The 331-kbp chlorovirusParamecium bursariachlorella 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 hostChlorella variabilisNC64A 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 (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.

Paramecium bursaria chlorella virus 1 (PBCV-1), the prototype of the genus Chlorovirus (family Phycodnaviridae), is a large, icosahedral (190 nm in diameter), plaque-forming virus that infects the unicellular, eukaryotic green alga Chlorella sp. strain NC64A. The PBCV-1 virion has a lipid membrane located inside an outer glycoprotein capsid. The 330-kb genome is a linear, nonpermutated, double-stranded DNA (dsDNA) molecule with covalently closed hairpin ends that has approximately 365 protein encoding genes (CDSs), as well as 11 tRNA encoding genes (reviewed in references 34, 39, and 40). The CDSs are evenly distributed on both strands and intergenic space is minimal (typically fewer than 100 nucleotides); the exception is a 1,788-bp sequence in the middle of the genome that encodes the tRNA genes. Approximately 35% of the 365 PBCV-1 gene products resemble proteins in the public databases.PBCV-1 initiates infection by attaching rapidly and specifically to the cell wall of its host (22), probably at a unique virus vertex (4, 26). Attachment is immediately followed by host cell wall degradation by a virus-packaged enzyme(s) at the point of contact. After wall degradation, the viral internal membrane presumably fuses with the host membrane, causing host membrane depolarization (9), potassium ion efflux (25), and an increase in the cytoplasm pH (2). These events are predicted to facilitate entry of the viral DNA and virion-associated proteins into the cell. PBCV-1 lacks a gene encoding a recognizable RNA polymerase or a subunit of it, and RNA polymerase activity is not detected in PBCV-1 virions. Therefore, viral DNA and virion-associated proteins are predicted to migrate to the nucleus, and early viral transcription is detected 5 to 10 min postinfection (p.i.), presumably by commandeering a host RNA polymerase(s) (possibly RNA polymerase II) (14, 29). Virus DNA synthesis begins 60 to 90 min p.i., followed by virus assembly at 3 to 5 h p.i. in localized regions of the cytoplasm, called virus assembly centers (21). At 6 to 8 h p.i., virusinduced host cell lysis occurs resulting in release of progeny virions (ϳ1,000 viruses/cell, ϳ25% of which are infectious). These events are depicted in Fig. 1.To initiate PBCV-1 transcription, the host RNA polymerase(s), possibly in combination with a virus transcription factor(s), must recognize virus DNA promoter sequences. Recently, three short nucleotide sequences were identified in putative virus promoter regions (150 bp upstream and 50 bp downstream of the ATG translation site) that are conserved in PBCV-1 and other Chlorovirus members (7). PBCV-1 CDSs are not spatially clustered on the genome by either temporal or functional class, suggesting that transcription regulation must occur via cis-and possible trans-acting regulatory elements.To understand the dynamics of PBCV-1 global gene expression during virus replication, we constructed a microarray containing 50-mer probes to each of the 365 PBCV-1 CDSs.

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.

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.