PUBLICATIONS

Processing of primary microRNA (pri-miRNA) stem-loops by the Drosha-DGCR8 complex is the initial step in miRNA maturation and crucial for miRNA function. Nonetheless, the underlying mechanism that determines the Drosha cleavage site of pri-miRNAs has remained unclear. Two prevalent but seemingly conflicting models propose that Drosha-DGCR8 anchors to and directs cleavage a fixed distance from either the basal single-stranded (ssRNA) or the terminal loop. However, recent studies suggest that the basal ssRNA and/or the terminal loop may influence the Drosha cleavage site dependent upon the sequence/structure of individual pri-miRNAs. Here, using a panel of closely related pri-miRNA variants, we further examine the role of pri-miRNA structures on Drosha cleavage site selection in cells. Our data reveal that both the basal ssRNA and terminal loop influence the Drosha cleavage site within three pri-miRNAs, the Simian Virus 40 (SV40) pri-miRNA, pri-miR-30a, and pri-miR-16. In addition to the flanking ssRNA regions, we show that an internal loop within the SV40 pri-miRNA stem strongly influences Drosha cleavage position and efficiency. We further demonstrate that the positions of the internal loop, basal ssRNA, and the terminal loop of the SV40 pri-miRNA cooperatively coordinate Drosha cleavage position and efficiency. Based on these observations, we propose that the pri-miRNA stem, defined by internal and flanking structural elements, guides the binding position of Drosha-DGCR8, which consequently determines the cleavage site. This study provides mechanistic insight into pri-miRNA processing in cells that has numerous biological implications and will assist in refining Drosha-dependent shRNA design.

Establishing lifelong infection and periodically shedding infectious progeny is a successful strategy employed by several persistent pathogens. In this issue of Cell Host & Microbe, Pan et al. (2014) demonstrate that a cell-type-specific host microRNA can restrict gene expression and pathogenicity of herpes simplex virus 1, thereby promoting long-term infection.

Transcripts possessing a 5'-triphosphate are a hallmark of viral transcription and can trigger the host antiviral response. 5'-triphosphates are also found on common host transcripts transcribed by RNA polymerase III (RNAP III), yet how these transcripts remain non-immunostimulatory is incompletely understood. Most microRNAs (miRNAs) are 5'-monophosphorylated as a result of sequential endonucleolytic processing by Drosha and Dicer from longer RNA polymerase II (RNAP II)-transcribed primary transcripts. In contrast, bovine leukemia virus (BLV) expresses subgenomic RNAP III transcripts that give rise to miRNAs independent of Drosha processing. Here, we demonstrate that each BLV pre-miRNA is directly transcribed by RNAP III from individual, compact RNAP III type II genes. Thus, similar to manmade RNAP III-generated short hairpin RNAs (shRNAs), the BLV pre-miRNAs are initially 5'-triphosphorylated. Nonetheless, the derivative 5p miRNAs and shRNA-generated 5p small RNAs (sRNAs) possess a 5'-monophosphate. Our enzymatic characterization and small RNA sequencing data demonstrate that BLV 5p miRNAs are co-terminal with 5'-triphosphorylated miRNA precursors (pre-miRNAs). Thus, these results identify a 5'-tri-phosphatase activity that is involved in the biogenesis of BLV miRNAs and shRNA-generated sRNAs. This work advances our understanding of retroviral miRNA and shRNA biogenesis and may have implications regarding the immunostimulatory capacity of RNAP III transcripts.

UNLABELLED: Several different polyomaviruses (PyVs) encode microRNAs (miRNAs) that regulate viral as well as host gene expression. However, the functions of polyomaviral miRNAs, particularly during in vivo infection, remain poorly understood. Here we identify rare naturally arising PyVs that are severely attenuated or null for miRNA expression. We identify hypomorphic or null strains for miRNA expression from rhesus macaque simian virus 40 (SV40) and human JC virus. These strains were isolated from immunocompromised hosts and derive from insertions or deletions in the viral DNA that preserve the amino acid reading frame of opposing-strand large T antigen gene. Characterization of the SV40 miRNA hypomorph, K661, shows that it is inhibited at the early miRNA biogenesis step of Drosha-mediated processing. Despite having a nonrearranged enhancer, which a previous study has shown renders some PyVs more susceptible to the autoregulatory activities of the miRNA, restoring miRNA expression to K661 has little effect on virus growth in either immortalized or primary monkey kidney cells. Thus, in addition to any effect of accompanying genomic elements, these results suggest that the cellular context also determines susceptibility to PyV miRNA-mediated effects. Combined, these results demonstrate that polyomaviruses lacking miRNAs can arise infrequently and that the functional importance of polyomaviral miRNAs is context dependent, consistent with an activity connected to the immune status of the host. IMPORTANCE: Diverse virus families encode miRNAs, yet much remains unknown about viral miRNA function and contribution to the infectious cycle. Polyomaviruses (PyVs) are small DNA viruses, long known to be important as etiological agents of rare diseases and valuable models of DNA virus infection. Here, in immunosuppressed hosts, we uncover rare naturally arising variants of different PyVs that have lost the ability to express miRNAs. This represents some of the only known natural viruses to have lost miRNA expression. By probing the biogenesis pathways of these variants, we uncover that miRNA expression is lost via small insertions or deletions that render the transcripts resistant to early steps of miRNA biogenesis while preserving the reading frame of the opposing T antigen transcripts. Overall, our study informs how miRNA genes evolve/devolve in viruses and suggests that miRNA function is exquisitely dependent not only on viral genomic context but also on the cellular and host environment.

MicroRNAs (miRNAs) play regulatory roles in diverse processes in both eukaryotic hosts and their viruses, yet fundamental questions remain about which viruses code for miRNAs and the functions that they serve. Simian foamy viruses (SFVs) of Old World monkeys and apes can zoonotically infect humans and, by ill-defined mechanisms, take up lifelong infections in their hosts. Here, we report that SFVs encode multiple miRNAs via a noncanonical mode of biogenesis. The primary SFV miRNA transcripts (pri-miRNAs) are transcribed by RNA polymerase III (RNAP III) and take multiple forms, including some that are cleaved by Drosha. However, these miRNAs are generated in a context-dependent fashion, as longer RNAP II transcripts spanning this region are resistant to Drosha cleavage. This suggests that the virus may avoid any fitness penalty that could be associated with viral genome/transcript cleavage. Two SFV miRNAs share sequence similarity and functionality with notable host miRNAs, the lymphoproliferative miRNA miR-155 and the innate immunity suppressor miR-132. These results have important implications regarding foamy virus biology, viral miRNAs, and the development of retroviral-based vectors. IMPORTANCE Fundamental questions remain about which viruses encode miRNAs and their associated functions. Currently, few natural viruses with RNA genomes have been reported to encode miRNAs. Simian foamy viruses are retroviruses that are prevalent in nonhuman host populations, and some can zoonotically infect humans who hunt primates or work as animal caretakers. We identify a cluster of miRNAs encoded by SFV. Characterization of these miRNAs reveals evolutionarily conserved, unconventional mechanisms to generate small RNAs. Several SFV miRNAs share sequence similarity and functionality with host miRNAs, including the oncogenic miRNA miR-155 and innate immunity suppressor miR-132. Strikingly, unrelated herpesviruses also tap into one or both of these same regulatory pathways, implying relevance to a broad range of viruses. These findings provide new insights with respect to foamy virus biology and vectorology.

Effects of polyomavirus SV40 microRNA on pathogenesis of viral infections in vivo are not known. Syrian golden hamsters are the small animal model for studies of SV40. We report here effects of SV40 microRNA and influence of the structure of the regulatory region on dynamics of SV40 DNA levels in vivo. Outbred young adult hamsters were inoculated by the intracardiac route with 1×10⁷ plaque-forming units of four different variants of SV40. Infected animals were sacrificed from 3 to 270 days postinfection and viral DNA loads in different tissues determined by quantitative real-time polymerase chain reaction assays. All SV40 strains displayed frequent establishment of persistent infections and slow viral clearance. SV40 had a broad tissue tropism, with infected tissues including liver, kidney, spleen, lung, and brain. Liver and kidney contained higher viral DNA loads than other tissues; kidneys were the preferred site for long-term persistent infection although detectable virus was also retained in livers. Expression of SV40 microRNA was demonstrated in wild-type SV40-infected tissues. MicroRNA-negative mutant viruses consistently produced higher viral DNA loads than wild-type SV40 in both liver and kidney. Viruses with complex regulatory regions displayed modestly higher viral DNA loads in the kidney than those with simple regulatory regions. Early viral transcripts were detected at higher levels than late transcripts in liver and kidney. Infectious virus was detected infrequently. There was limited evidence of increased clearance of microRNA-deficient viruses. Wild-type and microRNA-negative mutants of SV40 showed similar rates of transformation of mouse cells in vitro and tumor induction in weanling hamsters in vivo. This report identified broad tissue tropism for SV40 in vivo in hamsters and provides the first evidence of expression and function of SV40 microRNA in vivo. Viral microRNA dampened viral DNA levels in tissues infected by SV40 strains with simple or complex regulatory regions.

3' untranslated regions (UTRs) are known to play an important role in posttranscriptional regulation of gene expression. Here we map the 3' UTRs of Kaposi's sarcoma-associated herpesvirus (KSHV) using next-generation RNA sequencing, 3' rapid amplification of cDNA ends (RACE), and tiled microarray analyses. Chimeric reporters containing the KSHV 3' UTRs show a general trend toward reduced gene expression under conditions of latent infection. Those 3' UTRs with a higher GC content are more likely to be associated with reduced gene expression. KSHV transcripts display an extensive use of shared polyadenylation sites allowing for partially overlapping 3' UTRs and regulatory activities. In addition, a subset of KSHV 3' UTRs is sufficient to convey increased gene expression under conditions of lytic infection. These results suggest a role for viral 3' UTRs in contributing to differential gene expression during latent versus lytic infection.

Hundreds of virus-encoded microRNAs (miRNAs) have been uncovered, but an in-depth functional understanding is lacking for most. A major challenge for the field is separating those miRNA targets that are biologically relevant from those that are not advantageous to the virus. Here, we show that miRNAs from related variants of the polyomavirus simian vacuolating virus 40 (SV40) have differing host target repertoires (targetomes) while their direct autoregulatory activity on virus-encoded early gene products is completely preserved. These results underscore the importance of miRNA-mediated viral gene autoregulation in some polyomavirus life cycles. More broadly, these findings imply that some host targets of virus-encoded miRNAs are likely to be of little selective advantage to the virus, and our approach provides a strategy for prioritizing relevant targets.

RNA interference (RNAi) is an established antiviral defense mechanism in plants and invertebrates. Whether RNAi serves a similar function in mammalian cells remains unresolved. We find that in some cell types, mammalian RNAi activity is reduced shortly after viral infection via poly-ADP-ribosylation of the RNA-induced silencing complex (RISC), a core component of RNAi. Well-established antiviral signaling pathways, including RIG-I/MAVS and RNaseL, contribute to inhibition of RISC. In the absence of virus infection, microRNAs repress interferon-stimulated genes (ISGs) associated with cell death and proliferation, thus maintaining homeostasis. Upon detection of intracellular pathogen-associated molecular patterns, RISC activity decreases, contributing to increased expression of ISGs. Our results suggest that, unlike in lower eukaryotes, mammalian RISC is not antiviral in some contexts, but rather RISC has been co-opted to negatively regulate toxic host antiviral effectors via microRNAs.

The growing availability of large-scale functional networks has promoted the development of many successful techniques for predicting functions of genes. Here we extend these network-based principles and techniques to functionally characterize whole sets of genes. We present RIDDLE (Reflective Diffusion and Local Extension), which uses well developed guilt-by-association principles upon a human gene network to identify associations of gene sets. RIDDLE is particularly adept at characterizing sets with no annotations, a major challenge where most traditional set analyses fail. Notably, RIDDLE found microRNA-450a to be strongly implicated in ocular diseases and development. A web application is available at http://www.functionalnet.org/RIDDLE.

MicroRNAs (miRNAs) are small RNAs that play a regulatory role in numerous and diverse eukaryotic cellular processes. Virus-encoded miRNAs have garnered much interest, although the functions of most remain to be deciphered. To date, readily detectable, evolutionarily conserved natural miRNAs have only been identified from viruses with DNA genomes. Combined with the fact that most miRNAs are generated from endonucleolytic cleavage of longer transcripts, this finding has led to a common conception that naturally occurring RNA viruses will not encode miRNAs to avoid unproductive cleavage of their genomes or mRNAs. Here we demonstrate that the bovine leukemia virus (BLV), a retrovirus with an RNA genome, encodes a conserved cluster of miRNAs that are transcribed by RNA polymerase III (pol III). Thus, the BLV miRNAs avoid the conundrum of genome/mRNA cleavage because only the subgenomic pol III transcripts are efficiently processed into miRNAs. BLV infection is strongly associated with B-cell tumors in cattle. Because most cells in BLV-associated tumors express little viral mRNAs or proteins, exactly how BLV contributes to tumorigenesis has remained a decades-long unsolved mystery. One BLV miRNA, BLV-miR-B4, shares partial sequence identity and shared common targets with the host miRNA, miR-29. As miR-29 overexpression is associated with B-cell neoplasms that resemble BLV-associated tumors, our findings suggest a possible mechanism contributing to BLV-induced tumorigenesis.

MicroRNAs (miRNAs) are small RNAs that play important roles in the regulation of gene expression. First described as posttranscriptional gene regulators in eukaryotic hosts, virus-encoded miRNAs were later uncovered. It is now apparent that diverse virus families, most with DNA genomes, but at least some with RNA genomes, encode miRNAs. While deciphering the functions of viral miRNAs has lagged behind their discovery, recent functional studies are bringing into focus these roles. Some of the best characterized viral miRNA functions include subtle roles in prolonging the longevity of infected cells, evading the immune response, and regulating the switch to lytic infection. Notably, all of these functions are particularly important during persistent infections. Furthermore, an emerging view of viral miRNAs suggests two distinct groups exist. In the first group, viral miRNAs mimic host miRNAs and take advantage of conserved networks of host miRNA target sites. In the larger second group, viral miRNAs do not share common target sites conserved for host miRNAs, and it remains unclear what fraction of these targeted transcripts are beneficial to the virus. Recent insights from multiple virus families have revealed new ways of interacting with the host miRNA machinery including noncanonical miRNA biogenesis and new mechanisms of posttranscriptional cis gene regulation. Exciting challenges await the field, including determining the most relevant miRNA targets and parlaying our current understanding of viral miRNAs into new therapeutic strategies. To accomplish these goals and to better grasp miRNA function, new in vivo models that recapitulate persistent infections associated with viral pathogens are required.

microRNAs (miRNAs) of host and viral origin have been suggested to play important roles in the viral infectious cycle. The discovery of new viral miRNAs, and understanding how viral infection alters host miRNAs, has been greatly aided by Northern blot analysis. The Northern blot method is used to detect specific RNAs that have been separated by size and immobilized onto a membrane. This method can provide specific information regarding the size of a miRNA and possible precursor structures. Thus, it represents a valuable tool in the discovery and validation of new miRNAs. Viral infection can sometimes present special challenges to utilizing Northern blot analysis. These challenges may include low miRNA expression levels, high GC content, and abundant background signal from nonspecific RNA degradation fragments triggered by the stress of lytic infection. We present a protocol for small RNA Northern blot analysis that we have successfully used to detect viral miRNAs from cells undergoing lytic infection from members of the Herpes and Polyoma virus families. Included are optimization strategies and a protocol for using radiolabeled oligonucleotides to detect larger RNAs.

It is well-appreciated that viruses use host effectors for macromolecular synthesis and as regulators of viral gene expression. Viruses can encode their own regulators, but often use host-encoded factors to optimize replication. Here, we show that Drosha, an endoribonuclease best known for its role in the biogenesis of microRNAs (miRNAs), can also function to directly regulate viral gene expression. Kaposi's Sarcoma-associated Herpesvirus (KSHV) is associated with various tumors, and like all herpesviruses, has two modes of infection, latent and lytic, which are characterized by differential expression of viral gene products. Kaposin B (KapB) is a KSHV-encoded protein associated with cytokine production and cytotoxicity. We demonstrate that in addition to previously known transcriptional mechanisms, differences in Drosha levels contribute to low levels of KapB expression in latency and robust increases in expression during lytic replication. Thus, surprisingly, KSHV modulates Drosha activity differentially depending on the mode of replication. This regulation is dependent on Drosha-mediated cleavage, and KapB transcripts lacking the Drosha cleavage sites express higher levels of KapB, resulting in increased cell death. This work increases the known functions of Drosha and implies that tying viral gene expression to Drosha activity is advantageous for viruses.

Several different members of the Polyomaviridae, including some human pathogens, encode microRNAs (miRNAs) that lie antisense with respect to the early gene products, the tumor (T) antigens. These miRNAs negatively regulate T antigen expression by directing small interfering RNA (siRNA)-like cleavage of the early transcripts. miRNA mutant viruses of some members of the Polyomaviridae express increased levels of early proteins during lytic infection. However, the importance of miRNA-mediated negative regulation of the T antigens remains uncertain. Bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1) is associated with papillomas and carcinomas in the endangered marsupial the western barred bandicoot (Perameles bougainville). BPCV1 is the founding member of a new group of viruses that remarkably share distinct properties in common with both the polyomavirus and papillomavirus families. Here, we show that BPCV1 encodes, in the same orientation as the papillomavirus-like transcripts, a miRNA located within a long noncoding region (NCR) of the genome. Furthermore, this NCR serves the function of both promoter and template for the primary transcript that gives rise to the miRNA. Unlike the polyomavirus miRNAs, the BPCV1 miRNA is not encoded antisense to the T antigen transcripts but rather lies in a separate, proximal region of the genome. We have mapped the 3' untranslated region (UTR) of the BPCV1 large T antigen early transcript and identified a functional miRNA target site that is imperfectly complementary to the BPCV1 miRNA. Chimeric reporters containing the entire BPCV1 T antigen 3' UTR undergo negative regulation when coexpressed with the BPCV1 miRNA. Notably, the degree of negative regulation observed is equivalent to that of an identical reporter that is engineered to bind to the BPCV1 miRNA with perfect complementarity. We also show that this miRNA and this novel mode of early gene regulation are conserved with the related BPCV2. Finally, papillomatous lesions from a western barred bandicoot express readily detectable levels of this miRNA, stressing its likely importance in vivo. Combined, the alternative mechanisms of negative regulation of T antigen expression between the BPCVs and the polyomaviruses support the importance of miRNA-mediated autoregulation in the life cycles of some divergent polyomaviruses and polyomavirus-like viruses.

MicroRNAs (miRNAs) are the subject of enormous interest. They are small non-coding RNAs that play a regulatory role in numerous and diverse cellular processes such as immune function, apoptosis and tumorigenesis. Several virus families have been shown to encode miRNAs, and an appreciation for their roles in the viral infectious cycle continues to grow. Despite the identification of numerous (>225) viral miRNAs, an in depth functional understanding of most virus-encoded miRNAs is lacking. Here we focus on a few viral miRNAs with well-defined functions. We use these examples to extrapolate general themes of viral miRNA activities including autoregulation of viral gene expression, avoidance of host defenses, and a likely important role in maintaining latent and persistent infections. We hypothesize that although the molecular mechanisms and machinery are similar, the majority of viral miRNAs may utilize a target strategy that differs from host miRNAs. That is, many viral miRNAs may have evolved to regulate viral-encoded transcripts or networks of host genes that are unique to viral miRNAs. Included in this latter category is a likely abundant class of viral miRNAs that may regulate only one or a few principal host genes. Key steps forward for the field are discussed, including the need for additional functional studies that utilize surgical viral miRNA mutants combined with relevant models of infection.