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Viral lncRNA upregulates multiple A+U-richViral lncRNA upregulates multiple A+U-rich transcripts to promote cell motility and viral dissemination transcripts to promote cell motility and viral dissemination

ABSTRACT: Long non-coding RNAs are frequently associated with broad modulation of gene expression and thus provide the cell with the ability to synchronize entire metabolic processes. We used transcriptomic and proteomic approaches to investigate whether the most abundant human cytomegalovirus-encoded lncRNA, RNA2.7, has this characteristic. By comparing RNA2.7 deletion mutants with wild-type virus, RNA2.7 was found to regulate a large number of cellular genes late in infection. Pathway analysis indicated that >100 RNA2.7-regulated cellular genes are associated with promoting cell movement, and ten of the most highly regulated were verified in further experiments. Morphological analysis and live cell tracking showed that RNA2.7 promotes the movement and detachment of infected cells late in infection, and plaque assays using sparse cell monolayers demonstrated that RNA2.7 promotes cell-to-cell dissemination and spread of virus. Bioinformatic analysis indicated that RNA2.7-upregulated mRNAs are relatively A+U-rich, which is a trait associated with transcript instability, and that they are also enriched in motifs associated with mRNA instability. Experiments involving transcriptional inhibition showed that transcripts from four RNA2.7-regulated cellular genes were longer-lived in the presence of RNA2.7. These findings demonstrate that RNA2.7 promotes cell movement and viral dissemination late in infection and indicate that this may be due to general stabilization of A+U-rich transcripts.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human) Human Herpesvirus 5 Strain Merlin

SUBMITTER: Katie Nightingale

LAB HEAD: Andrew Davison

PROVIDER: PXD022277 | Pride | 2021-07-27

REPOSITORIES: Pride

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Project description:Paramecium bursaria chlorella virus (PBCV-1), a member of the family Phycodnaviridae, is a large dsDNA, plaque-forming virus that infects the unicellular green alga Chlorella NC64A. The 331 kb PBCV-1 genome is predicted to encode 365 proteins and 11 tRNAs. To follow global transcription during PBCV-1 replication, a microarray containing 50-mer probes to the PBCV-1 365 protein-encoding genes (CDS) was constructed. Competitive hybridization experiments were conducted employing cDNA from poly A-containing RNA obtained from cells at seven time points after virus infection. The results led to the following conclusions: i) the PBCV-1 replication cycle is temporally programmed and regulated; ii) 360 (99%) of the arrayed PBCV-1 CDSs are expressed at some time in the virus life cycle in the laboratory; iii) 227 (62%) of the CDSs are expressed before virus DNA synthesis begins; iv) these 227 CDSs were grouped into two classes: 127 transcripts disappear prior to initiation of virus DNA synthesis (considered early) and 100 transcripts are still detected after virus DNA synthesis begins (considered early/late); v) 133 (36%) of the CDSs are expressed after virus DNA synthesis begins (considered late); vi) expression of most late CDSs is inhibited by adding the DNA replication inhibitor aphidicolin prior to virus infection. This study provides the first comprehensive evaluation of virus gene expression during the PBCV-1 lifecycle. Time course analysis of chlorella virus PBCV-1: cDNAs from poly A-containing RNAs isolated from cells at seven times after PBCV-1 infection (20, 40, 60, 90, 120, 240, 360 min p.i.) were competitively hybridized against a reference sample on the microarrays. Three independent biological replica, four technical replica of the genome per array. Time course analysis of chlorella virus PBCV-1 gene expression in the presence of aphidicolin: cDNAs from poly A-containing RNAs isolated from aphidicolin-treated cells at seven times after PBCV-1 infection (20, 40, 60, 90, 120, 240, 360 min p.i.) were competitively hybridized against non-treated, infected samples from the same time points. Three independent biological replica, four technical replica of the genome per array.

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Viral lncRNA upregulates multiple A+U-richViral lncRNA upregulates multiple A+U-rich transcripts to promote cell motility and viral dissemination transcripts to promote cell motility and viral dissemination

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