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Many chloroviruses replicate in Chlorella variabilis algal strains that are ex-endosymbionts isolated from the protozoan Paramecium bursaria, including the NC64A and Syngen 2-3 strains. We noticed that indigenous water samples produced a higher number of plaque-forming viruses on C. variabilis Syngen 2-3 lawns than on C. variabilis NC64A lawns. These observed differences led to the discovery of viruses that replicate exclusively in Syngen 2-3 cells, named Only Syngen (OSy) viruses. Here, we demonstrate that OSy viruses initiate infection in the restricted host NC64A by synthesizing some early virus gene products and that approximately 20% of the cells produce a small number of empty virus capsids. However, the infected cells did not produce infectious viruses because the cells were unable to replicate the viral genome. This is interesting because all previous attempts to isolate host cells resistant to chlorovirus infection were due to changes in the host receptor for the virus. 

Viruses face many challenges on their road to successful replication, and they meet those challenges by reprogramming the intracellular environment. Two major issues challenging Paramecium bursaria chlorella virus 1 (PBCV-1, genus Chlorovirus, family Phycodnaviridae) at the level of DNA replication are (i) the host cell has a DNA G+C content of 66%, while the virus is 40%; and (ii) the initial quantity of DNA in the haploid host cell is approximately 50 fg, yet the virus will make approximately 350 fg of DNA within hours of infection to produce approximately 1000 virions per cell. Thus, the quality and quantity of DNA (and RNA) would seem to restrict replication efficiency, with the looming problem of viral DNA synthesis beginning in only 60–90 min. Our analysis includes (i) genomics and functional annotation to determine gene augmentation and complementation of the nucleotide biosynthesis pathway by the virus, (ii) transcriptional profiling of these genes, and (iii) metabolomics of nucleotide intermediates. The studies indicate that PBCV-1 reprograms the pyrimidine biosynthesis pathway to rebalance the intracellular nucleotide pools both qualitatively and quantitatively, prior to viral DNA amplification, and reflects the genomes of the progeny virus, providing a successful road to virus infection. 

Giant viruses are a large group of viruses that infect many eukaryotes. Although components that do not obey the overall icosahedral symmetry of their capsids have been observed and found to play critical roles in the viral life cycles, identities and high-resolution structures of these components remain unknown. Here, by determining a near-atomic-resolution, five-fold averaged structure of Paramecium bursaria chlorella virus 1, we unexpectedly found the viral capsid possesses up to five major capsid protein variants and a penton protein variant. These variants create varied capsid microenvironments for the associations of fibers, a vesicle, and previously unresolved minor capsid proteins. Our structure reveals the identities and atomic models of the capsid components that do not obey the overall icosahedral symmetry and leads to a model for how these components are assembled and initiate capsid assembly, and this model might be applicable to many other giant viruses.

 

Chloroviruses are large dsDNA, plaque-forming viruses that infect certain chlorella-like green algae; the algae are normally mutualistic endosymbionts of protists and metazoans and are often referred to as zoochlorellae. The viruses are ubiquitous in inland aqueous environments throughout the world and occasionally single types reach titers of thousands of plaque-forming units per ml of native water. The viruses are icosahedral in shape with a spike structure located at one of the vertices. They contain an internal membrane that is required for infectivity. The viral genomes are 290 to 370 kb in size, which encode up to 16 tRNAs and 330 to ~415 proteins, including many not previously seen in viruses. Examples include genes encoding DNA restriction and modification enzymes, hyaluronan and chitin biosynthetic enzymes, polyamine biosynthetic enzymes, ion channel and transport proteins, and enzymes involved in the glycan synthesis of the virus major capsid glycoproteins. The proteins encoded by many of these viruses are often the smallest or among the smallest proteins of their class. Consequently, some of the viral proteins are the subject of intensive biochemical and structural investigation. 

Paramecium bursaria chlorella virus-1 (PBCV-1) is a large double-stranded DNA (dsDNA) virus that infects the unicellular green alga Chlorella variabilis NC64A. Unlike many other viruses, PBCV-1 encodes most, if not all, of the enzymes involved in the synthesis of the glycans attached to its major capsid protein. Importantly, these glycans differ from those reported from the three domains of life in terms of structure and asparagine location in the sequon of the protein. Previous data collected from 20 PBCV-1 spontaneous mutants (or antigenic variants) suggested that the a064r gene encodes a glycosyltransferase (GT) with three domains, each with a different function. Here, we demonstrate that: domain 1 is a β- l -rhamnosyltransferase; domain 2 is an α- l -rhamnosyltransferase resembling only bacterial proteins of unknown function, and domain 3 is a methyltransferase that methylates the C-2 hydroxyl group of the terminal α- l -rhamnose (Rha) unit. We also establish that methylation of the C-3 hydroxyl group of the terminal α- l -Rha is achieved by another virus-encoded protein A061L, which requires an O-2 methylated substrate. This study, thus, identifies two of the glycosyltransferase activities involved in the synthesis of the N -glycan of the viral major capsid protein in PBCV-1 and establishes that a single protein A064R possesses the three activities needed to synthetize the 2-OMe-α- l -Rha-(1→2)-β- l -Rha fragment. Remarkably, this fragment can be attached to any xylose unit. 

Chloroviruses are large, plaque-forming, dsDNA viruses that infect chlorella-like green algae that live in a symbiotic relationship with protists. Chloroviruses have genomes from 290 to 370 kb, and they encode as many as 400 proteins. One interesting feature of chloroviruses is that they encode a potassium ion (K+) channel protein named Kcv. The Kcv protein encoded by SAG chlorovirus ATCV-1 is one of the smallest known functional K+ channel proteins consisting of 82 amino acids. The KcvATCV-1 protein has similarities to the family of two transmembrane domain K+ channel proteins; it consists of two transmembrane α-helixes with a pore region in the middle, making it an ideal model for studying K+ channels. To assess their genetic diversity, kcv genes were sequenced from 103 geographically distinct SAG chlorovirus isolates. Of the 103 kcv genes, there were 42 unique DNA sequences that translated into 26 new Kcv channels. The new predicted Kcv proteins differed from KcvATCV-1 by 1 to 55 amino acids. The most conserved region of the Kcv protein was the filter, the turret and the pore helix were fairly well conserved, and the outer and the inner transmembrane domains of the protein were the most variable. Two of the new predicted channels were shown to be functional K+ channels. 

ABSTRACT Chloroviruses exist in aquatic systems around the planet and they infect certain eukaryotic green algae that are mutualistic endosymbionts in a variety of protists and metazoans. Natural chlorovirus populations are seasonally dynamic, but the precise temporal changes in these populations and the mechanisms that underlie them have heretofore been unclear. We recently reported the novel concept that predator/prey-mediated virus activation regulates chlorovirus population dynamics, and in the current study, we demonstrate virus-packaged chemotactic modulation of prey behavior. IMPORTANCE Viruses have not previously been reported to act as chemotactic/chemoattractive agents. Rather, viruses as extracellular entities are generally viewed as non-metabolically active spore-like agents that await further infection events upon collision with appropriate host cells. That a virus might actively contribute to its fate via chemotaxis and change the behavior of an organism independent of infection is unprecedented.