Virus-host interactions: insights from the replication cycle of the large Paramecium bursaria chlorella virus.

The increasing interest in cytoplasmic factories generated by eukaryotic-infecting viruses stems from the realization that these highly ordered assemblies may contribute fundamental novel insights to the functional significance of order in cellular biology. Here, we report the formation process and structural features of the cytoplasmic factories of the large dsDNA virus Paramecium bursaria chlorella virus 1 (PBCV-1). By combining diverse imaging techniques, including scanning transmission electron microscopy tomography and focused ion beam technologies, we show that the architecture and mode of formation of PBCV-1 factories are significantly different from those generated by their evolutionary relatives Vaccinia and Mimivirus. Specifically, PBCV-1 factories consist of a network of single membrane bilayers acting as capsid templates in the central region, and viral genomes spread throughout the host cytoplasm but excluded from the membrane-containing sites. In sharp contrast, factories generated by Mimivirus have viral genomes in their core, with membrane biogenesis region located at their periphery. Yet, all viral factories appear to share structural features that are essential for their function. In addition, our studies support the notion that PBCV-1 infection, which was recently reported to result in significant pathological outcomes in humans and mice, proceeds through a bacteriophage-like infection pathway.

Chlorovirus ATCV-1 is part of the human oropharyngeal virome and is associated with changes in cognitive functions in humans and mice.

Chloroviruses (family Phycodnaviridae) are large DNA viruses known to infect certain eukaryotic green algae and have not been previously shown to infect humans or to be part of the human virome. We unexpectedly found sequences homologous to the chlorovirus Acanthocystis turfacea chlorella virus 1 (ATCV-1) in a metagenomic analysis of DNA extracted from human oropharyngeal samples. These samples were obtained by throat swabs of adults without a psychiatric disorder or serious physical illness who were participating in a study that included measures of cognitive functioning. The presence of ATCV-1 DNA was confirmed by quantitative PCR with ATCV-1 DNA being documented in oropharyngeal samples obtained from 40 (43.5%) of 92 individuals. The presence of ATCV-1 DNA was not associated with demographic variables but was associated with a modest but statistically significant decrease in the performance on cognitive assessments of visual processing and visual motor speed. We further explored the effects of ATCV-1 in a mouse model. The inoculation of ATCV-1 into the intestinal tract of 9-11-wk-old mice resulted in a subsequent decrease in performance in several cognitive domains, including ones involving recognition memory and sensory-motor gating. ATCV-1 exposure in mice also resulted in the altered expression of genes within the hippocampus. These genes comprised pathways related to synaptic plasticity, learning, memory formation, and the immune response to viral exposure.

Global analysis of Chlorella variabilis NC64A mRNA profiles during the early phase of Paramecium bursaria chlorella virus-1 infection.

The PBCV-1/Chlorella variabilis NC64A system is a model for studies on interactions between viruses and algae. Here we present the first global analyses of algal host transcripts during the early stages of infection, prior to virus replication. During the course of the experiment stretching over 1 hour, about a third of the host genes displayed significant changes in normalized mRNA abundance that either increased or decreased compared to uninfected levels. The population of genes with significant transcriptional changes gradually increased until stabilizing at 40 minutes post infection. Functional categories including cytoplasmic ribosomal proteins, jasmonic acid biosynthesis and anaphase promoting complex/cyclosomes had a significant excess in upregulated genes, whereas spliceosomal snRNP complexes and the shikimate pathway had significantly more down-regulated genes, suggesting that these pathways were activated or shut-down in response to the virus infection. Lastly, we examined the expression of C. varibilis RNA polymerase subunits, as PBCV-1 transcription depends on host RNA polymerases. Two subunits were up-regulated, RPB10 and RPC34, suggesting that they may function to support virus transcription. These results highlight genes and pathways, as well as overall trends, for further refinement of our understanding of the changes that take place during the early stages of viral infection.

Deep RNA sequencing reveals hidden features and dynamics of early gene transcription in Paramecium bursaria chlorella virus 1.

Paramecium bursaria chlorella virus 1 (PBCV-1) is the prototype of the genus Chlorovirus (family Phycodnaviridae) that infects the unicellular, eukaryotic green alga Chlorella variabilis NC64A. The 331-kb PBCV-1 genome contains 416 major open reading frames. A mRNA-seq approach was used to analyze PBCV-1 transcriptomes at 6 progressive times during the first hour of infection. The alignment of 17 million reads to the PBCV-1 genome allowed the construction of single-base transcriptome maps. Significant transcription was detected for a subset of 50 viral genes as soon as 7 min after infection. By 20 min post infection (p.i.), transcripts were detected for most PBCV-1 genes and transcript levels continued to increase globally up to 60 min p.i., at which time 41% or the poly (A+)-containing RNAs in the infected cells mapped to the PBCV-1 genome. For some viral genes, the number of transcripts in the latter time points (20 to 60 min p.i.) was much higher than that of the most highly expressed host genes. RNA-seq data revealed putative polyadenylation signal sequences in PBCV-1 genes that were identical to the polyadenylation signal AAUAAA of green algae. Several transcripts have an RNA fragment excised. However, the frequency of excision and the resulting putative shortened protein products suggest that most of these excision events have no functional role but are probably the result of the activity of misled splicesomes.

Structure of N-linked oligosaccharides attached to chlorovirus PBCV-1 major capsid protein reveals unusual class of complex N-glycans.

The major capsid protein Vp54 from the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) contains four Asn-linked glycans. The structure of the four N-linked oligosaccharides and the type of substitution at each glycosylation site was determined by chemical, spectroscopic, and spectrometric analyses. Vp54 glycosylation is unusual in many ways, including: (i) unlike most viruses, PBCV-1 encodes most, if not all, of the machinery to glycosylate its major capsid protein; (ii) the glycans are attached to the protein by a ß-glucose linkage; (iii) the Asn-linked glycans are not located in a typical N-X-(T/S) consensus site; and (iv) the process probably occurs in the cytoplasm. The four glycoforms share a common core structure, and the differences are related to the nonstoichiometric presence of two monosaccharides. The most abundant glycoform consists of nine neutral monosaccharide residues, organized in a highly branched fashion. Among the most distinctive features of the glycoforms are (i) a dimethylated rhamnose as the capping residue of the main chain, (ii) a hyperbranched fucose unit, and (iii) two rhamnose residues with opposite absolute configurations. These glycoforms differ from what has been reported so far in the three domains of life. Considering that chloroviruses and other members of the family Phycodnaviridae may have a long evolutionary history, we suggest that the chlorovirus glycosylation pathway is ancient, possibly existing before the development of the endoplasmic reticulum and Golgi pathway, and involves still unexplored mechanisms.

Evaluation of higher plant virus resistance genes in the green alga, Chlorella variabilis NC64A, during the early phase of infection with Paramecium bursaria chlorella virus-1.

With growing industrial interest in algae plus their critical roles in aquatic systems, the need to understand the effects of algal pathogens is increasing. We examined a model algal host-virus system, Chlorella variabilis NC64A and virus, PBCV-1. C. variabilis encodes 375 homologs to genes involved in RNA silencing and in response to virus infection in higher plants. Illumina RNA-Seq data showed that 325 of these homologs were expressed in healthy and early PBCV-1 infected (≤60min) cells. For each of the RNA silencing genes to which homologs were found, mRNA transcripts were detected in healthy and infected cells. C. variabilis, like higher plants, may employ certain RNA silencing pathways to defend itself against virus infection. To our knowledge this is the first examination of RNA silencing genes in algae beyond core proteins, and the first analysis of their transcription during virus infection.

Towards defining the chloroviruses: a genomic journey through a genus of large DNA viruses.

BACKGROUND: Giant viruses in the genus Chlorovirus (family Phycodnaviridae) infect eukaryotic green microalgae. The prototype member of the genus, Paramecium bursaria chlorella virus 1, was sequenced more than 15 years ago, and to date there are only 6 fully sequenced chloroviruses in public databases. Presented here are the draft genome sequences of 35 additional chloroviruses (287 - 348 Kb/319 - 381 predicted protein encoding genes) collected across the globe; they infect one of three different green algal species. These new data allowed us to analyze the genomic landscape of 41 chloroviruses, which revealed some remarkable features about these viruses. RESULTS: Genome colinearity, nucleotide conservation and phylogenetic affinity were limited to chloroviruses infecting the same host, confirming the validity of the three previously known subgenera. Clues for the existence of a fourth new subgenus indicate that the boundaries of chlorovirus diversity are not completely determined. Comparison of the chlorovirus phylogeny with that of the algal hosts indicates that chloroviruses have changed hosts in their evolutionary history. Reconstruction of the ancestral genome suggests that the last common chlorovirus ancestor had a slightly more diverse protein repertoire than modern chloroviruses. However, more than half of the defined chlorovirus gene families have a potential recent origin (after Chlorovirus divergence), among which a portion shows compositional evidence for horizontal gene transfer. Only a few of the putative acquired proteins had close homologs in databases raising the question of the true donor organism(s). Phylogenomic analysis identified only seven proteins whose genes were potentially exchanged between the algal host and the chloroviruses. CONCLUSION: The present evaluation of the genomic evolution pattern suggests that chloroviruses differ from that described in the related Poxviridae and Mimiviridae. Our study shows that the fixation of algal host genes has been anecdotal in the evolutionary history of chloroviruses. We finally discuss the incongruence between compositional evidence of horizontal gene transfer and lack of close relative sequences in the databases, which suggests that the recently acquired genes originate from a still largely un-sequenced reservoir of genomes, possibly other unknown viruses that infect the same hosts.

Paramecium bursaria chlorella virus 1 proteome reveals novel architectural and regulatory features of a giant virus.

The 331-kbp chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) genome was resequenced and annotated to correct errors in the original 15-year-old sequence; 40 codons was considered the minimum protein size of an open reading frame. PBCV-1 has 416 predicted protein-encoding sequences and 11 tRNAs. A proteome analysis was also conducted on highly purified PBCV-1 virions using two mass spectrometry-based protocols. The mass spectrometry-derived data were compared to PBCV-1 and its host Chlorella variabilis NC64A predicted proteomes. Combined, these analyses revealed 148 unique virus-encoded proteins associated with the virion (about 35% of the coding capacity of the virus) and 1 host protein. Some of these proteins appear to be structural/architectural, whereas others have enzymatic, chromatin modification, and signal transduction functions. Most (106) of the proteins have no known function or homologs in the existing gene databases except as orthologs with proteins of other chloroviruses, phycodnaviruses, and nuclear-cytoplasmic large DNA viruses. The genes encoding these proteins are dispersed throughout the virus genome, and most are transcribed late or early-late in the infection cycle, which is consistent with virion morphogenesis.

Paramecium bursaria chlorella virus 1 encodes a polyamine acetyltransferase.

Paramecium bursaria chlorella virus 1 (PBCV-1), a large DNA virus that infects green algae, encodes a histone H3 lysine 27-specific methyltransferase that functions in global transcriptional silencing of the host. PBCV-1 has another gene a654l that encodes a protein with sequence similarity to the GCN5 family histone acetyltransferases. In this study, we report a 1.5 Å crystal structure of PBCV-1 A654L in a complex with coenzyme A. The structure reveals a unique feature of A654L that precludes its acetylation of histone peptide substrates. We demonstrate that A654L, hence named viral polyamine acetyltransferase (vPAT), acetylates polyamines such as putrescine, spermidine, cadaverine, and homospermidine present in both PBCV-1 and its host through a reaction dependent upon a conserved glutamate 27. Our study suggests that as the first virally encoded polyamine acetyltransferase, vPAT plays a possible key role in the regulation of polyamine catabolism in the host during viral replication.

Functional HAK/KUP/KT-like potassium transporter encoded by chlorella viruses.

Chlorella viruses are a source of interesting membrane transport proteins. Here we examine a putative K(+) transporter encoded by virus FR483 and related chlorella viruses. The protein shares sequence and structural features with HAK/KUP/KT-like K(+) transporters from plants, bacteria and fungi. Yeast complementation assays and Rb(+) uptake experiments show that the viral protein, termed HAKCV (high-affinity K(+) transporter of chlorella virus), is functional, with transport characteristics that are similar to those of known K(+) transporters. Expression studies revealed that the protein is expressed as an early gene during viral replication, and proteomics data indicate that it is not packaged in the virion. The function of HAKCV is unclear, but the data refute the hypothesis that the transporter acts as a substitute for viral-encoded K(+) channels during virus infection.

Identification of an L-rhamnose synthetic pathway in two nucleocytoplasmic large DNA viruses.

Nucleocytoplasmic large DNA viruses (NCLDVs) are characterized by large genomes that often encode proteins not commonly found in viruses. Two species in this group are Acanthocystis turfacea chlorella virus 1 (ATCV-1) (family Phycodnaviridae, genus Chlorovirus) and Acanthamoeba polyphaga mimivirus (family Mimiviridae), commonly known as mimivirus. ATCV-1 and other chlorovirus members encode enzymes involved in the synthesis and glycosylation of their structural proteins. In this study, we identified and characterized three enzymes responsible for the synthesis of the sugar L-rhamnose: two UDP-D-glucose 4,6-dehydratases (UGDs) encoded by ATCV-1 and mimivirus and a bifunctional UDP-4-keto-6-deoxy-D-glucose epimerase/reductase (UGER) from mimivirus. Phylogenetic analysis indicated that ATCV-1 probably acquired its UGD gene via a recent horizontal gene transfer (HGT) from a green algal host, while an earlier HGT event involving the complete pathway (UGD and UGER) probably occurred between a protozoan ancestor and mimivirus. While ATCV-1 lacks an epimerase/reductase gene, its Chlorella host may encode this enzyme. Both UGDs and UGER are expressed as late genes, which is consistent with their role in posttranslational modification of capsid proteins. The data in this study provide additional support for the hypothesis that chloroviruses, and maybe mimivirus, encode most, if not all, of the glycosylation machinery involved in the synthesis of specific glycan structures essential for virus replication and infection.

Isolation of the phycodnavirus PBCV-1 by biological laser printing.

The Phycodnaviridae family of viruses is diverse genetically but similar morphologically. These viruses infect eukaryotic algal hosts from both fresh and marine waters, and are an important component of aqueous environments. They play important roles in the dynamics of algal blooms, nutrient cycling, algal community structure, and possibly gene transfer between organisms. As such, it is important to identify new viruses within the Phycodnaviridae family. Biological laser printing (BioLP) was used to isolate single virus particles from solution. BioLP prints droplets containing a single virus particle directly onto a host medium, thereby enabling viruses to be isolated from unmodified samples. This manuscript demonstrates how BioLP can be used as a single-step method to separate and possibly identify viruses from complex environmental specimens.

Chlorella viruses encode most, if not all, of the machinery to glycosylate their glycoproteins independent of the endoplasmic reticulum and Golgi.

In contrast to all other viruses that use the host machinery located in the endoplasmic reticulum and Golgi to glycosylate their glycoproteins, the large dsDNA-containing chlorella viruses encode most, if not all, of the components to glycosylate their major capsid proteins. Furthermore, all experimental results indicate that glycosylation occurs independent of the endoplasmic reticulum and Golgi.

Epigenetic transcriptional repression of cellular genes by a viral SET protein.

Viruses recruit host proteins to secure viral genome maintenance and replication. However, whether they modify host histones directly to interfere with chromatin-based transcription is unknown. Here we report that Paramecium bursaria chlorella virus 1 (PBCV-1) encodes a functional SET domain histone Lys methyltransferase (HKMTase) termed vSET, which is linked to rapid inhibition of host transcription after viral infection. We show that vSET is packaged in the PBCV-1 virion, and that it contains a nuclear localization signal and probably represses host transcription by methylating histone H3 at Lys 27 (H3K27), a modification known to trigger gene silencing in eukaryotes. We also show that vSET induces cell accumulation at the G2/M phase by recruiting the Polycomb repressive complex CBX8 to the methylated H3K27 site in a heterologous system, vSET-like proteins that have H3K27 methylation activity are conserved in chlorella viruses. Our findings suggest a viral mechanism to repress gene transcription by direct modification of chromatin by PBCV-1 vSET.

Chlorella viruses contain genes encoding a complete polyamine biosynthetic pathway.

Two genes encoding the putative polyamine biosynthetic enzymes agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (CPA) were cloned from the chloroviruses PBCV-1, NY-2A and MT325. They were expressed in Escherichia coli to form C-terminal (His)6-tagged proteins and the recombinant proteins were purified by Ni2+-binding affinity chromatography. The biochemical properties of the two enzymes are similar to AIH and CPA enzymes from Arabidopsis thaliana and Pseudomonas aeruginosa. Together with the previously known virus genes encoding ornithine/arginine decarboxlyase (ODC/ADC) and homospermidine synthase, the chloroviruses have genes that encode a complete set of functional enzymes that synthesize the rare polyamine homospermidine from arginine via agmatine, N-carbamoylputrescine and putrescine. The PBCV-1 aih and cpa genes are expressed early during virus infection together with the odc/adc gene, suggesting that biosynthesis of putrescine is important in early stages of viral replication. The aih and cpa genes are widespread in the chlorella viruses.

Atomic force microscopy investigation of a chlorella virus, PBCV-1.

A virus PBCV-1, which infects certain fresh water algae and has been shown by transmission and cryo-electron microscopy to exist as a triskaidecahedron, was imaged using atomic force microscopy (AFM). From AFM the particles have diameters of about 190nm and the overall structure is in all important respects consistent with existing models. The surface lattice of the virion is composed of trimeric capsid proteins distributed according to p3 symmetry to create a honeycomb arrangement of raised edges forming quasi-hexagonal cells. At the pentagonal vertices are five copies of a different protein forming an exact pentagon, and this has yet another unique protein in its center. The apical protein exhibits some unusual mechanical properties in that it can be made to retract into the virion interior when subjected to AFM tip pressure. When PBCV-1 virions degrade, they give rise to small, uniform, spherical, and virus like particles (VLP) consistent with T=1 or 3 icosahedral products. Also observed upon disintegration are strands of linear dsDNA. Fibers of unknown function are also occasionally seen associated with some virions.

Functional analysis of FAD-dependent thymidylate synthase ThyX from Paramecium bursaria Chlorella virus-1.

Sequence analysis of the 330-kb double-stranded DNA genome of Paramecium bursaria chlorella virus-1 revealed an open reading frame A674R that encodes a protein with up to 53% amino acid identity to a recently discovered new class of thymidylate synthases, called ThyX. Unlike the traditional thymidylate synthase, ThyA, that uses methylenetetrahydrofolate (CH(2)H(4)folate) as both a source of the methylene group and the reductant, CH(2)H(4)folate only supplies the methylene group in ThyX-catalyzed reactions. Furthermore, ThyX only catalyzes thymidylate (dTMP) formation in the presence of reduced pyridine nucleotides and oxidized FAD. The distribution and transcription patterns of the a674r gene in Chlorella viruses were examined. The a674r gene was cloned, and the protein was expressed in Escherichia coli. Biochemical characterization of the P. bursaria chlorella virus-1 recombinant ThyX protein indicates that it is more efficient at converting dUMP to dTMP than previously studied ThyX enzymes, thus allowing more detailed mechanistic studies of the enzyme. The ThyX-dUMP complexes with bound FAD function as efficient NAD(P)H oxidases, indicating that dUMP binds to the enzyme prior to NAD(P)H. This oxidation activity is directly linked to FAD reduction. Our results indicate that ThyX-specific inhibitors can be designed that do not affect ThyA enzymes. Finally, a model is proposed for the early stages of ThyX catalysis.

Genetic diversity in chlorella viruses flanking kcv, a gene that encodes a potassium ion channel protein.

The chlorella virus PBCV-1 encodes a 94-amino acid protein named Kcv that produces a K+-selective and slightly voltage-sensitive conductance when expressed in heterologous systems. As reported herein, (i) Northern analysis of kcv expression in PBCV-1-infected cells revealed a complicated pattern suggesting that the gene might be transcribed as a di- or tri-cistronic mRNA both at early and late times after virus infection. (ii) The protein kinase inhibitors H-89, A3, and staurosporine inhibited PBCV-1 Kcv activity in Xenopus oocytes, suggesting that Kcv activity might be controlled by phosphorylation or dephosphorylation. (iii) The PBCV-1 genomic sequence revealed a gene encoding a putative protein kinase (pkx) adjacent to kcv. These findings prompted us to examine the kcv flanking regions in 16 additional chlorella viruses and transcription in two of these viruses, as well as the effect of the three protein kinase inhibitors on two Kcv homologs in Xenopus oocytes. The results indicate (i) pkx is always located 5' to kcv, but the spacing between the two genes varies from 31 to 1588 nucleotides. More variation occurs in the kcv 3' flanking region of the 16 viruses. (ii) The kcv gene is expressed as a late mono-cistronic mRNA. (iii) Unlike the affect on PBCV-1 Kcv, the three protein kinase inhibitors have little or no effect on the activity of the two Kcv homologs in oocytes. (iv) A comparison of the kcv 5' upstream sequences from the 16 viruses identified a highly conserved 10-nucleotide sequence that is present in the promoter region of all of the viruses.

Characterization of a chlorella virus PBCV-1 encoded ribonuclease III.

Sequence analysis of the 330-kb genome of chlorella virus PBCV-1 revealed an open reading frame, A464R, which encodes a protein with 30-35% amino acid identity to ribonuclease III (RNase III) from many bacteria. The a464r gene was cloned and the protein was expressed in Escherichia coli using the chitin-binding intein system. The recombinant PBCV-1 RNase III cleaves model dsRNA substrates, in a Mg(2+)-dependent manner, into a defined set of products. The substrate cleavage specificity overlaps, but is nonidentical to that of E. coli RNase III. The a464r gene is expressed very early during PBCV-1 infection, within 5-10 min p.i. The RNase III protein appears at 15 min p.i. and disappears by 120 min p.i. The a464r gene is highly conserved among the chlorella viruses. Phylogenetic analyses indicate that the PBCV enzyme is most closely related to Mycoplasma pneumoniae RNase III.