ISG 15 functions as an interferon-mediated antiviral effector early in the murine norovirus life cycle Running title : ISG 15 is an antiviral effector during MNV-1 infection

Human noroviruses (HuNoV) are the leading cause of non-bacterial 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 viral 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 states of MNV-1 replication. on July 8, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Importance Type I IFNs (IFNs) are important in controlling murine norovirus (MNV-1) infections; however, the proteins induced by IFNs that restrict viral growth are largely unknown. This study reveals that the interferon-stimulated gene (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. on July 8, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Introduction Human noroviruses (HuNoV) are enteric pathogens responsible for the majority of epidemic, non-bacterial gastroenteritis worldwide (rober1, 2, 3). In the United States alone, approximately 23 million people a year are infected with HuNoV where most outbreaks occur in day care settings, nursing homes, cruise ships and at catered events (2-4). HuNoV transmission occurs predominantly via the fecal-oral route by ingestion of contaminated foods or water (3). Following a 12-48 hour incubation period, HuNoV infection leads to a multitude of symptoms including explosive vomiting and diarrhea, low-grade fever, headache, chills and malaise (2, 4). Little is known about the molecular mechanisms mediating HuNoV pathogenesis, as there is no immunocompetent small animal or tissue culture system of HuNoV infection to date. The discovery of murine noroviruses (MNV-1), the establishment of an MNV-1 animal model, as well as a tissue culture system for MNV-1 has significantly broadened our understanding of host and viral determinants of NoV pathogenesis (5-15). Murine noroviruses are positive-stranded, encapsidated viruses which enter cells using a pH-independent, cholesterol and dynamin II-dependent endocytic pathway (16, 17). MNV-1 is sensitive to the antiviral effects of both type I and type II interferons (IFN), as mice lacking IFN alpha, beta and gamma signaling succumb to MNV-1 infection (5, 7, 11-13). In tissue culture, MNV-1 replication in dendritic cells and macrophages are also sensitive to the antiviral effects of type I and type II IFN (7, 13, 18). In macrophages, type II IFN-mediated on July 8, 2017 by gest http/jvi.asm .rg/ D ow nladed fom anti-MNV-1 defenses require STAT1, the autophagy protein complex Atg5Atg12/Atg16L and the transcription factor interferon regulatory factor 1 (IRF-1) (11, 13). However, neither IRF-1 nor the Atg5-Atg12/ATG16L complex is required for type I IFN-mediated restriction of MNV-1 replication (11, 13). The type I IFN-mediated effectors that restrict MNV-1 replication are poorly characterized. Previous studies have shown that the transcription factors IRF3 and IRF-7 along with the viral sensor melanoma differentiation associated gene 5 (MDA5) play a critical role in inducing the type I IFN antiviral response against MNV-1 (12, 15, 19). Furthermore, the actions of type I IFN in bone marrow derived dendritic cells (BMDC) is at least partially dependent upon PKR, RNAse L and/or Mx-1 as the effect of IFN treatment on triply deficient cells was less robust than in WT cells (18). While the type I IFN effectors that restrict MNV1 replication are not well defined, utilization of IFNα/βR conditional knockout mice revealed that type I IFN responses in dendritic cells and macrophages/neutrophils mitigate acute MNV-1 replication in vivo (12). Therefore, identifying additional IFN effectors in dendritic cells and macrophages will further our understanding of type I IFN-mediated regulation of MNV-1. ISG15 is an ubiquitin-like protein strongly induced by type I IFN, which functions as both an antiviral and immunoregulatory molecule. Mice deficient in ISG15 are susceptible to numerous viruses including Influenza A and B, HSV-1, Sindbis and Chikungunya virus (CHIKV) (20-24). Consisting of two ubiquitin-like on July 8, 2017 by gest http/jvi.asm .rg/ D ow nladed fom domains separated by a peptide linker, ISG15 can covalently associate with numerous target proteins (ISGylation) via its C-terminal LRLRGG motif. It utilizes an E1 (UbE1L), E2 (UbcM6, UbcM8) and E3 (Herc5, Herc6, HHARI, TRIM25) dependent conjugation cascade (25-30), of which all members are induced by type I IFN. Similar to ubiquitination, ISGylation is a reversible process yielding unconjugated “free” forms of ISG15 found both inside and outside the cell (3134). Recent work has shown that, depending on the virus, ISG15 conjugation to target proteins and the actions of free ISG15 can suppress viral replication and regulate host responses to infection (20-24). While ISG15 has been implicated in type I IFN-dependent antiviral responses against numerous viruses, its mechanism(s) are not well defined. However, one study showed that ISG15 conjugation can inhibit Influenza A protein expression in virally infected human cells (35). Studies using human immunodeficiency virus (HIV), Ebola (EBOV) virus like particles (VLPs), and influenza A virus implicate ISG15 in disruption of viral egress (36-39). ISG15 overexpression was shown to inhibit HIV Gag and Tsg101 ubiquitination, which is important for Gag:Tsg101 association and subsequent release of infectious virions from the cell (37). Tsg101 is also important for influenza A trafficking. Recent studies have shown that influenza A hemagglutinin transport is inhibited by type I IFNs in a Tsg101and ISG15-dependent manner (39). Moreover, ISG15 was found to inhibit ubiquitination mediated by the Nedd4 ubiquitin ligase (36, 38). Disruption of Nedd4 activity inhibited EBOV VP40 ubiquitination and on July 8, 2017 by gest http/jvi.asm .rg/ D ow nladed fom was shown to disrupt EBOV VLP release. These studies demonstrate that ISG15 can act as an antiviral effector capable of disrupting viral protein translation and viral egress. In this study, we identify ISG15 as a type I IFN-mediated effector molecule that restricts MNV-1 replication early in the viral life cycle. Utilizing mice and primary cells deficient in ISG15 and/or UbE1L, we showed that the ISG15 pathway contributes to type I IFN-mediated control of MNV-1. In both ISG15 and UbE1L cells treated with IFN, we observed increased MNV-1 replication compared to WT cells indicating that ISG15 conjugation mediates this antiviral activity. ISG15 and UbE1L mice infected with MNV-1 also displayed increased viral replication early during the course of the infection. Further analysis revealed that ISG15 conjugation inhibited an early step in the MNV-1 life cycle, preceding genome transcription. The ability to bypass this inhibition following the transfection of MNV-1 RNA into mouse embryonic fibroblast (MEFs) and BMDC implicate that the MNV-1 entry/uncoating step in the life cycle is being inhibited by ISG15. These results identify ISG15 as an IFN effector protein regulating MNV-1 pathogenesis a step preceding genome replication. on July 8, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Materials and Methods Mice. Mice were bred and housed at Washington University School of Medicine in accordance with all federal and university guidelines, under specificpathogen-free conditions (40). Wild type C57BL/6 mice (Catalog no. 000664) were purchased from Jackson laboratory (Bar Harbor, ME), bred and maintained in our facilities. ISG15 mice (provided by Dr. Klaus-Peter Knobeloch; University Clinic Freiburg, Germany) and UbE1L mice (provided by Dr. Dong-Er Zhang; University of California San Diego School of Medicine) were generated as previously described (41, 42). UbE1L and ISG15 mice were fully backcrossed [(>99% identity to C57BL/6; by congenic SNP analysis through Taconic Laboratories (Hudson, NY)]. ISG15 x UbE1L (DKO) mice were generated by breeding ISG15 and UbE1L mice to obtain F1 progeny, which were then brother and sister mated. Cells. Bone marrow derived macrophages (BMM) were generated as described previously (13) using BMM media supplemented with 2% supernatant derived from CMG14-12 cells, as a source of M-CSF (43). Bone marrow derived dendritic cells (BMDC) were produced by flushing bone marrow from femurs and lysing cells in RBC lysis buffer (Sigma, Saint Louis). Cells were seeded at 9 x 10 cells in T175 flasks and cultured for seven days in BMDC media and 2% supernatant derived from GM-CSF producing cells, which were a gift from Marco Colonna (Washington University, St. Louis, MO; (44). Cells were fed on day 4 and were harvested and plated for experiments on day 7. BMDC were rested for on July 8, 2017 by gest http/jvi.asm .rg/ D ow nladed fom 24hr