Search Results (13)

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. Full article

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. Full article

The Chlorovirus genus of the Phycodnaviridae family includes large viruses with a double-stranded DNA genome. Chloroviruses are widely distributed in freshwater bodies around the world and have been isolated from freshwater sources in Europe, Asia, Australia, and North and South America. One representative of chloroviruses is Acanthocystis turfacea chlorella virus 1 (ATCV-1), which is hosted by Chlorella heliozoae. A few publications in the last ten years about the potential effects of ATCV-1 on the human brain sparked interest among specialists in the field of human infectious pathology. The goal of our viewpoint was to compile the scant research on the effects of ATCV-1 on the human body, to demonstrate the role of chloroviruses as new possible infectious agents for human health, and to indicate potential routes of virus transmission. We believe that ATCV-1 transmission routes remain unexplored. We also question whether chlorella-based nutritional supplements are dangerous for ATCV-1 infections. Further research will help to identify the routes of infection, the cell types in which ATCV-1 can persist, and the pathological mechanisms of the virus’s effect on the human body. Full article

Investigation of virus-induced microalgal host lysis and the associated infection dynamics typically requires sampling of infected cultures at multiple timepoints, visually monitoring the state of infected cells, or determining virus titration within the culture media. Such approaches require intensive effort and are prone to low sensitivity and high error rates. Furthermore, natural physiological variations can become magnified by poor environmental control, which is often compounded by variability in virus stock efficacy and relatively long infection cycles. We introduce a new method that closely monitors host health and integrity to learn about the infection strategy of Chloroviruses. Our approach combines aspects of spectrometry, plaque assays, and infection dose assessment to monitor algal cells under conditions more representative of the natural environment. Our automated method exploits the continuous monitoring of infected microalgae cultures in highly controlled lab-scale photobioreactors that provide the opportunity for environmental control, technical replication, and intensive culture monitoring without external intervention or culture disruption. This approach has enabled the development of a protocol to investigate molecular signalling impacting the virus life cycle and particle release, accurate determination of virus lysis time under multiple environmental conditions, and assessment of the functional diversity of multiple virus isolates. Full article

Chloroviruses are large viruses that replicate in chlorella-like green algae and normally exist as mutualistic endosymbionts (referred to as zoochlorellae) in protists such as Paramecium bursaria. Chlorovirus populations rise and fall in indigenous waters through time; however, the factors involved in these virus fluctuations are still under investigation. Chloroviruses attach to the surface of P. bursaria but cannot infect their zoochlorellae hosts because the viruses cannot reach the zoochlorellae as long as they are in the symbiotic phase. Predators of P. bursaria, such as copepods and didinia, can bring chloroviruses into contact with zoochlorellae by disrupting the paramecia, which results in an increase in virus titers in microcosm experiments. Here, we report that another predator of P. bursaria, Bursaria truncatella, can also increase chlorovirus titers. After two days of foraging on P. bursaria, B. truncatella increased infectious chlorovirus abundance about 20 times above the controls. Shorter term foraging (3 h) resulted in a small increase of chlorovirus titers over the controls and more foraging generated more chloroviruses. Considering that B. truncatella does not release viable zoochlorellae either during foraging or through fecal pellets, where zoochlorellae could be infected by chlorovirus, we suggest a third pathway of predator virus catalysis. By engulfing the entire protist and digesting it slowly, virus replication can occur within the predator and some of the virus is passed out through a waste vacuole. These results provide additional support for the hypothesis that predators of P. bursaria are important drivers of chlorovirus population sizes and dynamics. Full article

Chloroviruses are unusual among viruses infecting eukaryotic organisms in that they must, like bacteriophages, penetrate a rigid cell wall to initiate infection. Chlorovirus PBCV-1 infects its host, Chlorella variabilis NC64A by specifically binding to and degrading the cell wall of the host at the point of contact by a virus-packaged enzyme(s). However, PBCV-1 does not use any of the five previously characterized virus-encoded polysaccharide degrading enzymes to digest the Chlorella host cell wall during virus entry because none of the enzymes are packaged in the virion. A search for another PBCV-1-encoded and virion-associated protein identified protein A561L. The fourth domain of A561L is a 242 amino acid C-terminal domain, named A561L^D4, with cell wall degrading activity. An A561L^D4 homolog was present in all 52 genomically sequenced chloroviruses, infecting four different algal hosts. A561L^D4 degraded the cell walls of all four chlorovirus hosts, as well as several non-host Chlorella spp. Thus, A561L^D4 was not cell-type specific. Finally, we discovered that exposure of highly purified PBCV-1 virions to A561L^D4 increased the specific infectivity of PBCV-1 from about 25–30% of the particles forming plaques to almost 50%. We attribute this increase to removal of residual host receptor that attached to newly replicated viruses in the cell lysates. Full article

The structures of the four N-linked glycans from the prototype chlorovirus PBCV-1 major capsid protein do not resemble any other glycans in the three domains of life. All known chloroviruses and antigenic variants (or mutants) share a unique conserved central glycan core consisting of five sugars, except for antigenic mutant virus P1L6, which has four of the five sugars. A combination of genetic and structural analyses indicates that the protein coded by PBCV-1 gene a111/114r, conserved in all chloroviruses, is a glycosyltransferase with three putative domains of approximately 300 amino acids each. Here, in addition to in silico sequence analysis and protein modeling, we measured the hydrolytic activity of protein A111/114R. The results suggest that domain 1 is a galactosyltransferase, domain 2 is a xylosyltransferase and domain 3 is a fucosyltransferase. Thus, A111/114R is the protein likely responsible for the attachment of three of the five conserved residues of the core region of this complex glycan, and, if biochemically corroborated, it would be the second three-domain protein coded by PBCV-1 that is involved in glycan synthesis. Importantly, these findings provide additional support that the chloroviruses do not use the canonical host endoplasmic reticulum–Golgi glycosylation pathway to glycosylate their glycoproteins; instead, they perform glycosylation independent of cellular organelles using virus-encoded enzymes. Full article

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 tRNA^Arg 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 tRNA^Met 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. Full article

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 Kcv_ATCV-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 Kcv_ATCV-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. Full article

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. Full article

Chloroviruses (family Phycodnaviridae) are dsDNA viruses found throughout the world’s inland waters. The open reading frames in the genomes of 41 sequenced chloroviruses (330 ± 40 kbp each) representing three virus types were analyzed for evidence of evolutionarily conserved local genomic “contexts”, the organization of biological information into units of a scale larger than a gene. Despite a general loss of synteny between virus types, we informatically detected a highly conserved genomic context defined by groups of three or more genes that we have termed “gene gangs”. Unlike previously described local genomic contexts, the definition of gene gangs requires only that member genes be consistently co-localized and are not constrained by strand, regulatory sites, or intervening sequences (and therefore represent a new type of conserved structural genomic element). An analysis of functional annotations and transcriptomic data suggests that some of the gene gangs may organize genes involved in specific biochemical processes, but that this organization does not involve their coordinated expression. Full article

Chloroviruses are large double-stranded DNA (dsDNA) viruses that infect certain isolates of chlorella-like green algae. They contain up to approximately 400 protein-encoding genes and 16 transfer RNA (tRNA) genes. This review summarizes the unexpected finding that many of the chlorovirus genes encode proteins involved in manipulating carbohydrates. These include enzymes involved in making extracellular polysaccharides, such as hyaluronan and chitin, enzymes that make nucleotide sugars, such as GDP-L-fucose and GDP-D-rhamnose and enzymes involved in the synthesis of glycans attached to the virus major capsid proteins. This latter process differs from that of all other glycoprotein containing viruses that traditionally use the host endoplasmic reticulum and Golgi machinery to synthesize and transfer the glycans. Full article

Glycosylation of surface molecules is a key feature of several eukaryotic viruses, which use the host endoplasmic reticulum/Golgi apparatus to add carbohydrates to their nascent glycoproteins. In recent years, a newly discovered group of eukaryotic viruses, belonging to the Nucleo-Cytoplasmic Large DNA Virus (NCLDV) group, was shown to have several features that are typical of cellular organisms, including the presence of components of the glycosylation machinery. Starting from initial observations with the chlorovirus PBCV-1, enzymes for glycan biosynthesis have been later identified in other viruses; in particular in members of the Mimiviridae family. They include both the glycosyltransferases and other carbohydrate-modifying enzymes and the pathways for the biosynthesis of the rare monosaccharides that are found in the viral glycan structures. These findings, together with genome analysis of the newly-identified giant DNA viruses, indicate that the presence of glycogenes is widespread in several NCLDV families. The identification of autonomous viral glycosylation machinery leads to many questions about the origin of these pathways, the mechanisms of glycan production, and eventually their function in the viral replication cycle. The scope of this review is to highlight some of the recent results that have been obtained on the glycosylation systems of the large DNA viruses, with a special focus on the enzymes involved in nucleotide-sugar production. Full article