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Bovine Leukemia Virus Small Noncoding RNAs Are Functional Elements That Regulate Replication and Contribute to Oncogenesis In Vivo.

ABSTRACT: Retroviruses are not expected to encode miRNAs because of the potential problem of self-cleavage of their genomic RNAs. This assumption has recently been challenged by experiments showing that bovine leukemia virus (BLV) encodes miRNAs from intragenomic Pol III promoters. The BLV miRNAs are abundantly expressed in B-cell tumors in the absence of significant levels of genomic and subgenomic viral RNAs. Using deep RNA sequencing and functional reporter assays, we show that miRNAs mediate the expression of genes involved in cell signaling, cancer and immunity. We further demonstrate that BLV miRNAs are essential to induce B-cell tumors in an experimental model and to promote efficient viral replication in the natural host.

SUBMITTER: Gillet NA

PROVIDER: S-EPMC4849745 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Bovine Leukemia Virus Small Noncoding RNAs Are Functional Elements That Regulate Replication and Contribute to Oncogenesis In Vivo.

Gillet Nicolas A NA Hamaidia Malik M de Brogniez Alix A Gutiérrez Gerónimo G Renotte Nathalie N Reichert Michal M Trono Karina K Willems Luc L

PLoS pathogens 20160428 4

Retroviruses are not expected to encode miRNAs because of the potential problem of self-cleavage of their genomic RNAs. This assumption has recently been challenged by experiments showing that bovine leukemia virus (BLV) encodes miRNAs from intragenomic Pol III promoters. The BLV miRNAs are abundantly expressed in B-cell tumors in the absence of significant levels of genomic and subgenomic viral RNAs. Using deep RNA sequencing and functional reporter assays, we show that miRNAs mediate the expre ...[more]

PMID: 27123579

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Project description:UNLABELLED:Many viruses express noncoding RNAs (ncRNAs). The gammaherpesviruses (?HVs), including Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, and murine ?HV68, each contain multiple ncRNA genes, including microRNAs (miRNAs). While these ncRNAs can regulate multiple host and viral processes in vitro, the genetic contribution of these RNAs to infection and pathogenesis remains largely unknown. To study the functional contribution of these RNAs to ?HV infection, we have used ?HV68, a small-animal model of ?HV pathogenesis. ?HV68 encodes eight small hybrid ncRNAs that contain both tRNA-like elements and functional miRNAs. These genes are transcribed by RNA polymerase III and are referred to as the ?HV68 TMERs (tRNA-miRNA-encoded RNAs). To determine the total concerted genetic contribution of these ncRNAs to ?HV acute infection and pathogenesis, we generated and characterized a recombinant ?HV68 strain devoid of all eight TMERs. TMER-deficient ?HV68 has wild-type levels of lytic replication in vitro and normal establishment of latency in B cells early following acute infection in vivo. In contrast, during acute infection of immunodeficient mice, TMER-deficient ?HV68 has reduced virulence in a model of viral pneumonia, despite having an enhanced frequency of virus-infected cells. Strikingly, expression of a single viral tRNA-like molecule, in the absence of all other virus-encoded TMERs and miRNAs, reverses both attenuation in virulence and enhanced frequency of infected cells. These data show that ?HV ncRNAs play critical roles in acute infection and virulence in immunocompromised hosts and identify these RNAs as a new potential target to modulate ?HV-induced infection and pathogenesis. IMPORTANCE:The gammaherpesviruses (?HVs) are a subfamily of viruses associated with chronic inflammatory diseases and cancer, particularly in immunocompromised individuals. These viruses uniformly encode multiple types of noncoding RNAs (ncRNAs) that are not translated into proteins. It remains unclear how virus-expressed ncRNAs influence the course and outcome of infection in vivo. Here, we generated a mouse ?HV that lacks the expression of multiple ncRNAs. Notably, this mutant virus is critically impaired in the ability to cause disease in immunocompromised hosts yet shows a paradoxical increase in infected cells early during infection in these hosts. While the original mouse virus encodes multiple ncRNAs, the expression of a single domain of one ncRNA can partially reverse the defects of the mutant virus. These studies demonstrate that ?HV ncRNAs can directly contribute to virus-induced disease in vivo and that these RNAs may be multifunctional, allowing the opportunity to specifically interfere with different functional domains of these RNAs.

Project description:In sheep infected with bovine leukemia virus (BLV), transcription of structural, enzymatic, and accessory genes is silenced. However, the BLV provirus transcribes a series of non-coding RNAs that remain undetected by the host immune response. Specifically, three RNAs (AS1-L, AS1-S, and AS2) are consistently expressed from the antisense strand, originating from transcriptional initiation at the 3'-LTR. To investigate the role of these non-coding RNAs in viral replication and pathogenesis, a reverse genetics approach was devised, capitalizing on a mechanistic disparity in transcription initiation between the 5' and 3' promoters. A two-nucleotide mutation (GG>TA) in the TFIIB-recognition element (BRE) impaired antisense transcription originating from the 3'-LTR. In the context of the provirus, this A1 mutation significantly diminished the expression of antisense RNAs, while not notably affecting sense transcription. When inoculated to sheep, the A1 provirus was infectious but exhibited reduced replication levels, shedding light on the role of antisense transcription in vivo. In comparison to lymphoid organs in sheep infected with a wild-type (WT) provirus, the A1 mutant demonstrated alterations in both the spatial distribution and rates of cell proliferation in the lymph nodes and the spleen. Analysis through RNA sequencing and RT-qPCR unveiled an upregulation of the Hmcn1 gene in B-lymphocytes from A1-infected sheep. Further examination via confocal microscopy and immunohistochemistry revealed an increase in the amount of hemicentin-1 protein encoded by Hmcn1 in peripheral blood mononuclear cells (PBMCs) and lymphoid organs of sheep infected with the A1 mutant. RNA interference targeting Hmcn1 expression impacted the migration of ovine kidney (OVK) cells in vitro. In contrast to the WT provirus, the A1 mutant lacked oncogenic activity when inoculated into sheep. Collectively, this study underscores the essential role of antisense transcription in BLV replication and pathogenicity. These findings may offer valuable insights into understanding the relevance of antisense transcription in the context of human T-cell leukemia virus (HTLV-1).

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Bovine Leukemia Virus Small Noncoding RNAs Are Functional Elements That Regulate Replication and Contribute to Oncogenesis In Vivo.

Publications

Bovine Leukemia Virus Small Noncoding RNAs Are Functional Elements That Regulate Replication and Contribute to Oncogenesis In Vivo.

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