Microarray Analysis of Paramecium bursaria Chlorella Virus 1 Transcription

Abstract

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.

Giant virus with a remarkable complement of genes infects marine zooplankton

“…This percentage is comparable with the observed expression of 65% of viral genes during infection of the marine phytoplankter Emiliania huxleyi by EhV-86 (35). However, recent gene expression studies in PBCV-1 and Mimivirus validated transcription for nearly all of their predicted genes (3,36). It seems therefore likely that our microarray data underestimated the true extent of transcriptional activity in CroV.…”

Classification and Determination of Possible Origins of ORFans through Analysis of Nucleocytoplasmic Large DNA Viruses

“…Thus, a percentage of ORFans might be true functional genes with the same expression potential as non-ORFans, undergoing an equal evolution rate. This conclusion is consistent with different reports showing that 95, 96, 97 and 99% of predicted coding sequences are respectively expressed in Vaccinia, Red Sea bream iridovirus, SGIV, and Paramecium bursaria Chlorella virus-1 at some time during the virus infectious cycle [43][44][45][46][47] . Thus, ORFans represent a high proportion of potentially coding sequences in the current databases, but very few viral ORFans have been experimentally characterized [48] .…”

Chlorovirus PBCV-1 Encodes an Active Copper-Zinc Superoxide Dismutase

“…(43), the cvSOD-encoding gene a245r was expressed as a late gene. A microarray experiment also indicated that the a245r gene was expressed as a late gene (44). The hybridized RNA band was ϳ1.3 kb in size (see Fig.…”