Akers, T.G., and C.H. Cunningham. 1968. Replication and cytOpathogenicity of avian infectious bronch- itis virus in chicken embryo kidney cells. Arch. ges. Virusforsch. 25:30-37. Almeida, J.D., D.M. Berry, C.H. Cunningham, D. Hamre, M.S. Hofstad, L. Mallucci, K. McIntosh, and D.A.J. Tyrrell. Coronaviruses. Nature 220:650. Almeida, J.D., and D.A.J. Tyrrell. 1967. The mor- phology of three previously uncharacterized human respiratory viruses that grow in organ culture. J. Gen. Virol. 1:175-178. Bablanian, R. 1968. The prevention of early vaccinia- virus-induced cytopathic effects by inhibition of protein synthesis. J. Gen. Virol. 3:51-61. Baltimore, D., A.S. Huang, and M. Stampfer. 1970. Ribonucleic acid synthesis of vesicular stomatitis virus. II. An RNA polymerase in the virion. Proc. Natl. Acad. Sci. U.S. 66:572-576. Becker, W.B., K. McIntosh, J.H. Dees, and R.M. Chanock. 1967. Morphogenesis of avian infectious bronchitis virus and a related human virus (strain 229E). J. Virol. ‘1:1019-1027. Berry, D.M., and J.D. Almeida. 1968. The morphologi— cal and biological effects of various antisera on avian infectious bronchitis virus. J. Gen. Virol. 3:97-102. Bishop, D.H.L., J.F. Obijeski, and R.W. Simpson. 1971. Transcription of the influenza ribonucleic acid genome by a virion polymerase. I. Optimal condi- tions for in_vitro activity of the RNA-dependent RNA polymerase. J. Virol. 8:66—73. 30 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 31 Bishop, D.H.L., J.F. Obijeski, and R.W. Simpson. 1971. Transcription of the influenza ribonucleic acid genome by a virion polymerase. II. Nature of the in vitro polymerase product. J. Virol. 8:74-80. Bradburne, A.F. 1970. Antigenic relationships amongst Coronaviruses. Arch. ges. Virusforsch. 31:362-364. Brown, J.L., and C.H. Cunningham. Immunofluorescence of avian infectious bronchitis virus and Newcastle disease virus in singly and dually infected cell cultures. Avian Diseases (In press). Chow, N., and R.W. Simpson. 1971. RNA-dependent RNA polymerase activity associated with virions and subviral components of myxoviruses. Proc. Natl. Acad. Sci. U.S. 685752-756. Cunningham, C.H. 1966. A laboratory guide in virology, 6th ed. Burgess Publishing Co., Minneapolis. Cunningham, D.H. 1966. Newer information on the properties of infectious bronchitis virus. Proc. XIII World's Poultry Congr., Kiev, Russia. Cunningham, C.H. 1970. Avian infectious bronchitis. Adv. Vet. Sci. 14:105-148. Cunningham, C.H., and M.P. Spring. 1965. Some studies of infectious bronchitis virus in cell cultures. Avian Diseases 9:182-193. Fenner, F. 1970. The genetics of animal viruses. Ann. Rev. Microbiol. 24:297-334. Flanagan, J.F., and H.S. Ginsberg. 1964. Role of ribonucleic acid biosynthesis in multiplication of type 5 adenovirus. J. Bact. 813977-987. Gottesman, M.E. 1967. Reaction of ribosome-bound peptidyl tRNA.with aminoacyl tRNA of puromycin. J. Biol. Chem. 242:5564-5571. Handschumacher, R.E. 1960. Orotydilic acid decar- boxylase: inhibition studies with azauridine 5'-phosphate. J. Biol. Chem. 235:2917-2919. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 32 Hershey, J.W.B., and R.E. Thach. 1967. Role of guanosine 5'-triphosphate in the initiation of peptide synthesis. I. Synthesis of formylmethionyl- puromycin. Proc. Natl. Acad. Sci. U.S. 51:759-766. Hille, M.E., M.J. Miller, K. Iwasaki, and A.J. Wahba. 1967. Translation of the genetic message. VI. The role of ribosomal subunits in binding of formylmethionyl tRNA and its reaction with puro- mycin. Proc. Natl. Acad. Sci. U.S. 58:1652-1654. Huang, A.S., D. Baltimore, and M.A. Bratt. 1971. Ribonucleic acid polymerase in virions of Newcastle disease virus: comparison with the vesicular stomatitis virus polymerase. J. Virol. 1:389-394. Kaye, H.S., J.C. Hierholzer, and W.R. Dowdle. 1970. Purification and further characterization of an "IBV-like" virus (Coronavirus). Proc. Soc. Expmtl. Biol. Med. 135:457-463. Kornberg, A.I., I. Lieberman, and E.S. Sims. 1955. Enzymatic synthesis and properties of 5-phosphoribo- sylperphosphate. J. Biol. Chem. 215:389-402. Levintow, L., M.M. Thoren, J.E. Darnell, Jr., and J.L. Hooper. 1962. Effect of p-fluorophenyla- lanine and puromycin on the replication of polio- virus. Virol. 16:220-2229. Lieberman, 1., A. Kornberg, and E.S. Sims. 1955. Enzymatic synthesis of pyrimidine nucleotides orotidine-5'-phosphate and uridine-5'-phosphate. J. Biol. Chem. 215:403-415. Lukert, P.D. 1965. Comparative sensitivities of embryonating chicken's eggs and primary chicken embryo kidney and liver cell cultures to infectious bronchitis virus. Avian Diseases 9:308-316. Lukert, P.D. 1966. Immunofluorescence of avian infectious bronchitis virus in primary chicken embryo kidney, liver, lung, and fibroblast cell cultures. Arch. ges. Virusforsch. 19:265-272. McIntosh, K., J.H. Dees, W.B. Becker, A.Z. Kapikan, and R.M. Chanock. 1967. Recovery in tracheal cultures of novel viruses from patients with respiratory disease. Proc. Natl. Acad. Sci. U.S. 51:933-940. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 33 Melnick, J.L. 1970. Summary of classification of anima1,viruses, 1970. Prog. Med. Virol. 12:337-341. Mohanty, S.B., and S.C. Chang. 1963. Development and ether sensitivity of infectious bronchitis virus of chickens in cell cultures. Amer. J. Vet. Res. 24:822-826. Monro, R.E., and K.A. Marcker. 1967. Ribosome- catalyzed reaction of puromycin with a formyl- methionine-containing oligonucleotide. J. Mol. Nathans, D. 1964. Puromycin inhibition of protein synthesis: incorporation of puromycin into peptide chains. Proc. Natl. Acad. Sci. U.S. 51:585-592. Nathans, D. 1964. Inhibition of protein synthesis by puromycin. Fed. Proc. 23:984-989. Nazerian, K., and C.H. Cunningham. 1968. Morpho- genesis of avian infectious bronchitis virus in chicken embryo fibroblasts. J. Gen. Virol. ‘3:469-470. Pasternak, C.A., and R.E. Handschumacher. 1959. The biochemical activity of 6-azauridine: interference with pyrimidine metabolism in transplantable mouse tumors. J. Biol. Chem. 234:2992-2997. Robinson, W.S. 1971. Ribonucleic acid polymerase activity in Sendai virions and nucleocapsid. J. Virol. 8:81-86. Shalk, A.F., and M.C. Hawn. 1939. An apparently new respiratory disease of baby chicks. J. Amer. Vet. Med. Assoc. 18:413-422. Smith, J.D., R.R. Traut, G.M. Blackburn, and R.E. Monro. 1965. Action of puromycin in polyadenylic acid- directed polylycine synthesis. J. Mol. Biol. 13: 617- 628. Stollar, V., T.M. Stevens, and R.W. Schlesinger. 1966. Studies on the nature of Dengue viruses. II. Characterization of viral RNA and effects of RNA synthesis. Virol. 30:303-312. 42. 43. 44. 45. 46. 34 Stone, H.O., A. Porter, and D.W. Kingsbury. 1971. Ribonucleic acid transcriptases in Sendai virions and in infected cells. J. Virol. 8:174- 180. Traut, R.R., and R.E. Monro. 1964. The puromycin reaction and its relation to protein synthesis. J. Mol. Biol. 10:63-72. Watson, J.D. 1970. Molecular biology of the gene. W.A. Benjamin, Inc., New York. Yarmolinsky, M.B., and G.L. de la Haba. 1959. In- hibition by puromycin of amino acid incorporation into protein. Proc. Natl. Acad. Sci. U.S. 45:1721-1729. Zamir, A., P. Leder, and D. Elson. 1966. A ribo- some catalyzed reaction between N-formylmethionyl- tRNA and puromycin. Proc. Natl. Acad. Sci. U.S. 56:1794-1801.