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MCMV infection of cultured mouse cells induces expression of miR-7a.

ABSTRACT: One goal of viral infection is to reprogram the host cell to optimize viral replication. As part of this process, viral miRNAs may compete for components of the miRNA/siRNA pathway as well as regulate cellular targets. Mouse Cytomegalovirus has been described to generate large numbers of viral miRNAs during lytic infection and was therefore used to analyze the impact of viral miRNAs on the host cell small RNA system as well as to check for sorting of viral small RNAs into specific Ago-proteins. Deep sequencing analysis of MCMV infected cells revealed that viral miRNAs represent only app. 13% of all detected miRNAs. All previously described MCMV miRNAs with the exception of miR-m88-1* were confirmed and for the MCMV miR-m01-1 hairpin an additional miRNA, designated miR-m01-1-3p, was found. Its presence was confirmed by qPCR and Northern Blot. Deep sequencing after RISC IP with antibodies specific for either Ago1 or Ago2 showed that all MCMV miRNAs are loaded into both RISC complexes. The ratio of MCMV to mouse miRNAs was not increased after immunoprecipitation of Ago-proteins. Viral miRNAs therefore do not overwhelm the host miRNA processing system nor are they preferentially incorporated into RISC. We found that 3 mouse miRNAs showed an altered expression due to MCMV infection. Down-regulation of miR-27a, as previously described, could be confirmed. In addition, miR-26a was down-regulated and an up-regulation of miR-7a dependent on viral protein expression could be observed.

ORGANISM(S): Mus musculus

PROVIDER: GSE36919 | GEO | 2012/07/01

SECONDARY ACCESSION(S): PRJNA157095

REPOSITORIES: GEO

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Project description:Small non-coding RNAs have emerged as key players in modulation of viral infection. A unique example is the critical dependence of hepatitis C virus (HCV) on the liver-specific microRNA (miRNA), miR-122, which has surfaced as therapeutic target. Here, we used crosslinking immunoprecipitation (CLIP) of the Argonaute (AGO) protein to characterize strengths and specificities of miRNA interactions across 15 viral genomes. Intriguingly, replication of pestiviruses, which are major threats to milk and meat industry, critically depends on cellular miR-17 and let-7 interactions with the viral 3âUTR. Like HCV, miRNA binding enhanced translation and prevented viral RNA degradation. On the cellular transcriptome, pestiviral miR-17 sequestration in vitro and ex vivo conferred reduced AGO binding and functional mRNA de-repression for miR-17 targets. These findings generalize the concept of RNA virus dependence on cellular miRNAs, highlight such interactions as therapeutic targets, and connect functional regulation of the transcriptome in primary cells to miRNA sequestration. Several subseries of analyses were performed. For each sample, subseries to which it belongs are indicated. In the âVirus AGO-CLIPâ subseries, AGO-CLIP was performed on cells infected with virus as indicated. Processed reads were aligned to the host genome (hg18 or BosTau7) and to the respective viral genome. The primary data of interest from this subseries concerns AGO binding to viral RNA, and is given as processed data file âTable S1â. In the âVirus AGO-CLIP: BVDV vs. Mockâ subseries, AGO-CLIP was performed on four replicates each of BVDV and mock infected MDBK cells. CLIP reads aligned to BosTau7 were clustered, and differential analysis was performed on binding to each cluster. In addition, differential analysis of AGO bound miRNAs was performed. The associated data is given as processed data file âTable S3 and S4â. In the âAGO-CLIP: tinyLNA-17 vs. Mockâ AGO-CLIP was performed on four replicates each of tinyLNA-17 and mock treated MDBK cells. CLIP reads aligned to BosTau7 were clustered, and differential analysis was performed on binding to each cluster. The associated data is given as processed data file âTable S5â. In the âRNA-seq: BVDV vs. Mockâ subseries, mRNA-seq was performed on two replicates each of MDBK cells infected with different biotypes of BVDV or with miR-17 seed site mutant BVDV. The latter (incl. mock controls) were trans-complemented with the corresponding mutated miR-17. Differential gene expression analysis was performed between selected conditions. The associated data is given as processed data file âTable S6â.

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MCMV infection of cultured mouse cells induces expression of miR-7a.

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