Deep RNA Sequencing Reveals Hidden Features and Dynamics of Early Gene Transcription in Paramecium bursaria Chlorella Virus 1

Guillaume Blanc,David D Dunigan,Jean-Jack Riethoven,Janet M Rowe,James L Van Etten,Irina V Agarkova,Yuannan Xia,Michael Mozar,Giane Yanai-Balser,James R Gurnon

doi:10.1371/journal.pone.0090989

Abstract

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.

Highlights

Viruses in the family Phycodnaviridae, together with those in the Poxviridae, Iridoviridae, Ascoviridae, Asfarviridae, and the Mimiviridae are believed to have a common evolutionary ancestor and are referred to as nucleocytoplasmic large DNA viruses (NCLDV) [1,2,3]
We cannot rule out the possibility that significant changes in these ratios occur between the early and late phases of virus replication but we cannot investigate this hypothesis at the present time because the experiment did not span the entire replication cycle. This RNA-seq study investigated the dynamic mRNA abundance profiles of both the algal host and virus Paramecium bursaria chlorella virus 1 (PBCV-1) during the earliest stages of virus infection. This analysis extends a previous study on PBCV-1 gene transcription that used a microarray approach, which revealed a temporal regulation of PBCV-1 genes [18]
In this study we focused on early events and discovered that by 7 min p.i., virus transcripts comprised,2% of the poly(A+)-containing RNAs in the cell

Summary

Introduction

Viruses in the family Phycodnaviridae, together with those in the Poxviridae, Iridoviridae, Ascoviridae, Asfarviridae, and the Mimiviridae are believed to have a common evolutionary ancestor and are referred to as nucleocytoplasmic large DNA viruses (NCLDV) [1,2,3]. Members of the Phycodnaviridae consist of a genetically diverse, but morphologically similar, group of large dsDNA-containing viruses (160 to 560 kb) that infect eukaryotic algae [4,5]. These large viruses are common in both terrestrial and marine waters throughout the world. Paramecium bursaria chlorella virus 1 (PBCV-1), the prototype of the Phycodnaviridae family (genus Chlorovirus), is a large, icosahedral (190 nm in diameter) virus that infects the unicellular, eukaryotic green alga Chlorella variabilis NC64A. 40% of the 416 PBCV-1 gene products resemble proteins in the public databases

Methods

Results

Conclusion