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Effector-triggered immunity involves "guarded" host processes that, when perturbed by pathogen factors, prompt a secondary response. A recent study published in Nature by Gaidt et al. demonstrates that MORC3 serves as both the guard and the guarded antiviral host factor-creating a "heads, I win; tails, you lose!" scenario.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that causes both debilitating acute and chronic disease. Previous work has shown that type I interferons (IFNs) play a critical role in limiting CHIKV pathogenesis and that interferon alpha (IFN-α) and interferon beta (IFN-β) control acute CHIKV infection by distinct mechanisms. However, the role of type I IFNs, especially specific subtypes, during chronic CHIKV disease is unclear. To address this gap in knowledge, we evaluated chronic CHIKV pathogenesis in mice lacking IFN-α or IFN-β. We found that IFN-α was the dominant subtype that controls chronic disease. Despite detecting a varying type I IFN response throughout the course of disease, IFN-α acts within the first few days of infection to control the levels of persistent CHIKV RNA. In addition, using a novel CHIKV-3′-Cre tdTomato reporter system that fate maps CHIKV-infected cells, we showed that IFN-α limits the number of cells that survive CHIKV at sites of dissemination, particularly dermal fibroblasts and immune cells. Though myofibers play a significant role in CHIKV disease, they were not impacted by the loss of IFN-α. Our studies highlight that IFN-α and IFN-β play divergent roles during chronic CHIKV disease through events that occur early in infection and that not all cell types are equally dependent on type I IFNs for restricting viral persistence. IMPORTANCE Chikungunya virus (CHIKV) is a reemerging global pathogen with no effective vaccine or antiviral treatment for acute or chronic disease, and the mechanisms underlying chronic disease manifestations remain poorly defined. The significance of our research is in defining IFN-α, but not IFN-β, as an important host regulator of chronic CHIKV pathogenesis that acts within the first 48 hours of infection to limit persistent viral RNA and the number of cells that survive CHIKV infection 1 month post-infection. Loss of IFN-α had a greater impact on immune cells and dermal fibroblasts than myofibers, highlighting the need to delineate cell-specific responses to type I IFNs. Altogether, our work demonstrates that very early events of acute CHIKV infection influence chronic disease. Continued efforts to delineate early host-pathogen interactions may help stratify patients who are at risk for developing chronic CHIKV symptoms and identify therapeutics that may prevent progression to chronic disease altogether.

A recently emerged betacoronavirus, SARS-CoV-2, has led to a global health crisis that calls for the identification of effective therapeutics for COVID-19 disease. Coronavirus papain-like protease (PLpro) is an attractive drug target as it is essential for viral polyprotein cleavage and for deconjugation of ISG15, an antiviral ubiquitin-like protein. We show here that 6-Thioguanine (6-TG) inhibits SARS-CoV-2 PLpro-catalyzed viral polyprotein cleavage and ISG15 deconjugation in cells and inhibits replication of SARS-CoV-2 in Vero-E6 cells and Calu3 cells at submicromolar levels. As a well-characterized FDA-approved orally delivered drug, 6-TG represents a promising therapeutic for COVID-19 and other emerging coronaviruses.One Sentence SummaryA repurposed drug that targets an essential enzymatic activity of SARS-CoV-2 represents a promising COVID-19 therapeutic.

The emergence of SARS-CoV-2 has led to a global health crisis that, in addition to vaccines and immunomodulatory therapies, calls for the identification of antiviral therapeutics. The papain-like protease (PLpro) activity of nsp3 is an attractive drug target as it is essential for viral polyprotein cleavage and for deconjugation of ISG15, an antiviral ubiquitin-like protein. We show here that 6-Thioguanine (6-TG), an orally available and widely available generic drug, inhibits SARS-CoV-2 replication in Vero-E6 cells with an EC50 of approximately 2 μM. 6-TG also inhibited PLpro-catalyzed polyprotein cleavage and de-ISGylation in cells and inhibited proteolytic activity of the purified PLpro domain in vitro. We therefore propose that 6-TG is a direct-acting antiviral that could potentially be repurposed and incorporated into the set of treatment and prevention options for COVID-19., Graphical abstract, Therapeutics; Drugs; Virology

Type I interferons (IFNs) are key mediators of the innate immune response. Although members of this family of cytokines signal through a single shared receptor, biochemical and functional variation exists in response to different IFN subtypes. While previous work has demonstrated that type I IFNs are essential to control infection by chikungunya virus (CHIKV), a globally emerging alphavirus, the contributions of individual IFN subtypes remain undefined. To address this question, we evaluated CHIKV pathogenesis in mice lacking IFN-β (IFN-β knockout [IFN-β-KO] mice or mice treated with an IFN-β-blocking antibody) or IFN-α (IFN regulatory factor 7 knockout [IRF7-KO] mice or mice treated with a pan-IFN-α-blocking antibody). Mice lacking either IFN-α or IFN-β developed severe clinical disease following infection with CHIKV, with a marked increase in foot swelling compared to wild-type mice. Virological analysis revealed that mice lacking IFN-α sustained elevated infection in the infected ankle and in distant tissues. In contrast, IFN-β-KO mice displayed minimal differences in viral burdens within the ankle or at distal sites and instead had an altered cellular immune response. Mice lacking IFN-β had increased neutrophil infiltration into musculoskeletal tissues, and depletion of neutrophils in IFN-β-KO but not IRF7-KO mice mitigated musculoskeletal disease caused by CHIKV. Our findings suggest disparate roles for the IFN subtypes during CHIKV infection, with IFN-α limiting early viral replication and dissemination and IFN-β modulating neutrophil-mediated inflammation. IMPORTANCE Type I interferons (IFNs) possess a range of biological activity and protect against a number of viruses, including alphaviruses. Despite signaling through a shared receptor, there are established biochemical and functional differences among the IFN subtypes. The significance of our research is in demonstrating that IFN-α and IFN-β both have protective roles during acute chikungunya virus (CHIKV) infection but do so by distinct mechanisms. IFN-α limits CHIKV replication and dissemination, whereas IFN-β protects from CHIKV pathogenesis by limiting inflammation mediated by neutrophils. Our findings support the premise that the IFN subtypes have distinct biological activities in the antiviral response.

Influenza B viruses (IBVs) cause annual outbreaks of respiratory illness in humans and are increasingly recognized as a major cause of influenza-associated morbidity and mortality. Studying influenza viruses requires the use of secondary methodologies to identify virus-infected cells. To this end, replication-competent influenza A viruses (IAVs) expressing easily traceable fluorescent proteins have been recently developed. In contrast, similar approaches for IBV are mostly lacking. In this report, we describe the generation and characterization of replication-competent influenza B/Brisbane/60/2008 viruses expressing fluorescent mCherry or GFP fused to the C-terminal of the viral non-structural 1 (NS1) protein. Fluorescent-expressing IBVs display similar growth kinetics and plaque phenotype to wild-type IBV, while fluorescent protein expression allows for the easy identification of virus-infected cells. Without the need of secondary approaches to monitor viral infection, fluorescent-expressing IBVs represent an ideal approach to study the biology of IBV and an excellent platform for the rapid identification and characterization of antiviral therapeutics or neutralizing antibodies using high-throughput screening approaches. Lastly, fluorescent-expressing IBVs can be combined with the recently described reporter-expressing IAVs for the identification of novel therapeutics to combat these two important human respiratory pathogens.

ISG15 is a diubiquitin-like modifier and one of the most rapidly induced genes upon type I interferon stimulation. Hundreds of host proteins and a number of viral proteins have been shown to be ISGylated, and understanding how these modifications affect the interferon response and virus replication has been of considerable interest. ISG15 −/− mice exhibit increased susceptibility to viral infection, and in the case of influenza B virus and vaccinia virus, ISG15 conjugation has been shown to restrict virus replication in vivo . A number of studies have also found that ISG15 is capable of antagonizing replication of some viruses in tissue culture. However, recent findings have demonstrated that ISG15 can protect mice from Chikungunya virus infection without affecting the virus burden. In order to better understand the function of ISG15 in vivo , we characterized the pathogenesis of influenza A virus and Sendai virus in ISG15 −/− mice. We found that ISG15 protects mice from virus induced lethality by a conjugation-dependent mechanism in both of these models. However, surprisingly, we found that ISG15 had minimal effect on virus replication and did not have an obvious role in the modulation of the acute immune response to infection. Instead, we observed an increase in the number of diseased small airways in mice lacking ISG15. This ability of ISG15 to protect mice in a conjugation-dependent, but nonantiviral, manner from respiratory virus infection represents a previously undescribed role for ISG15 and demonstrates the importance of further characterization of ISG15 in vivo . IMPORTANCE It has previously been demonstrated that ISG15 −/− mice are more susceptible to a number of viral infections. Since ISG15 is one of the most strongly induced genes after type I interferon stimulation, analysis of ISG15 function has largely focused on its role as an antiviral molecule during acute infection. Although a number of studies have shown that ISG15 does have a small effect on virus replication in tissue culture, few studies have confirmed this mechanism of protection in vivo . In these studies we have found that while ISG15 −/− mice are more susceptible to influenza A virus and Sendai virus infections, ISGylation does not appear to mediate this protection through the direct inhibition of virus replication or the modulation of the acute immune response. Thus, in addition to showing a novel mode of ISG15 mediated protection from virus infection, this study demonstrates the importance of studying the role of ISG15 in vivo .

SummaryThe host immune system functions constantly to maintain chronic commensal and pathogenic organisms in check. The consequences of these immune responses on host physiology are as yet unexplored, and may have long-term implications in health and disease. We show that chronic viral infection increases epithelial turnover in multiple tissues, and the antiviral cytokines type I interferons (IFNs) mediate this response. Using a murine model with persistently elevated type I IFNs in the absence of exogenous viral infection, the Irgm1−/− mouse, we demonstrate that type I IFNs act through nonepithelial cells, including macrophages, to promote increased epithelial turnover and wound repair. Downstream of type I IFN signaling, the highly related IFN-stimulated genes Apolipoprotein L9a and b activate epithelial proliferation through ERK activation. Our findings demonstrate that the host immune response to chronic viral infection has systemic effects on epithelial turnover through a myeloid-epithelial circuit.

Re-emergence of chikungunya virus, a mosquito-transmitted pathogen, is of serious public health concern. In the past 15 years, after decades of infrequent, sporadic outbreaks, the virus has caused major epidemic outbreaks in Africa, Asia, the Indian Ocean, and more recently the Caribbean and the Americas. Chikungunya virus is mainly transmitted by Aedes aegypti mosquitoes in tropical and subtropical regions, but the potential exists for further spread because of genetic adaptation of the virus to Aedes albopictus, a species that thrives in temperate regions. Chikungunya virus represents a substantial health burden to affected populations, with symptoms that include severe joint and muscle pain, rashes, and fever, as well as prolonged periods of disability in some patients. The inflammatory response coincides with raised levels of immune mediators and infiltration of immune cells into infected joints and surrounding tissues. Animal models have provided insights into disease pathology and immune responses. Although host innate and adaptive responses have a role in viral clearance and protection, they can also contribute to virus-induced immune pathology. Understanding the mechanisms of host immune responses is essential for the development of treatments and vaccines. Inhibitory compounds targeting key inflammatory pathways, as well as attenuated virus vaccines, have shown some success in animal models, including an attenuated vaccine strain based on an isolate from La Reunion incorporating an internal ribosome entry sequence that prevents the virus from infecting mosquitoes and a vaccine based on virus-like particles expressing envelope proteins. However, immune correlates of protection, as well as the safety of prophylactic and therapeutic candidates, are important to consider for their application in chikungunya infections. In this Review, we provide an update on chikungunya virus with regard to its epidemiology, molecular virology, virus-host interactions, immunological responses, animal models, and potential antiviral therapies and vaccines.

To combat emerging coronaviruses, developing safe and efficient platforms to evaluate viral protease activities and the efficacy of protease inhibitors is a high priority. Here, we exploit a biosafety level 2 (BSL-2) chimeric Sindbis virus system to evaluate protease activities and the efficacy of inhibitors directed against the papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus (SARS-CoV), a biosafety level 3 (BSL-3) pathogen. We engineered Sindbis virus to coexpress PLpro and a substrate, murine interferon-stimulated gene 15 (ISG15), and found that PLpro mediates removal of ISG15 (deISGylation) from cellular proteins. Mutation of the catalytic cysteine residue of PLpro or addition of a PLpro inhibitor blocked deISGylation in virus-infected cells. Thus, deISGylation is a marker of PLpro activity. Infection of alpha/beta interferon receptor knockout (IFNAR −/− ) mice with these chimeric viruses revealed that PLpro deISGylation activity removed ISG15-mediated protection during viral infection. Importantly, administration of a PLpro inhibitor protected these mice from lethal infection, demonstrating the efficacy of a coronavirus protease inhibitor in a mouse model. However, this PLpro inhibitor was not sufficient to protect the mice from lethal infection with SARS-CoV MA15, suggesting that further optimization of the delivery and stability of PLpro inhibitors is needed. We extended the chimeric-virus platform to evaluate the papain-like protease/deISGylating activity of Middle East respiratory syndrome coronavirus (MERS-CoV) to provide a small-animal model to evaluate PLpro inhibitors of this recently emerged pathogen. This platform has the potential to be universally adaptable to other viral and cellular enzymes that have deISGylating activities. IMPORTANCE Evaluating viral protease inhibitors in a small-animal model is a critical step in the path toward antiviral drug development. We modified a biosafety level 2 chimeric virus system to facilitate evaluation of inhibitors directed against highly pathogenic coronaviruses. We used this system to demonstrate the in vivo efficacy of an inhibitor of the papain-like protease of severe acute respiratory syndrome coronavirus. Furthermore, we demonstrate that the chimeric-virus system can be adapted to study the proteases of emerging human pathogens, such as Middle East respiratory syndrome coronavirus. This system provides an important tool to rapidly assess the efficacy of protease inhibitors targeting existing and emerging human pathogens, as well as other enzymes capable of removing ISG15 from cellular proteins.

Chikungunya virus (CHIKV) is an alphavirus transmitted by mosquitoes that is known to cause severe arthritis and myositis in affected patients. The ongoing epidemic began in eastern Africa in 2004 and then spread to islands of the Indian Ocean, India, and Southeast Asia, ultimately afflicting millions. During this outbreak, more severe disease manifestations, including fatalities, have been documented. The reasons for this change in pathogenesis are multifactorial but likely include mutations that have arisen in the viral genome which could alter disease pathogenesis. To test this hypothesis, we used a murine model of CHIKV to compare the disease pathogeneses of two recombinant strains of CHIKV, the first derived from the La Reunion outbreak in 2006 (LR2006 OPY1) and the second isolated from Senegal in 1983 (37997). While the two strains exhibited similar growth in mammalian cells in vitro , we observed more severe clinical disease and pathology in mice infected with the LR2006 OPY1 strain of CHIKV, which included prolonged viremia and elevated viral titers and persistence in the muscle, resulting in devastating myonecrosis. Both CHIKV strains infected connective tissue fibroblasts of the muscle, but only the LR2006 OPY1 strain replicated within myofibers in vivo , despite similar growth of the two strains in these cell types in vitro . However, when the 37997 strain was administered directly into muscle, myofiber infection was comparable to that in LR2006 OPY1-infected mice. These results indicate that differences in the ability of the strain of CHIKV to establish infection in myofibers may contribute to the increased disease severity. IMPORTANCE CHIKV is an emerging pathogen that causes significant morbidity. Little is known about the pathogenesis of the disease, and this study suggests that the ability of a recent epidemic strain to infect myofibers results in increased disease severity. Better understanding of how CHIKV causes disease contributes to the ultimate goal of creating therapeutics to alleviate the impact of this debilitating virus.

Viruses have long been studied not only for their pathology and associated disease but also as model systems for understanding cellular and immunological processes. Rodent herpesvirus Peru (RHVP) is a recently characterized rhadinovirus related to murine gammaherpesvirus 68 (MHV68) and Kaposi's sarcoma-associated herpesvirus (KSHV) that establishes acute and latent infection in laboratory mice. RHVP encodes numerous unique proteins that we hypothesize might facilitate host immune evasion during infection. We report here that open reading frame (ORF) R17 encodes a high-affinity chemokine binding protein that broadly recognizes human and murine CC and C chemokines. The interaction of R17 with chemokines is generally characterized by rapid association kinetics, and in the case of CCL3, CCL4, CCL5, CCL24, and XCL1, extremely stable complexes are formed. Functionally, R17 potently inhibited CCL2-driven chemotaxis of the human monocytic cell line THP-1, CCL3-driven chemotaxis of peripheral blood mononuclear cells, and CCL2-mediated calcium flux. Our studies also reveal that R17 binds to glycosaminoglycans (GAGs) in a process dependent upon two BBXB motifs and that chemokine and GAG binding can occur simultaneously at distinct sites. Collectively, these studies suggest that R17 may play a role in RHVP immune evasion through the targeted sabotage of chemokine-mediated immune surveillance.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that acutely causes fever as well as severe joint and muscle pain. Chronic musculoskeletal pain persists in a substantial fraction of patients for months to years after the initial infection, yet we still have a poor understanding of what promotes chronic disease. While replicating virus has not been detected in joint-associated tissues of patients with persistent arthritis nor in various animal models at convalescent time points, viral RNA is detected months after acute infection. To identify the cells that might contribute to pathogenesis during this chronic phase, we developed a recombinant CHIKV that expresses Cre recombinase (CHIKV-3ʹ-Cre). CHIKV-3ʹ-Cre replicated in myoblasts and fibroblasts, and it induced arthritis during the acute phase in mice. Importantly, it also induced chronic disease, including persistent viral RNA and chronic myositis and synovitis similar to wild-type virus. CHIKV-3ʹ-Cre infection of tdTomato reporter mice resulted in a population of tdTomato+ cells that persisted for at least 112 days. Immunofluorescence and flow cytometric profiling revealed that these tdTomato+ cells predominantly were myofibers and dermal and muscle fibroblasts. Treatment with an antibody against Mxra8, a recently defined host receptor for CHIKV, reduced the number of tdTomato+ cells in the chronic phase and diminished the levels of chronic viral RNA, implicating these tdTomato+ cells as the reservoir of chronic viral RNA. Finally, isolation and flow cytometry-based sorting of the tdTomato+ fibroblasts from the skin and ankle and analysis for viral RNA revealed that the tdTomato+ cells harbor most of the persistent CHIKV RNA at chronic time points. Therefore, this CHIKV-3ʹ-Cre and tdTomato reporter mouse system identifies the cells that survive CHIKV infection in vivo and are enriched for persistent CHIKV RNA. This model represents a useful tool for studying CHIKV pathogenesis in the acute and chronic stages of disease., Author summary Chikungunya virus (CHIKV) is spread by mosquitoes and causes severe joint and muscle pain. Approximately 30 to 60 percent of people infected with CHIKV continue to experience joint pain for months to years after the initial infection. However, the cause of this persistent joint pain is unclear, as replicating virus cannot be detected during the chronic phase. We have developed a reporter virus system to permanently mark cells infected by CHIKV. Using this system, we show in mice that marked cells surviving CHIKV infection are present for at least 112 days after initial virus inoculation. The marked cells are a mixture of muscle and skin cells. We also show that treatment of mice with an antibody that blocks receptor engagement and CHIKV infection reduces the number of marked cells in the muscle and skin. Finally, we have demonstrated that surviving tdTomato+ cells contain most of the persistent CHIKV RNA. This reporter virus system represents a useful tool for identifying and isolating cells that harbor chronic viral RNA in order to study chronic disease pathogenesis.

Type I interferons (IFNs) exert their effects through the induction of hundreds of IFN-stimulated genes (ISGs), many of which function by inhibiting viral replication and modulating immune responses. ISG15, a di-ubiquitin-like protein, is one of the most abundantly induced ISGs and is critical for control of certain viral and bacterial infections. Like ubiquitin, ISG15 is covalently conjugated to target proteins. In addition, free unconjugated ISG15 is present both intra- and extracellularly. Although much remains to be learned about conjugated ISG15, even less is known about the 2 free forms of ISG15. This article focuses on the role that ISG15 plays during the host response to pathogen challenge, in particular on the recent observations describing the immunomodulatory properties of free ISG15 and its potential implication in disease pathogenesis.

ISG15 is an interferon (IFN)-α/β-induced ubiquitin-like protein. It exists as a free molecule, intracellularly and extracellularly, and conjugated to target proteins. Studies in mice have demonstrated a role for Isg15 in antiviral immunity. By contrast, human ISG15 was shown to have critical immune functions, but not in antiviral immunity. Namely, free extracellular ISG15 is crucial in IFN-γ-dependent antimycobacterial immunity, while free intracellular ISG15 is crucial for USP18-mediated downregulation of IFN-α/β signalling. Here we describe ISG15-deficient patients who display no enhanced susceptibility to viruses in vivo, in stark contrast to Isg15-deficient mice. Furthermore, fibroblasts derived from ISG15-deficient patients display enhanced antiviral protection, and expression of ISG15 attenuates viral resistance to WT control levels. The species-specific gain-of-function in antiviral immunity observed in ISG15 deficiency is explained by the requirement of ISG15 to sustain USP18 levels in humans, a mechanism not operating in mice., ISG15 is a ubiquitin-like protein which has important immune-related functions in mice and humans. Here the authors demonstrate that, unlike in mice, human ISG15 stabilizes UPS18 and that ISG15-deficient human cells are more resistant to viral infection.

Mycobacterium tuberculosis infection results in 1.5 million deaths annually. Type I interferon (IFN) signaling through its receptor IFNAR correlates with increased severity of disease, although how this increases susceptibility to M. tuberculosis remains uncertain. ISG15 is one of the most highly induced interferon stimulated genes (ISGs) during M. tuberculosis infection. ISG15 functions by conjugation to target proteins (ISGylation), by noncovalent association with intracellular proteins, and by release from the cell. Recent studies indicated that ISG15 can function via conjugation-independent mechanisms to suppress the type I IFN response. These data raised the question of whether ISG15 may have diverse and sometimes opposing functions during M. tuberculosis infection. To address this, we analyzed ISGylation during M. tuberculosis infection and show that ISGylated proteins accumulate following infection in an IFNAR-dependent manner. Type I IFN and ISG15 both play transient roles in promoting bacterial replication. However, as the disease progresses, ISGylation deviates from the overall effect of type I IFN and, ultimately, mice deficient in ISGylation are significantly more susceptible than IFNAR mice. Our data demonstrate that ISGs can both protect against and promote disease and are the first to report a role for ISGylation during M. tuberculosis infection.

The type I interferon system plays a critical role in limiting the spread of viral infection. Viruses induce the production of interferon (IFN), which after binding to the IFN-α/β receptor (IFNAR), and triggering of the JAK/STAT signaling cascade, results in the induction of interferon-stimulated genes (ISGs). These ISGs function to inhibit viral replication and to regulate the host immune response. Among these ISGs, the ubiquitin-like molecule, ISG15, is one of the most strongly induced proteins. Similar to ubiquitin, through an IFN induced conjugation cascade, ISG15 is covalently linked to a variety of cellular proteins, suggesting regulation of different cellular processes. Studies performed over the past several years have shown that ISG15 plays a central role in the host’s antiviral response against many viruses. Mice lacking ISG15 display increased susceptibility to multiple viruses. Furthermore, several viruses have developed immune evasion strategies that directly target the ISG15 pathway. Work is now underway to determine the mechanism by which ISG15 functions as an antiviral molecule, such that therapies targeting this pathway can be developed in the future.

Interferon-stimulated expression and conjugation of the ubiquitin-like modifier ISG15 restricts replication of several viruses. Here, we established complete E1-activating, E2-conjugating, and E3 ligase-dependent expression systems for assaying both human and mouse ISGylation. We confirm that human HerC5, but not human HerC6, has ISG15 E3 ligase activity and identify mouse HerC6 as a bona fide ISG15 E3 ligase. Furthermore, we demonstrate that influenza B virus NS1 protein potently antagonizes human but not mouse ISGylation, a property dependent on B/NS1 binding the N-terminal domain of human but not mouse ISG15. Using chimeric human/mouse ISG15 constructs, we show that the B/NS1:ISG15 interaction is both necessary and sufficient to inhibit ISGylation regardless of the ligation machinery used. Inability to block ISGylation in certain species may contribute to limiting influenza B virus host range.

ISG15 functions as a critical antiviral molecule against influenza virus, with infection inducing both the conjugation of ISG15 to target proteins and production of free ISG15. Here, we report that mice lacking the ISG15 E1 enzyme UbE1L fail to form ISG15 conjugates. Both UbE1L−/−and ISG15−/−mice display increased susceptibility to influenza B virus infection, including non-mouse-adapted strains. Finally, we demonstrate that ISG15 controls influenza B virus infection through its action within radioresistant stromal cells and not bone marrow-derived cells. Thus, the conjugation of ISG15 to target proteins within stromal cells is critical to its activity against influenza virus.

Human noroviruses (HuNoV) are the leading cause of nonbacterial gastroenteritis worldwide. Similar to HuNoV, murine noroviruses (MNV) are enteric pathogens spread via the fecal-oral route and have been isolated from numerous mouse facilities worldwide. Type I and type II interferons (IFN) restrict MNV-1 replication; however, the antiviral effectors impacting MNV-1 downstream of IFN signaling are largely unknown. Studies using dendritic cells, macrophages, and mice deficient in free and conjugated forms of interferon-stimulated gene 15 (ISG15) revealed that ISG15 conjugation contributes to protection against MNV-1 both in vitro and in vivo . ISG15 inhibited a step early in the viral life cycle upstream of viral genome transcription. Directly transfecting MNV-1 RNA into IFN-stimulated mouse embryonic fibroblasts (MEFs) and bone marrow-derived dendritic cells (BMDC) lacking ISG15 conjugates bypassed the antiviral activity of ISG15, further suggesting that ISG15 conjugates restrict the MNV-1 life cycle at the viral entry/uncoating step. These results identify ISG15 as the first type I IFN effector regulating MNV-1 infection both in vitro and in vivo and for the first time implicate the ISG15 pathway in the regulation of early stages of MNV-1 replication. IMPORTANCE Type I IFNs are important in controlling murine norovirus 1 (MNV-1) infections; however, the proteins induced by IFNs that restrict viral growth are largely unknown. This report reveals that interferon-stimulated gene 15 (ISG15) mitigates MNV-1 replication both in vitro and in vivo . In addition, it shows that ISG15 inhibits MNV-1 replication by targeting an early step in the viral life cycle, MNV-1 entry and/or uncoating. These results identify ISG15 as the first type I IFN effector regulating MNV-1 infection both in vitro and in vivo and for the first time implicate the ISG15 pathway in the regulation of viral entry/uncoating.

In this study, we have identified a unique mechanism in which human cytomegalovirus (HCMV) protein pUL79 acts as an elongation factor to direct cellular RNA polymerase II for viral transcription during late times of infection. We and others previously reported that pUL79 and its homologues are required for viral transcript accumulation after viral DNA synthesis. We hypothesized that pUL79 represented a unique mechanism to regulate viral transcription at late times during HCMV infection. To test this hypothesis, we analyzed the proteome associated with pUL79 during virus infection by mass spectrometry. We identified both cellular transcriptional factors, including multiple RNA polymerase II (RNAP II) subunits, and novel viral transactivators, including pUL87 and pUL95, as protein binding partners of pUL79. Co-immunoprecipitation (co-IP) followed by immunoblot analysis confirmed the pUL79-RNAP II interaction, and this interaction was independent of any other viral proteins. Using a recombinant HCMV virus where pUL79 protein is conditionally regulated by a protein destabilization domain ddFKBP, we showed that this interaction did not alter the total levels of RNAP II or its recruitment to viral late promoters. Furthermore, pUL79 did not alter the phosphorylation profiles of the RNAP II C-terminal domain, which was critical for transcriptional regulation. Rather, a nuclear run-on assay indicated that, in the absence of pUL79, RNAP II failed to elongate and stalled on the viral DNA. pUL79-dependent RNAP II elongation was required for transcription from all three kinetic classes of viral genes (i.e. immediate-early, early, and late) at late times during virus infection. In contrast, host gene transcription during HCMV infection was independent of pUL79. In summary, we have identified a novel viral mechanism by which pUL79, and potentially other viral factors, regulates the rate of RNAP II transcription machinery on viral transcription during late stages of HCMV infection., Author Summary In this study, we report a novel mechanism used by human cytomegalovirus (HCMV) to regulate the elongation rate of RNA polymerase II (RNAP II) to facilitate viral transcription during late stages of infection. Recently, we and others have identified several viral factors that regulate gene expression during late infection. These factors are functionally conserved among beta- and gamma- herpesviruses, suggesting a unique transcriptional regulation shared by viruses of these two subfamilies. However, the mechanism remains elusive. Here we show that HCMV pUL79, one of these factors, interacts with RNAP II as well as other viral factors involved in late gene expression. We have started to elucidate the nature of the pUL79-RNAP II interaction, finding that pUL79 does not alter the protein levels of RNAP II or its recruitment to viral promoters. However, during late times of infection, pUL79 helps RNAP II efficiently elongate along the viral DNA template to transcribe HCMV genes. Host genes are not regulated by this pUL79-mediated mechanism. Therefore, our study discovers a previously uncharacterized mechanism where RNAP II activity is modulated by viral factor pUL79, and potentially other viral factors as well, for coordinated viral transcription.

Bone marrow stromal Ag 2 (BST2) is a transmembrane protein that prevents virus release from infected cells. It was also reported that BST2 inhibits type I IFN production by plasmacytoid dendritic cells. To determine BST2 impact on antiviral responses in vivo, we generated BST2−/− mice. Following infection with a murine retrovirus, BST2−/− mice had slightly elevated viral loads; however, infection with other enveloped viruses revealed unexpected roles of BST2. BST2−/− mice showed reduced type I IFN production by plasmacytoid dendritic cells. Moreover, BST2−/− mice had lower viral titers in lungs following intranasal infection with vesicular stomatitis virus expressing OVA and influenza B and increased numbers of virus-specific CD8 T cells in the lungs, suggesting that BST2 may facilitate entry and/or replication of enveloped viruses and modulate priming of CD8 T cells. These findings suggest complex roles of BST2 beyond retroviral control in vivo, possibly reflecting the involvement of BST2 in endocytosis and intracellular trafficking of viruses, viral nucleic acids, and Ags.

Chikungunya virus (CHIKV) is a re-emerging alphavirus that has caused significant disease in the Indian Ocean region since 2005. During this outbreak, in addition to fever, rash and arthritis, severe cases of CHIKV infection have been observed in infants. Challenging the notion that the innate immune response in infants is immature or defective, we demonstrate that both human infants and neonatal mice generate a robust type I interferon (IFN) response during CHIKV infection that contributes to, but is insufficient for, the complete control of infection. To characterize the mechanism by which type I IFNs control CHIKV infection, we evaluated the role of ISG15 and defined it as a central player in the host response, as neonatal mice lacking ISG15 were profoundly susceptible to CHIKV infection. Surprisingly, UbE1L−/− mice, which lack the ISG15 E1 enzyme and therefore are unable to form ISG15 conjugates, displayed no increase in lethality following CHIKV infection, thus pointing to a non-classical role for ISG15. No differences in viral loads were observed between wild-type (WT) and ISG15−/− mice, however, a dramatic increase in proinflammatory cytokines and chemokines was observed in ISG15−/− mice, suggesting that the innate immune response to CHIKV contributes to their lethality. This study provides new insight into the control of CHIKV infection, and establishes a new model for how ISG15 functions as an immunomodulatory molecule in the blunting of potentially pathologic levels of innate effector molecules during the host response to viral infection., Author Summary Type I interferon plays a critical role in the host defense to viral infection. Signaling through the type I IFN receptor allows for the induction of hundreds of interferon stimulated genes (ISGs) that generate an antiviral state within host cells. The ubiquitin-like molecule ISG15 has been shown to play an important role during multiple viral infections, including influenza virus infection. To date, the ability of ISG15 to protect against viral infection has been shown to be dependent on its ability to covalently bind (or conjugate) to target proteins, including the binding of viral proteins. We investigated the importance of the type I interferon response and ISG15 conjugation in a neonatal model of Chikungunya virus infection, a re-emerging human pathogen in the Indian Ocean region. Remarkably, the role of ISG15 during CHIKV infection appears to be conjugation independent, suggesting a non-classical role for ISG15 during viral infection. Our data also suggests that ISG15 plays an immunoregulatory role, as opposed to having direct antiviral function. Our CHIKV model may provide an opportunity to identify a novel mechanism by which ISG15 contributes to the innate immune response to viral infection.

Interferon (IFN)-stimulated gene 15 (ISG15) is a ubiquitin-like molecule that conjugates to target proteins via a C-terminal LRLRGG motif and has antiviral function in vivo. We used structural modeling to predict human ISG15 (hISG15) residues important for interacting with its E1 enzyme, UbE1L. Kinetic analysis revealed that mutation of arginine 153 to alanine (R153A) ablated hISG15-hUbE1L binding and transthiolation of UbcH8. Mutation of other predicted UbE1L-interacting residues had minimal effects on the transfer of ISG15 from UbE1L to UbcH8. The capacity of hISG15 R153A to form protein conjugates in 293T cells was markedly diminished. Mutation of the homologous residue in mouse ISG15 (mISG15), arginine 151, to alanine (R151A) also attenuated protein ISGylation following transfection into 293T cells. We assessed the role of ISG15-UbE1L interactions in control of virus infection by constructing double subgenomic Sindbis viruses that expressed the mISG15 R151A mutant. While expression of mISG15 protected alpha/beta-IFN-receptor-deficient (IFN-αβR−/−) mice from lethality following Sindbis virus infection, expression of mISG15 R151A conferred no survival benefit. The R151A mutation also attenuated ISG15's ability to decrease Sindbis virus replication in IFN-αβR−/−mice or prolong survival of ISG15−/−mice. The importance of UbE1L was confirmed by demonstrating that mice lacking this ISG15 E1 enzyme were highly susceptible to Sindbis virus infection. Together, these data support a role for protein conjugation in the antiviral effects of ISG15.

Type I interferons (IFNs) play an essential role in the host response to viral infection through the induction of numerous IFN-stimulated genes (ISGs), including important antiviral molecules such as PKR, RNase L, Mx, and iNOS. Yet, additional antiviral ISGs likely exist. IFN-stimulated gene 15 (ISG15) is a ubiquitin homolog that is rapidly up-regulated after viral infection, and it conjugates to a wide array of host proteins. Although it has been hypothesized that ISG15 functions as an antiviral molecule, the initial evaluation of ISG15-deficient mice revealed no defects in their responses to vesicular stomatitis virus or lymphocytic choriomeningitis virus, leaving open the important question of whether ISG15 is an antiviral moleculein vivo. Here we demonstrate that ISG15 is critical for the host response to viral infection. ISG15−/−mice are more susceptible to influenza A/WSN/33 and influenza B/Lee/40 virus infections. ISG15−/−mice also exhibited increased susceptibility to both herpes simplex virus type 1 and murine gammaherpesvirus 68 infection and to Sindbis virus infection. The increased susceptibility of ISG15−/−mice to Sindbis virus infection was rescued by expressing wild-type ISG15, but not a mutant form of ISG15 that cannot form conjugates, from the Sindbis virus genome. The demonstration of ISG15 as a novel antiviral molecule with activity against both RNA and DNA viruses provides a target for the development of therapies against important human pathogens.

The innate immune response, and in particular the alpha/beta interferon (IFN-α/β) system, plays a critical role in the control of viral infections. Interferons α and β exert their antiviral effects through the induction of hundreds of interferon-induced (or -stimulated) genes (ISGs). While several of these ISGs have characterized antiviral functions, their actions alone do not explain all of the effects mediated by IFN-α/β. To identify additional IFN-induced antiviral molecules, we utilized a recombinant chimeric Sindbis virus to express selected ISGs in IFN-α/β receptor (IFN-α/βR)−/−mice and looked for attenuation of Sindbis virus infection. Using this approach, we identified a ubiquitin homolog, interferon-stimulated gene 15 (ISG15), as having antiviral activity. ISG15 expression protected against Sindbis virus-induced lethality and decreased Sindbis virus replication in multiple organs without inhibiting the spread of virus throughout the host. We establish that, much like ubiquitin, ISG15 requires its C-terminal LRLRGG motif to form intracellular conjugates. Finally, we demonstrate that ISG15's LRLRGG motif is also required for its antiviral activity. We conclude that ISG15 can be directly antiviral.

SummaryUbiquitin (Ub) and interferon-stimulated gene product 15 (ISG15) reversibly conjugate to proteins and mediate important innate antiviral responses. The ovarian tumor (OTU) domain represents a superfamily of predicted proteases found in eukaryotic, bacterial, and viral proteins, some of which have Ub-deconjugating activity. We show that the OTU domain-containing proteases from nairoviruses and arteriviruses, two unrelated groups of RNA viruses, hydrolyze Ub and ISG15 from cellular target proteins. This broad activity contrasts with the target specificity of known mammalian OTU domain-containing proteins. Expression of a viral OTU domain-containing protein antagonizes the antiviral effects of ISG15 and enhances susceptibility to Sindbis virus infection in vivo. We also show that viral OTU domain-containing proteases inhibit NF-κB-dependent signaling. Thus, the deconjugating activity of viral OTU proteases represents a unique viral strategy to inhibit Ub- and ISG15-dependent antiviral pathways.