PARP14 is pro- and anti-viral host factor that promotes IFN production and affects the replication of multiple viruses.

PARP14 is a 203 kDa multi-domain protein that is primarily known as an ADP-ribosyltransferase, and is involved in a variety of cellular functions including DNA damage, microglial activation, inflammation, and cancer progression. In addition, PARP14 is upregulated by interferon (IFN), indicating a role in the antiviral response. Furthermore, PARP14 has evolved under positive selection, again indicating that it is involved in host-pathogen conflict. We found that PARP14 is required for increased IFN-I production in response to coronavirus infection lacking ADP-ribosylhydrolase (ARH) activity and poly(I:C), however, whether it has direct antiviral function remains unclear. Here we demonstrate that the catalytic activity of PARP14 enhances IFN-I and IFN-III responses and restricts ARH-deficient murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. To determine if PARP14's antiviral functions extended beyond CoVs, we tested the ability of herpes simplex virus 1 (HSV-1) and several negative-sense RNA viruses, including vesicular stomatitis virus (VSV), Ebola virus (EBOV), and Nipah virus (NiV), to infect A549 PARP14 knockout (KO) cells. HSV-1 had increased replication in PARP14 KO cells, indicating that PARP14 restricts HSV-1 replication. In contrast, PARP14 was critical for the efficient infection of VSV, EBOV, and NiV, with EBOV infectivity at less than 1% of WT cells. A PARP14 active site inhibitor had no impact on HSV-1 or EBOV infection, indicating that its effect on these viruses was independent of its catalytic activity. These data demonstrate that PARP14 promotes IFN production and has both pro- and anti-viral functions targeting multiple viruses.

A Pseudomonas aeruginosa small RNA regulates chronic and acute infection.

The ability to switch between different lifestyles allows bacterial pathogens to thrive in diverse ecological niches1,2. However, a molecular understanding of their lifestyle changes within the human host is lacking. Here, by directly examining bacterial gene expression in human-derived samples, we discover a gene that orchestrates the transition between chronic and acute infection in the opportunistic pathogen Pseudomonas aeruginosa. The expression level of this gene, here named sicX, is the highest of the P. aeruginosa genes expressed in human chronic wound and cystic fibrosis infections, but it is expressed at extremely low levels during standard laboratory growth. We show that sicX encodes a small RNA that is strongly induced by low-oxygen conditions and post-transcriptionally regulates anaerobic ubiquinone biosynthesis. Deletion of sicX causes P. aeruginosa to switch from a chronic to an acute lifestyle in multiple mammalian models of infection. Notably, sicX is also a biomarker for this chronic-to-acute transition, as it is the most downregulated gene when a chronic infection is dispersed to cause acute septicaemia. This work solves a decades-old question regarding the molecular basis underlying the chronic-to-acute switch in P. aeruginosa and suggests oxygen as a primary environmental driver of acute lethality.

Interpreting and de-noising genetically engineered barcodes in a DNA virus.

The concept of a nucleic acid barcode applied to pathogen genomes is easy to grasp and the many possible uses are straightforward. But implementation may not be easy, especially when growing through multiple generations or assaying the pathogen long-term. The potential problems include: the barcode might alter fitness, the barcode may accumulate mutations, and construction of the marked pathogens may result in unintended barcodes that are not as designed. Here, we generate approximately 5,000 randomized barcodes in the genome of the prototypic small DNA virus murine polyomavirus. We describe the challenges faced with interpreting the barcode sequences obtained from the library. Our Illumina NextSeq sequencing recalled much greater variation in barcode sequencing reads than the expected 5,000 barcodes-necessarily stemming from the Illumina library processing and sequencing error. Using data from defined control virus genomes cloned into plasmid backbones we develop a vetted post-sequencing method to cluster the erroneous reads around the true virus genome barcodes. These findings may foreshadow problems with randomized barcodes in other microbial systems and provide a useful approach for future work utilizing nucleic acid barcoded pathogens.

Characterization of ALTO-encoding circular RNAs expressed by Merkel cell polyomavirus and trichodysplasia spinulosa polyomavirus.

Circular RNAs (circRNAs) are a conserved class of RNAs with diverse functions, including serving as messenger RNAs that are translated into peptides. Here we describe circular RNAs generated by human polyomaviruses (HPyVs), some of which encode variants of the previously described alternative large T antigen open reading frame (ALTO) protein. Circular ALTO RNAs (circALTOs) can be detected in virus positive Merkel cell carcinoma (VP-MCC) cell lines and tumor samples. CircALTOs are stable, predominantly located in the cytoplasm, and N6-methyladenosine (m6A) modified. The translation of MCPyV circALTOs into ALTO protein is negatively regulated by MCPyV-generated miRNAs in cultured cells. MCPyV ALTO expression increases transcription from some recombinant promoters in vitro and upregulates the expression of multiple genes previously implicated in MCPyV pathogenesis. MCPyV circALTOs are enriched in exosomes derived from VP-MCC lines and circALTO-transfected 293T cells, and purified exosomes can mediate ALTO expression and transcriptional activation in MCPyV-negative cells. The related trichodysplasia spinulosa polyomavirus (TSPyV) also expresses a circALTO that can be detected in infected tissues and produces ALTO protein in cultured cells. Thus, human polyomavirus circRNAs are expressed in human tumors and infected tissues and express proteins that have the potential to modulate the infectious and tumorigenic properties of these viruses.

DUSP11-mediated control of 5'-triphosphate RNA regulates RIG-I sensitivity.

Deciphering the mechanisms that regulate the sensitivity of pathogen recognition receptors is imperative to understanding infection and inflammation. Here we demonstrate that the RNA triphosphatase dual-specificity phosphatase 11 (DUSP11) acts on both host and virus-derived 5'-triphosphate RNAs rendering them less active in inducing a RIG-I-mediated immune response. Reducing DUSP11 levels alters host triphosphate RNA packaged in extracellular vesicles and induces enhanced RIG-I activation in cells exposed to extracellular vesicles. Virus infection of cells lacking DUSP11 results in a higher proportion of triphosphorylated viral transcripts and attenuated virus replication, which is rescued by reducing RIG-I expression. Consistent with the activity of DUSP11 in the cellular RIG-I response, mice lacking DUSP11 display lower viral loads, greater sensitivity to triphosphorylated RNA, and a signature of enhanced interferon activity in select tissues. Our results reveal the importance of controlling 5'-triphosphate RNA levels to prevent aberrant RIG-I signaling and demonstrate DUSP11 as a key effector of this mechanism.

Comparison of media and standards for SARS-CoV-2 RT-qPCR without prior RNA preparation.

Since the emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there have been demands on the testing infrastructure that have strained testing capacity. As a simplification of method, we confirm the efficacy of RNA extraction-free RT-qPCR and saline as an alternative patient sample storage buffer. In addition, amongst potential reagent shortages, it has sometimes been difficult to obtain inactivated viral particles. We have therefore also characterized armored SARS-CoV-2 RNA from Asuragen as an alternative diagnostic standard to ATCC genomic SARS-CoV-2 RNA and heat inactivated virions and provide guidelines for its use in RT-qPCR.

The coronavirus macrodomain is required to prevent PARP-mediated inhibition of virus replication and enhancement of IFN expression.

ADP-ribosylation is a ubiquitous post-translational addition of either monomers or polymers of ADP-ribose to target proteins by ADP-ribosyltransferases, usually by interferon-inducible diphtheria toxin-like enzymes known as PARPs. While several PARPs have known antiviral activities, these activities are mostly independent of ADP-ribosylation. Consequently, less is known about the antiviral effects of ADP-ribosylation. Several viral families, including Coronaviridae, Togaviridae, and Hepeviridae, encode for macrodomain proteins that bind to and hydrolyze ADP-ribose from proteins and are critical for optimal replication and virulence. These results suggest that macrodomains counter cellular ADP-ribosylation, but whether PARPs or, alternatively, other ADP-ribosyltransferases cause this modification is not clear. Here we show that pan-PARP inhibition enhanced replication and inhibited interferon production in primary macrophages infected with macrodomain-mutant but not wild-type coronavirus. Specifically, knockdown of two abundantly expressed PARPs, PARP12 and PARP14, led to increased replication of mutant but did not significantly affect wild-type virus. PARP14 was also important for the induction of interferon in mouse and human cells, indicating a critical role for this PARP in the regulation of innate immunity. In summary, these data demonstrate that the macrodomain is required to prevent PARP-mediated inhibition of coronavirus replication and enhancement of interferon production.

RNA triphosphatase DUSP11 enables exonuclease XRN-mediated restriction of hepatitis C virus.

Seventy percent of people infected with hepatitis C virus (HCV) will suffer chronic infection, putting them at risk for liver disease, including hepatocellular carcinoma. The full range of mechanisms that render some people more susceptible to chronic infection and liver disease is still being elucidated. XRN exonucleases can restrict HCV replication and may help to resolve HCV infections. However, it is unknown how 5' triphosphorylated HCV transcripts, primary products of the viral polymerase, become susceptible to attack by 5' monophosphate-specific XRNs. Here, we show that the 5' RNA triphosphatase DUSP11 acts on HCV transcripts, rendering them susceptible to XRN-mediated attack. Cells lacking DUSP11 show substantially enhanced HCV replication, and this effect is diminished when XRN expression is reduced. MicroRNA-122 (miR-122), a target of current phase II anti-HCV drugs, is known to protect HCV transcripts against XRNs. We show that HCV replication is less dependent on miR-122 in cells lacking DUSP11. Combined, these results implicate DUSP11 as an important component of XRN-mediated restriction of HCV.

Identification of virus-encoded microRNAs in divergent Papillomaviruses.

MicroRNAs (miRNAs) are small RNAs that regulate diverse biological processes including multiple aspects of the host-pathogen interface. Consequently, miRNAs are commonly encoded by viruses that undergo long-term persistent infection. Papillomaviruses (PVs) are capable of undergoing persistent infection, but as yet, no widely-accepted PV-encoded miRNAs have been described. The incomplete understanding of PV-encoded miRNAs is due in part to lack of tractable laboratory models for most PV types. To overcome this, we have developed miRNA Discovery by forced Genome Expression (miDGE), a new wet bench approach to miRNA identification that screens numerous pathogen genomes in parallel. Using miDGE, we screened over 73 different PV genomes for the ability to code for miRNAs. Our results show that most PVs are unlikely to code for miRNAs and we conclusively demonstrate a lack of PV miRNA expression in cancers associated with infections of several high risk HPVs. However, we identified five different high-confidence or highly probable miRNAs encoded by four different PVs (Human PVs 17, 37, 41 and a Fringilla coelebs PV (FcPV1)). Extensive in vitro assays confirm the validity of these miRNAs in cell culture and two FcPV1 miRNAs are further confirmed to be expressed in vivo in a natural host. We show that miRNAs from two PVs (HPV41 & FcPV1) are able to regulate viral transcripts corresponding to the early region of the PV genome. Combined, these findings identify the first canonical PV miRNAs and support that miRNAs of either host or viral origin are important regulators of the PV life cycle.

The Murine Polyomavirus MicroRNA Locus Is Required To Promote Viruria during the Acute Phase of Infection.

Polyomaviruses (PyVs) can cause serious disease in immunosuppressed hosts. Several pathogenic PyVs encode microRNAs (miRNAs), small RNAs that regulate gene expression via RNA silencing. Despite recent advances in understanding the activities of PyV miRNAs, the biological functions of PyV miRNAs during in vivo infections are mostly unknown. The studies presented here used murine polyomavirus (MuPyV) as a model to assess the roles of the PyV miRNAs in a natural host. This analysis revealed that a MuPyV mutant that is unable to express miRNAs has enhanced viral DNA loads in select tissues at late times after infection. This is consistent with the PyV miRNAs functioning to reduce viral replication during the persistent phase of infection in a natural host. Additionally, the MuPyV miRNA locus promotes viruria during the acute phase of infection as evidenced by a defect in shedding during infection with the miRNA mutant virus. The viruria defect of the miRNA mutant virus could be rescued by infecting Rag2-/- mice. These findings implicate the miRNA locus as functioning in both the persistent and acute phases of infection and suggest a role for MuPyV miRNA in evading the adaptive immune response.IMPORTANCE MicroRNAs are expressed by diverse viruses, but for only a few is there any understanding of their in vivo function. PyVs can cause serious disease in immunocompromised hosts. Therefore, increased knowledge of how these viruses interact with the immune response is of clinical relevance. Here we show a novel activity for a viral miRNA locus in promoting virus shedding. This work indicates that in addition to any role for the PyV miRNA locus in long-term persistence, it also has biological activity during the acute phase. As this mutant phenotype is alleviated by infection of mice lacking an adaptive immune response, our work also connects the in vivo activity of the PyV miRNA locus to the immune response. Given that PyV-associated disease is associated with alterations in the immune response, our findings help to better understand how the balance between PyVs and the immune response becomes altered in pathogenic states.

MMTV does not encode viral microRNAs but alters the levels of cancer-associated host microRNAs.

Mouse mammary tumor virus (MMTV) induces breast cancer in mice in the absence of known virally-encoded oncogenes. Tumorigenesis by MMTV is thought to occur primarily through insertional mutagenesis, leading to the activation of cellular proto-oncogenes and outgrowth of selected cells. Here we investigated whether MMTV encodes microRNAs (miRNAs) and/or modulates host miRNAs that could contribute to tumorigenesis. High throughput small RNA sequencing analysis of MMTV-infected cells and MMTV-induced mammary tumors demonstrates that MMTV does not encode miRNAs. However, infected tissues have altered levels of several host miRNAs, including increased expression of members of the oncogenic miRNA cluster, miR-17-92. Notably, similar changes in miRNA levels have been previously reported in human breast cancers. Combined, our results demonstrate that virally encoded miRNAs do not contribute to MMTV-mediated tumorigenesis, but that changes in specific host miRNAs in infected cells may contribute to virus replication and tumor biology.

Expression of short hairpin RNAs using the compact architecture of retroviral microRNA genes.

Short hairpin RNAs (shRNAs) are effective in generating stable repression of gene expression. RNA polymerase III (RNAP III) type III promoters (U6 or H1) are typically used to drive shRNA expression. While useful for some knockdown applications, the robust expression of U6/H1-driven shRNAs can induce toxicity and generate heterogeneous small RNAs with undesirable off-target effects. Additionally, typical U6/H1 promoters encompass the majority of the ∼270 base pairs (bp) of vector space required for shRNA expression. This can limit the efficacy and/or number of delivery vector options, particularly when delivery of multiple gene/shRNA combinations is required. Here, we develop a compact shRNA (cshRNA) expression system based on retroviral microRNA (miRNA) gene architecture that uses RNAP III type II promoters. We demonstrate that cshRNAs coded from as little as 100 bps of total coding space can precisely generate small interfering RNAs (siRNAs) that are active in the RNA-induced silencing complex (RISC). We provide an algorithm with a user-friendly interface to design cshRNAs for desired target genes. This cshRNA expression system reduces the coding space required for shRNA expression by >2-fold as compared to the typical U6/H1 promoters, which may facilitate therapeutic RNAi applications where delivery vector space is limiting.

DUSP11 - An RNA phosphatase that regulates host and viral non-coding RNAs in mammalian cells.

Dual-specificity phosphatase 11 (DUSP11) is a conserved protein tyrosine phosphatase (PTP) in metazoans. The cellular substrates and physiologic activities of DUSP11 remain largely unknown. In nematodes, DUSP11 is required for normal development and RNA interference against endogenous RNAs (endo-RNAi) via molecular mechanisms that are not well understood. However, mammals lack analogous endo-RNAi pathways and consequently, a role for DUSP11 in mammalian RNA silencing was unanticipated. Recent work from our laboratory demonstrated that DUSP11 activity alters the silencing potential of noncanonical viral miRNAs in mammalian cells. Our studies further uncovered direct cellular substrates of DUSP11 and suggest that DUSP11 is part of regulatory pathway that controls the abundance of select triphosphorylated noncoding RNAs. Here, we highlight recent findings and present new data that advance understanding of mammalian DUSP11 during gene silencing and discuss the emerging biological activities of DUSP11 in mammalian cells.

DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs.

RNA silencing is a conserved eukaryotic gene expression regulatory mechanism mediated by small RNAs. In Caenorhabditis elegans, the accumulation of a distinct class of siRNAs synthesized by an RNA-dependent RNA polymerase (RdRP) requires the PIR-1 phosphatase. However, the function of PIR-1 in RNAi has remained unclear. Since mammals lack an analogous siRNA biogenesis pathway, an RNA silencing role for the mammalian PIR-1 homolog (dual specificity phosphatase 11 [DUSP11]) was unexpected. Here, we show that the RNA triphosphatase activity of DUSP11 promotes the RNA silencing activity of viral microRNAs (miRNAs) derived from RNA polymerase III (RNAP III) transcribed precursors. Our results demonstrate that DUSP11 converts the 5' triphosphate of miRNA precursors to a 5' monophosphate, promoting loading of derivative 5p miRNAs into Argonaute proteins via a Dicer-coupled 5' monophosphate-dependent strand selection mechanism. This mechanistic insight supports a likely shared function for PIR-1 in C. elegans Furthermore, we show that DUSP11 modulates the 5' end phosphate group and/or steady-state level of several host RNAP III transcripts, including vault RNAs and Alu transcripts. This study shows that steady-state levels of select noncoding RNAs are regulated by DUSP11 and defines a previously unknown portal for small RNA-mediated silencing in mammals, revealing that DUSP11-dependent RNA silencing activities are shared among diverse metazoans.

Lessons Learned from In Vivo Studies of a Viral Noncoding RNA.

Despite increasing interest in the biology of noncoding RNAs (ncRNAs), few functions have been uncovered for viral ncRNAs in vivo. In their recent article in mSphere, Feldman and colleagues [E. R. Feldman et al., mSphere 1(2):e00105-15, 2016, doi:10.1128/mSphere.00105-15] demonstrate a highly specific activity of a gammaherpesviral ncRNA in viral dissemination and establishment of latent infection. Their work highlights several interesting features that should be informative to future studies of viral ncRNA.

Identification, validation, and characterization of noncanonical miRNAs.

Many eukaryotes and some viruses encode microRNAs (miRNAs), small RNAs that post-transcriptionally regulate gene expression. While most miRNAs are generated through the activity of RNA Polymerase II (RNAP II) and subsequent processing by Drosha and Dicer, some viral miRNAs utilize alternative pathways of biogenesis. Some members of the herpesvirus and retrovirus families can direct synthesis of miRNAs through RNAP III transcription rather than RNAP II and can utilize atypical enzymes to generate miRNAs. Though the advantages of alternative miRNA biogenesis remain unclear for herpesviruses, the retroviral miRNA biogenesis routes allow the RNAP II transcribed retroviral genome to escape Drosha cleavage while still expressing abundant, biologically-active miRNAs. These RNAP III-derived miRNAs have unique characteristics that allow for their identification and characterization. In this article, we describe procedures to predict, validate, and characterize RNAP III-transcribed miRNAs and other small RNAs, while providing resources that are also useful for canonical miRNAs.