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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. 

ABSTRACT Chloroviruses (family Phycodnaviridae ) are large double-stranded DNA (dsDNA) viruses that infect unicellular green algae present in inland waters. These viruses have been isolated using three main chlorella-like green algal host cells, traditionally called NC64A, SAG, and Pbi, revealing extensive genetic diversity. In this study, we performed a functional genomic analysis on 36 chloroviruses that infected the three different hosts. Phylogenetic reconstruction based on the DNA polymerase B family gene clustered the chloroviruses into three distinct clades. The viral pan-genome consists of 1,345 clusters of orthologous groups of genes (COGs), with 126 COGs conserved in all viruses. Totals of 368, 268, and 265 COGs are found exclusively in viruses that infect NC64A, SAG, and Pbi algal hosts, respectively. Two-thirds of the COGs have no known function, constituting the “dark pan-genome” of chloroviruses, and further studies focusing on these genes may identify important novelties. The proportions of functionally characterized COGs composing the pan-genome and the core-genome are similar, but those related to transcription and RNA processing, protein metabolism, and virion morphogenesis are at least 4-fold more represented in the core genome. Bipartite network construction evidencing the COG sharing among host-specific viruses identified 270 COGs shared by at least one virus from each of the different host groups. Finally, our results reveal an open pan-genome for chloroviruses and a well-established core genome, indicating that the isolation of new chloroviruses can be a valuable source of genetic discovery. IMPORTANCE Chloroviruses are large dsDNA viruses that infect unicellular green algae distributed worldwide in freshwater environments. They comprise a genetically diverse group of viruses; however, a comprehensive investigation of the genomic evolution of these viruses is still missing. Here, we performed a functional pan-genome analysis comprising 36 chloroviruses associated with three different algal hosts in the family Chlorellaceae , referred to as zoochlorellae because of their endosymbiotic lifestyle. We identified a set of 126 highly conserved genes, most of which are related to essential functions in the viral replicative cycle. Several genes are unique to distinct isolates, resulting in an open pan-genome for chloroviruses. This profile is associated with generalist organisms, and new insights into the evolution and ecology of chloroviruses are presented. Ultimately, our results highlight the potential for genetic diversity in new isolates. 

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.

 

Viruses rely on their host’s translation machinery for the synthesis of their own proteins. Problems belie viral translation when the host has a codon usage bias (CUB) that is different from an infecting virus due to differences in the GC content between the host and virus genomes. Here, we examine the hypothesis that chloroviruses adapted to host CUB by acquisition and selection of tRNAs that at least partially favor their own CUB. The genomes of 41 chloroviruses comprising three clades, each infecting a different algal host, have been sequenced, assembled and annotated. All 41 viruses not only encode tRNAs, but their tRNA genes are located in clusters. While differences were observed between clades and even within clades, seven tRNA genes were common to all three clades of chloroviruses, including the tRNAArg gene, which was found in all 41 chloroviruses. By comparing the codon usage of one chlorovirus algal host, in which the genome has been sequenced and annotated (67% GC content), to that of two of its viruses (40% GC content), we found that the viruses were able to at least partially overcome the host’s CUB by encoding tRNAs that recognize AU-rich codons. Evidence presented herein supports the hypothesis that a chlorovirus tRNA cluster was present in the most recent common ancestor (MRCA) prior to divergence into three clades. In addition, the MRCA encoded a putative isoleucine lysidine synthase (TilS) that remains in 39/41 chloroviruses examined herein, suggesting a strong evolutionary pressure to retain the gene. TilS alters the anticodon of tRNAMet that normally recognizes AUG to then recognize AUA, a codon for isoleucine. This is advantageous to the chloroviruses because the AUA codon is 12–13 times more common in the chloroviruses than their host, further helping the chloroviruses to overcome CUB. Among large DNA viruses infecting eukaryotes, the presence of tRNA genes and tRNA clusters appear to be most common in the Phycodnaviridae and, to a lesser extent, in the Mimiviridae. 

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. 

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.