Dropping acid makes you see stars : Samba virus as a model system for studying giant virus genome release

"As their name implies, giant viruses (GV) are viruses of immense size. These viruses tend to have capsids larger than 300 nm and genomes that encode for over 1000 open reading frames. These viruses dwarf more common viruses, such as the human rhinovirus (common cold) that has a particle size of 30 nm and encodes for only 11 proteins. Some GV genomes even contain introns, a feature not typically associated with viruses as they were thought to have evolved towards simplicity. The discovery of these viruses challenged the canonical view of the virus as a small and simple biological entity and has cast some doubt on our current understanding of the definitions of life. GV have been isolated from every continent on the planet, yet most share several conserved structural features. These conserved features include an internal lipid membrane that contains the dsDNA genome as well as a seal complex that closes the capsid prior to genome release. In icosahedral GV (Mimivirus-like GV), this seal complex sits atop the capsid at one specific vertex, the stargate vertex, which opens to facilitate genome release. The mechanisms that trigger release of the seal complex in vivo remain unknown. To fill some of the gaps in our knowledge of the GV life cycle, I have developed an in vitro system for studying GV genome release using Samba virus (SMBV), an icosahedral GV isolated from a tributary of the Amazon River in Brazil. First, I developed a method to visualize SMBV using cryo-electron microscopy (cryo-EM), cryo-electron tomography (cryo-ET), and scanning electron microscopy (SEM). I then investigated the molecular forces responsible for maintaining the structural integrity of the SMBV external seal complex, treating SMBV particles with conditions known to disrupt viral capsids. Following each treatment, we determined the percentage of open SMBV particles, looking for conditions that induced a marked increase in open SMBV capsids. Both low pH (at or below pH 3) and high temperature (100 °C) triggered an increase in open SMBV particles, suggesting that electrostatic interactions and entropy, respectively, play a role in maintaining the structural integrity of the SMBV external seal complex. The role of these forces in maintaining external seal complex integrity is conserved throughout the icosahedral GV as three other GV shared similar structural responses to these conditions.Following low pH treatment small cracks appear in the GV capsid, mimicking the initiation of the genome release process and facilitating release of infection-related proteins. I separated the released proteins from the remaining capsid via centrifugation and analyzed the two populations via differential mass spectrometry. Through these analyses we identified 3030300 proteins that are released from SMBV and/or Tupanvirus soda lake, a GV isolated from an alkaline lake in Brazil, capsids during the initial stages of the infection process. These findings provide some of the first molecular information on the GV genome release process and hint at what triggers this process in vivo. This work also provides the first in vitro system capable of mimicking stages of the GV infection process, paving the way for future structural and biochemical studies of the GV life cycle."--Pages ii-iii.