Murine Coronavirus Infection Activates the Aryl Hydrocarbon Receptor in an Indoleamine 2,3-Dioxygenase-Independent Manner, Contributing to Cytokine Modulation and Proviral TCDD-Inducible-PARP Expression.

The aryl hydrocarbon receptor (AhR) is a cytoplasmic receptor/transcription factor that modulates several cellular and immunological processes following activation by pathogen-associated stimuli, though its role during virus infection is largely unknown. Here, we show that AhR is activated in cells infected with mouse hepatitis virus (MHV), a coronavirus (CoV), and contributes to the upregulation of downstream effector TCDD-inducible poly(ADP-ribose) polymerase (TiPARP) during infection. Knockdown of TiPARP reduced viral replication and increased interferon expression, suggesting that TiPARP functions in a proviral manner during MHV infection. We also show that MHV replication induced the expression of other genes known to be downstream of AhR in macrophages and dendritic cells and in livers of infected mice. Further, we found that chemically inhibiting or activating AhR reciprocally modulated the expression levels of cytokines induced by infection, specifically, interleukin 1ß (IL-1ß), IL-10, and tumor necrosis factor alpha (TNF-α), consistent with a role for AhR activation in the host response to MHV infection. Furthermore, while indoleamine 2,3-dioxygenase (IDO1) drives AhR activation in other settings, MHV infection induced equal expression of downstream genes in wild-type (WT) and IDO1-/- macrophages, suggesting an alternative pathway of AhR activation. In summary, we show that coronaviruses elicit AhR activation by an IDO1-independent pathway, contributing to upregulation of downstream effectors, including the proviral factor TiPARP, and to modulation of cytokine gene expression, and we identify a previously unappreciated role for AhR signaling in CoV pathogenesis.IMPORTANCE Coronaviruses are a family of positive-sense RNA viruses with human and agricultural significance. Characterizing the mechanisms by which coronavirus infection dictates pathogenesis or counters the host immune response would provide targets for the development of therapeutics. Here, we show that the aryl hydrocarbon receptor (AhR) is activated in cells infected with a prototypic coronavirus, mouse hepatitis virus (MHV), resulting in the expression of several effector genes. AhR is important for modulation of the host immune response to MHV and plays a role in the expression of TiPARP, which we show is required for maximal viral replication. Taken together, our findings highlight a previously unidentified role for AhR in regulating coronavirus replication and the immune response to the virus.

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.

The coronavirus nucleocapsid protein is ADP-ribosylated.

ADP-ribosylation is a common post-translational modification, although how it modulates RNA virus infection is not well understood. While screening for ADP-ribosylated proteins during coronavirus (CoV) infection, we detected a ~55kDa ADP-ribosylated protein in mouse hepatitis virus (MHV)-infected cells and in virions, which we identified as the viral nucleocapsid (N) protein. The N proteins of porcine epidemic diarrhea virus (PEDV), severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV were also ADP-ribosylated. ADP-ribosylation of N protein was also observed in cells exogenously expressing N protein by transduction using Venezuelan equine encephalitis virus replicon particles (VRPs). However, plasmid-derived N protein was not ADP-ribosylated following transient transfection but was ADP-ribosylated after MHV infection, indicating that this modification requires virus infection. In conclusion, we have identified a novel post-translation modification of the CoV N protein that may play a regulatory role for this important structural protein.

Selective Packaging in Murine Coronavirus Promotes Virulence by Limiting Type I Interferon Responses.

Selective packaging is a mechanism used by multiple virus families to specifically incorporate genomic RNA (gRNA) into virions and exclude other types of RNA. Lineage A betacoronaviruses incorporate a 95-bp stem-loop structure, the packaging signal (PS), into the nsp15 locus of ORF1b that is both necessary and sufficient for the packaging of RNAs. However, unlike other viral PSs, where mutations generally resulted in viral replication defects, mutation of the coronavirus (CoV) PS results in large increases in subgenomic RNA packaging with minimal effects on gRNA packaging in vitro and on viral titers. Here, we show that selective packaging is also required for viral evasion of the innate immune response and optimal pathogenicity. We engineered two distinct PS mutants in two different strains of murine hepatitis virus (MHV) that packaged increased levels of subgenomic RNAs, negative-sense genomic RNA, and even cellular RNAs. All PS mutant viruses replicated normally in vitro but caused dramatically reduced lethality and weight loss in vivo PS mutant virus infection of bone marrow-derived macrophages resulted in increased interferon (IFN) production, indicating that the innate immune system limited the replication and/or pathogenesis of PS mutant viruses in vivo PS mutant viruses remained attenuated in MAVS-/- and Toll-like receptor 7-knockout (TLR7-/-) mice, two well-known RNA sensors for CoVs, but virulence was restored in interferon alpha/beta receptor-knockout (IFNAR-/-) mice or in MAVS-/- mice treated with IFNAR-blocking antibodies. Together, these data indicate that coronaviruses promote virulence by utilizing selective packaging to avoid innate immune detection.IMPORTANCE Coronaviruses (CoVs) produce many types of RNA molecules during their replication cycle, including both positive- and negative-sense genomic and subgenomic RNAs. Despite this, coronaviruses selectively package only positive-sense genomic RNA into their virions. Why CoVs selectively package their genomic RNA is not clear, as disruption of the packaging signal in MHV, which leads to loss of selective packaging, does not affect genomic RNA packaging or virus replication in cultured cells. This contrasts with other viruses, where disruption of selective packaging generally leads to altered replication. Here, we demonstrate that in the absence of selective packaging, the virulence of MHV was significantly reduced. Importantly, virulence was restored in the absence of interferon signaling, indicating that selective packaging is a mechanism used by CoVs to escape innate immune detection.