Identification of Novel, Highly Expressed Retroviral MicroRNAs in Cells Infected by Bovine Foamy Virus

Abstract

While numerous viral microRNAs (miRNAs) expressed by DNA viruses, especially herpesvirus family members, have been reported, there have been very few reports of miRNAs derived from RNA viruses. Here we describe three miRNAs expressed by bovine foamy virus (BFV), a member of the spumavirus subfamily of retroviruses, in both BFV-infected cultured cells and BFV-infected cattle. All three viral miRNAs are initially expressed in the form of an ϳ122-nucleotide (nt) pri-miRNA, encoded within the BFV long terminal repeat U3 region, that is subsequently cleaved to generate two pre-miRNAs that are then processed to yield three distinct, biologically active miRNAs. The BFV pri-miRNA is transcribed by RNA polymerase III, and the three resultant mature miRNAs were found to contribute a remarkable ϳ70% of all miRNAs expressed in BFV-infected cells. These data document the second example of a retrovirus that is able to express viral miRNAs by using embedded proviral RNA polymerase III promoters. IMPORTANCE Foamy viruses are a ubiquitous family of nonpathogenic retroviruses that have potential as gene therapy vectors in humans.Here we demonstrate that bovine foamy virus (BFV) expresses high levels of three viral microRNAs (miRNAs) in BFV-infected cells in culture and also in infected cattle. The BFV miRNAs are unusual in that they are initially transcribed by RNA polymerase III as a single, ϳ122-nt pri-miRNA that is subsequently processed to release three fully functional miRNAs. The observation that BFV, a foamy virus, is able to express viral miRNAs in infected cells adds to emerging evidence that miRNA expression is a common, albeit clearly not universal, property of retroviruses and suggests that these miRNAs may exert a significant effect on viral replication in vivo. M icroRNAs (miRNAs) are a diverse class of ϳ22-nucleotide (nt) regulatory RNAs that are expressed by all known multicellular eukaryotes (1). Cellular miRNAs are normally transcribed by RNA polymerase II (Pol II) to give rise to a long primiRNA that is cleaved in the nucleus by the microprocessor complex, consisting of the RNase III enzyme Drosha and the RNA-binding cofactor DGCR8, to release the ϳ60-nt pre-miRNA intermediate, which contains an ϳ22-bp imperfect stem with an ϳ2-nt 3= overhang (2). The pre-miRNA is then exported to the cytoplasm, where it is cleaved by a second RNase III enzyme, Dicer, which removes the terminal loop, leaving a second ϳ2-nt 3= overhang.

Hepatitis B Virus-Encoded MicroRNA Controls Viral Replication

“…The first report of virus-encoded miRNA was of Epstein-Barr virus-encoded miRNA (6). Subsequently, it was found that miRNAs were encoded by simian virus 40 (SV40) (7), herpes simplex virus (HSV) (8), human cytomegalovirus (HCMV) (9), Kaposi's sarcoma-associated herpesvirus (KSHV) (10), Marek's disease virus (MDV) (11), and retrovirus families (12)(13)(14). Although the functions of the majority of viral miRNAs have not been elucidated, emerging studies have demonstrated that some viral miRNAs regulate the viral life cycle itself, including the maintenance/establishment of persistent infection and productive infection (15).…”

Identification, validation, and characterization of noncanonical miRNAs

“…In contrast to the canonical pathway of miRNA biogenesis, multiple viruses from the herpesvirus [14][15][16][17][18] and retrovirus [19][20][21] families are known to utilize alternative pathways to generate miRNAs. The Herpesvirus saimiri (HVS) pre-miRNAs are generated by the integrator complex, which is better known for its role in small nuclear RNA processing [22].…”

Viral noncoding RNAs: more surprises

“…All three bovine foamy virus (BFV) miRNAs are embedded in RNA Pol III transcripts structurally similar to SFV pri-miRNAs (Whisnant et al 2014). Whether Drosha is involved in their processing requires further investigation.…”

“…It is now clear that at least four retroviruses generate viral miRNAs (Kincaid et al 2012Whisnant et al 2014;Yao et al 2014). Surprisingly, only avian leukosis virus (ALV) uses the canonical biogenesis pathway to express its one miRNA (Yao et al 2014).…”

“…The other three viruses use RNA Pol III to transcribe pri-miRNAs from the proviral genome. These are then processed by the noncanonical pathways described below, which avoid potentially detrimental cleavage of the viral genome or mRNA by Drosha (Kincaid et al 2012Whisnant et al 2014). …”