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A viral nuclear noncoding RNA binds re-localized poly(A) binding protein and is required for late KSHV gene expression.

ABSTRACT: During the lytic phase of infection, the gamma herpesvirus Kaposi's Sarcoma-Associated Herpesvirus (KSHV) expresses a highly abundant, 1.1 kb nuclear noncoding RNA of unknown function. We observe that this polyadenylated nuclear (PAN) RNA avidly binds host poly(A)-binding protein C1 (PABPC1), which normally functions in the cytoplasm to bind the poly(A) tails of mRNAs, regulating mRNA stability and translation efficiency. During the lytic phase of KSHV infection, PABPC1 is re-localized to the nucleus as a consequence of expression of the viral shutoff exonuclease (SOX) protein; SOX also mediates the host shutoff effect in which host mRNAs are downregulated while viral mRNAs are selectively expressed. We show that whereas PAN RNA is not required for the host shutoff effect or for PABPC1 re-localization, SOX strongly upregulates the levels of PAN RNA in transient transfection experiments. This upregulation is destroyed by the same SOX mutation that ablates the host shutoff effect and PABPC1 nuclear re-localization or by removal of the poly(A) tail of PAN. In cells induced into the KSHV lytic phase, depletion of PAN RNA using RNase H-targeting antisense oligonucleotides reveals that it is necessary for the production of late viral proteins from mRNAs that are themselves polyadenylated. Our results add to the repertoire of functions ascribed to long noncoding RNAs and suggest a mechanism of action for nuclear noncoding RNAs in gamma herpesvirus infection.

SUBMITTER: Borah S

PROVIDER: S-EPMC3192849 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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A viral nuclear noncoding RNA binds re-localized poly(A) binding protein and is required for late KSHV gene expression.

Borah Sumit S Darricarrère Nicole N Darnell Alicia A Myoung Jinjong J Steitz Joan A JA

PLoS pathogens 20111013 10

During the lytic phase of infection, the gamma herpesvirus Kaposi's Sarcoma-Associated Herpesvirus (KSHV) expresses a highly abundant, 1.1 kb nuclear noncoding RNA of unknown function. We observe that this polyadenylated nuclear (PAN) RNA avidly binds host poly(A)-binding protein C1 (PABPC1), which normally functions in the cytoplasm to bind the poly(A) tails of mRNAs, regulating mRNA stability and translation efficiency. During the lytic phase of KSHV infection, PABPC1 is re-localized to the nu ...[more]

PMID: 22022268

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Project description:Human cytomegalovirus (HCMV) is a prototypic betaherpesvirus which causes severe manifestations in individuals with impaired or immature immunity. To investigate cytomegalovirus-induced pathogenesis and virus-specific immune responses, mouse cytomegalovirus (MCMV) infections in mice are employed as accepted small animal model. MCMV and HCMV share co-linear genomes and encode several homologous proteins. Due to the size and complexity of CMV genomes, the molecular functions of numerous cytomegaloviral gene products remain to be elucidated. While the essential nature of viral genes highlights their biological relevance, it renders functional studies particularly cumbersome by precluding experiments in the infection context. The HCMV-encoded protein pUL34 binds the HCMV genome and regulates viral gene expression (e.g., of US3). Several groups provided compelling evidence that UL34 is essential for HCMV replication. MCMV encodes the homologous protein pM34 (34% identical and 55% similar). Based on unsuccessful attempts to reconstitute M34-deficient virus from a bacterial artificial chromosome (BAC), M34 was previously classified as essential for MCMV replication. To characterize pM34 during viral infection, we engineered and analyzed an MCMV mutant expressing an HA-epitope-tagged pM34 which was expressed with early-late kinetics and localized in the nucleus. Additionally, we generated an M34-deficient ("ΔM34") MCMV-BAC by replacing the entire M34 coding sequence by a kanamycin resistance cassette. The deletion of M34 was confirmed by Southern blot and PCR. Unexpectedly, we could reconstitute replicating ΔM34-MCMV upon transfection of the BAC DNA into mouse embryonic fibroblasts. The absence of M34 from the genome of the replicating ΔM34-MCMV was also confirmed. Accordingly, a ΔM34-MCMV, in which the kanamycin cassette was excised by frt/Flp-mediated recombination, was also replication competent. In order to corroborate the absence of pM34 protein, the M34 deletion was recapitulated on the background of M34HA, which yielded replicating virus devoid of detectable pM34HA protein. The replication of MCMVs lacking M34 was found to be 10- to 100-fold reduced as compared to wt-MCMV which might explain previous unsuccessful reconstitution attempts conducted by others. Taken together, our findings reveal that MCMV remains replication competent despite the absence of M34, enabling functional studies in the infection context.

Dataset Information

A viral nuclear noncoding RNA binds re-localized poly(A) binding protein and is required for late KSHV gene expression.

Publications

A viral nuclear noncoding RNA binds re-localized poly(A) binding protein and is required for late KSHV gene expression.

Similar Datasets

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