Joseph M. Luna

EBV and human microRNAs co-target oncogenic and apoptotic viral and human genes during latency.

Epstein–Barr virus (EBV) controls gene expression to transform human B cells and maintain viral latency. High‐throughput sequencing and crosslinking immunoprecipitation (HITS‐CLIP) identified mRNA targets of 44 EBV and 310 human microRNAs (miRNAs) in Jijoye (Latency III) EBV‐transformed B cells. While 25% of total cellular miRNAs are viral, only three viral mRNAs, all latent transcripts, are targeted. Thus, miRNAs do not control the latent/lytic switch by targeting EBV lytic genes. Unexpectedly, 90% of the 1664 human 3′‐untranslated regions targeted by the 12 most abundant EBV miRNAs are also targeted by human miRNAs via distinct binding sites. Half of these are targets of the oncogenic miR‐17∼92 miRNA cluster and associated families, including mRNAs that regulate transcription, apoptosis, Wnt signalling, and the cell cycle. Reporter assays confirmed the functionality of several EBV and miR‐17 family miRNA‐binding sites in EBV latent membrane protein 1 (LMP1), EBV BHRF1, and host CAPRIN2 mRNAs. Our extensive list of EBV and human miRNA targets implicates miRNAs in the control of EBV latency and illuminates viral miRNA function in general.

miRNA–target chimeras reveal miRNA 3′-end pairing as a major determinant of Argonaute target specificity

microRNAs (miRNAs) act as sequence-specific guides for Argonaute (AGO) proteins, which mediate posttranscriptional silencing of target messenger RNAs. Despite their importance in many biological processes, rules governing AGO–miRNA targeting are only partially understood. Here we report a modified AGO HITS-CLIP strategy termed CLEAR (covalent ligation of endogenous Argonaute-bound RNAs)-CLIP, which enriches miRNAs ligated to their endogenous mRNA targets. CLEAR-CLIP mapped ∼130,000 endogenous miRNA–target interactions in mouse brain and ∼40,000 in human hepatoma cells. Motif and structural analysis define expanded pairing rules for over 200 mammalian miRNAs. Most interactions combine seed-based pairing with distinct, miRNA-specific patterns of auxiliary pairing. At some regulatory sites, this specificity confers distinct silencing functions to miRNA family members with shared seed sequences but divergent 3′-ends. This work provides a means for explicit biochemical identification of miRNA sites in vivo, leading to the discovery that miRNA 3′-end pairing is a general determinant of AGO binding specificity.

Mouse models of acute and chronic hepacivirus infection

New York City rats provide a gift to virologists Despite the development of curative drugs for hepatitis C virus (HCV) infection, global eradication of HCV will likely require a prophylactic vaccine. Progress toward a vaccine has been impeded by the absence of mouse models suitable for studying the immune response to HCV. Billerbeck et al. found that a HCV-related virus isolated from New York City rats produces an infection in laboratory mice that shares several immunological features with human infections (see the Perspective by Klenerman and Barnes). Their initial analyses of the infected mice revealed that acute clearance of the virus was dependent on T cells but not on natural killer cells. Science, this issue p. 204; see also p. 129 A mouse model may provide mechanistic insights into the immune response to hepatitis C virus infection. An estimated 71 million people worldwide are infected with hepatitis C virus (HCV). The lack of small-animal models has impeded studies of antiviral immune mechanisms. Here we show that an HCV-related hepacivirus discovered in Norway rats can establish high-titer hepatotropic infections in laboratory mice with immunological features resembling those seen in human viral hepatitis. Whereas immune-compromised mice developed persistent infection, immune-competent mice cleared the virus within 3 to 5 weeks. Acute clearance was T cell dependent and associated with liver injury. Transient depletion of CD4+ T cells before infection resulted in chronic infection, characterized by high levels of intrahepatic regulatory T cells and expression of inhibitory molecules on intrahepatic CD8+ T cells. Natural killer cells controlled early infection but were not essential for viral clearance. This model may provide mechanistic insights into hepatic antiviral immunity, a prerequisite for the development of HCV vaccines.

miRNA independent hepacivirus variants suggest a strong evolutionary pressure to maintain miR-122 dependence

Hepatitis C virus (HCV) requires the liver specific micro-RNA (miRNA), miR-122, to replicate. This was considered unique among RNA viruses until recent discoveries of HCV-related hepaciviruses prompting the question of a more general miR-122 dependence. Among hepaciviruses, the closest known HCV relative is the equine non-primate hepacivirus (NPHV). Here, we used Argonaute cross-linking immunoprecipitation (AGO-CLIP) to confirm AGO binding to the single predicted miR-122 site in the NPHV 5’UTR in vivo. To study miR-122 requirements in the absence of NPHV-permissive cell culture systems, we generated infectious NPHV/HCV chimeric viruses with the 5’ end of NPHV replacing orthologous HCV sequences. These chimeras were viable even in cells lacking miR-122, although miR-122 presence enhanced virus production. No other miRNAs bound this region. By random mutagenesis, we isolated HCV variants partially dependent on miR-122 as well as robustly replicating NPHV/HCV variants completely independent of any miRNAs. These miRNA independent variants even replicate and produce infectious particles in non-hepatic cells after exogenous delivery of apolipoprotein E (ApoE). Our findings suggest that miR-122 independent HCV and NPHV variants have arisen and been sampled during evolution, yet miR-122 dependence has prevailed. We propose that hepaciviruses may use this mechanism to guarantee liver tropism and exploit the tolerogenic liver environment to avoid clearance and promote chronicity.

Viral genome imaging of hepatitis C virus to probe heterogeneous viral infection and responses to antiviral therapies

Hepatitis C virus (HCV) is a positive single-stranded RNA virus of enormous global health importance, with direct-acting antiviral therapies replacing an immunostimulatory interferon-based regimen. The dynamics of HCV positive and negative-strand viral RNAs (vRNAs) under antiviral perturbations have not been studied at the single-cell level, leaving a gap in our understanding of antiviral kinetics and host-virus interactions. Here, we demonstrate quantitative imaging of HCV genomes in multiple infection models, and multiplexing of positive and negative strand vRNAs and host antiviral RNAs. We capture the varying kinetics with which antiviral drugs with different mechanisms of action clear HCV infection, finding the NS5A inhibitor daclatasvir to induce a rapid decline in negative-strand viral RNAs. We also find that the induction of host antiviral genes upon interferon treatment is positively correlated with viral load in single cells. This study adds smFISH to the toolbox available for analyzing the treatment of RNA virus infections.

Mopping up miRNA: An integrated HBV transcript disrupts liver homeostasis by sequestering miR-122

As the third leading cause of global cancer deaths, hepatocellular carcinoma (HCC) remains a significant health burden exacerbated by numerous routes to disease. Among the largest risk factors for HCC progression are chronic infections with hepatitis B (HBV) or hepatitis C (HCV) viruses. While the mechanisms underlying HCC development and progression due to these viruses are not completely understood, they are presumed attributable to similar programs of immune-mediated chronic inflammation. As a result, much work has focused on commonalities that may influence carcinogenic progression, such as alterations in known oncogenic pathways elicited by viral gene products, the role of viral replication in promoting inflammation, and the cascade of robust or blunted innate immune responses in response to each virus [1,2]. At the same time, the vastly contrasting lifecycles between HBV and HCV has revealed unique cellular requirements for each virus where many open questions remain as to how these differences specifically contribute to disease progression. For example, the unique role of ER-derived membranous structures for HCV RNA replication can be contrasted with the role of nuclear DNA repair for HBV cccDNA synthesis. The duality between the broad pathogenic similarities amidst stark molecular differences for HBV and HCV is perhaps best highlighted by miR-122, a cellular microRNA (miRNA) implicated in both virus lifecycles but in apparently opposing ways (Fig. 1). miRNAs are small (21–22 nt) non-coding RNAs that post-transcriptionally regulate gene expression by repressing specific mRNA targets. As the predominant miRNA in the liver, miR-122 regulates numerous hepatic processes such as cholesterol and lipid metabolism, circadian rhythm control, and mitochondrial function [3–5]. Strikingly, miR-122 is an essential host factor

Present and not reporting for duty: dsRNAi in mammalian cells

Double‐stranded RNA interference (dsRNAi) represents a primary means of anti‐viral defense in plants, worms, and insects, yet appears mostly supplanted by the protein‐based interferon (IFN) response in vertebrates such as mammals. The degree to which dsRNAi is anti‐viral in mammals has been contentious. Maillard et al ( ) find that dsRNAi retains sequence‐specific silencing in mammalian cells incapable of triggering an IFN response, suggesting that dsRNAi is inhibited by the action of interferon‐stimulated genes. Importantly, they observe that while dsRNA can “vaccinate” against the incoming cognate virus though dsRNAi silencing, no dsRNAi is observed with viral infection alone, suggesting that this evolutionarily conserved anti‐viral pathway is present but functionally elusive in the cell types studied thus far.

Global mapping of miRNA-target interactions in cattle ( Bos taurus )

With roles in development, cell proliferation and disease, micro-RNA (miRNA) biology is of great importance and a potential therapeutic target. Here we used cross-linking immunoprecipitation (CLIP) and ligation of miRNA-target chimeras on the Argonaute (AGO) protein to globally map miRNA interactions in the cow. The interactome is the deepest reported to date. miRNA targeting principles are consistent with observations in other species, but with expanded pairing rules. Experimental mapping robustly predicted functional miR-17 regulatory sites. From miRNA-specific targeting for >5000 mRNAs we determined gene ontologies (GO). This confirmed repression of genes important for embryonic development and cell cycle progress by the let-7 family, and repression of those involved in cell cycle arrest by the miR-17 family, but also suggested a number of unappreciated miRNA functions. Our results provide a significant resource for understanding of bovine and species-conserved miRNA regulation, and demonstrate the power of experimental methods for establishing comprehensive interaction maps.

Male germ cells support long-term propagation of Zika virus

Evidence of male-to-female sexual transmission of Zika virus (ZIKV) and viral RNA in semen and sperm months after infection supports a potential role for testicular cells in ZIKV propagation. Here, we demonstrate that germ cells (GCs) are most susceptible to ZIKV. We found that only GCs infected by ZIKV, but not those infected by dengue virus and yellow fever virus, produce high levels of infectious virus. This observation coincides with decreased expression of interferon-stimulated gene Ifi44l in ZIKV-infected GCs, and overexpression of Ifi44l results in reduced ZIKV production. Using primary human testicular tissue, we demonstrate that human GCs are also permissive for ZIKV infection and production. Finally, we identified berberine chloride as a potent inhibitor of ZIKV infection in both murine and human testes. Together, these studies identify a potential cellular source for propagation of ZIKV in testes and a candidate drug for preventing sexual transmission of ZIKV.Zika virus (ZIKV) can persist for months in semen and sperm. Here, the authors show that germ cells, compared to other cell types in the reproductive tract, are most susceptible to ZIKV and produce high levels of progeny virus, which coincides with decreased expression of the interferon-stimulated gene Ifi44l.

End game: Getting the most out of microRNAs

Viruses are notorious for their ability to usurp cellular pathways for their own benefit, often by exploiting host factors in new and unusual ways. Hepatitis C virus (HCV), a causative agent of chronic liver disease, is no exception. As well as using host proteins and lipids, the virus is known to enlist an abundant liver-specific microRNA, miR-122, to aid in its replication (1). MicroRNAs are short (≈22-nt) sequences that typically bind, through complementary ≈6-nt “seed” sites, to the 3′ noncoding regions (NCRs) of certain cellular mRNAs. By recruiting the RNA-induced silencing factor complex (RISC), microRNA binding generally leads to translational suppression and/or degradation of the target transcript. HCV interactions with microRNAs, however, seem to defy all conventions. miR-122 binds the viral genome at not one but two sites. These tandem target sequences are located in the 5′ NCR rather than at the 3′ end, and recruitment of miR-122 does not repress translation but enhances viral replication through as-yet-unclear mechanisms. In PNAS, Machlin et al. (2) uncover a further unique feature of the miR-122–HCV association. Their work reveals that miR-122 binds the viral genome in a complex structure that requires not only the seed sequence but also functionally important associations beyond the seed site. The complex RNA interaction seems to be indispensable for HCV replication but is not required for the actions of miR-122 on cellular mRNAs. This model opens up new potential mechanisms for miR-122 in the HCV life cycle.