Activation of RNase L by Murine Coronavirus in Myeloid Cells Is Dependent on Basal Oas Gene Expression and Independent of Virus-Induced Interferon.

UNLABELLED: The oligoadenylate synthetase (OAS)-RNase L pathway is a potent interferon (IFN)-induced antiviral activity. Upon sensing double-stranded RNA, OAS produces 2',5'-oligoadenylates (2-5A), which activate RNase L. Murine coronavirus (mouse hepatitis virus [MHV]) nonstructural protein 2 (ns2) is a 2',5'-phosphodiesterase (PDE) that cleaves 2-5A, thereby antagonizing RNase L activation. PDE activity is required for robust replication in myeloid cells, as a mutant of MHV (ns2(H126R)) encoding an inactive PDE fails to antagonize RNase L activation and replicates poorly in bone marrow-derived macrophages (BMM), while ns2(H126R) replicates to high titer in several types of nonmyeloid cells, as well as in IFN receptor-deficient (Ifnar1(-/-)) BMM. We reported previously that myeloid cells express significantly higher basal levels of OAS transcripts than nonmyeloid cells. Here, we investigated the contributions of Oas gene expression, basal IFN signaling, and virus-induced IFN to RNase L activation. Infection with ns2(H126R) activated RNase L in Ifih1(-/-) BMM to a similar extent as in wild-type (WT) BMM, despite the lack of IFN induction in the absence of MDA5 expression. However, ns2(H126R) failed to induce RNase L activation in BMM treated with IFNAR1-blocking antibody, as well as in Ifnar1(-/-) BMM, both expressing low basal levels of Oas genes. Thus, activation of RNase L does not require virus-induced IFN but rather correlates with adequate levels of basal Oas gene expression, maintained by basal IFN signaling. Finally, overexpression of RNase L is not sufficient to compensate for inadequate basal OAS levels. IMPORTANCE: The oligoadenylate synthetase (OAS)-RNase L pathway is a potent antiviral activity. Activation of RNase L during murine coronavirus (mouse hepatitis virus [MHV]) infection of myeloid cells correlates with high basal Oas gene expression and is independent of virus-induced interferon secretion. Thus, our data suggest that cells with high basal Oas gene expression levels can activate RNase L and thereby inhibit virus replication early in infection upon exposure to viral double-stranded RNA (dsRNA) before the induction of interferon and prior to transcription of interferon-stimulated antiviral genes. These findings challenge the notion that activation of the OAS-RNase L pathway requires virus to induce type I IFN, which in turn upregulates OAS gene expression, as well as to provide dsRNA to activate OAS. Our data further suggest that myeloid cells may serve as sentinels to restrict viral replication, thus protecting other cell types from infection.

MDA5 Is Critical to Host Defense during Infection with Murine Coronavirus.

UNLABELLED: Infection with the murine coronavirus mouse hepatitis virus (MHV) activates the pattern recognition receptors melanoma differentiation-associated gene 5 (MDA5) and Toll-like receptor 7 (TLR7) to induce transcription of type I interferon. Type I interferon is crucial for control of viral replication and spread in the natural host, but the specific contributions of MDA5 signaling to this pathway as well as to pathogenesis and subsequent immune responses are largely unknown. In this study, we use MHV infection of the liver as a model to demonstrate that MDA5 signaling is critically important for controlling MHV-induced pathology and regulation of the immune response. Mice deficient in MDA5 expression (MDA5(-/-) mice) experienced more severe disease following MHV infection, with reduced survival, increased spread of virus to additional sites of infection, and more extensive liver damage than did wild-type mice. Although type I interferon transcription decreased in MDA5(-/-) mice, the interferon-stimulated gene response remained intact. Cytokine production by innate and adaptive immune cells was largely intact in MDA5(-/-) mice, but perforin induction by natural killer cells and levels of interferon gamma, interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) in serum were elevated. These data suggest that MDA5 signaling reduces the severity of MHV-induced disease, at least in part by reducing the intensity of the proinflammatory cytokine response. IMPORTANCE: Multicellular organisms employ a wide range of sensors to detect viruses and other pathogens. One such sensor, MDA5, detects viral RNA and triggers induction of type I interferons, chemical messengers that induce inflammation and help regulate the immune responses. In this study, we sought to determine the role of MDA5 during infection with mouse hepatitis virus, a murine coronavirus used to model viral hepatitis as well as other human diseases. We found that mice lacking the MDA5 sensor were more susceptible to infection than were mice with MDA5 and experienced decreased survival. Viral replication in the liver was similar in mice with and without MDA5, but liver damage was increased in MDA5(-/-) mice, suggesting that the immune response is causing the damage. Production of several proinflammatory cytokines was elevated in MDA5(-/-) mice, suggesting that MDA5 may be responsible for keeping pathological inflammatory responses in check.

Cell-type-specific activation of the oligoadenylate synthetase-RNase L pathway by a murine coronavirus.

Previous studies have demonstrated that the murine coronavirus mouse hepatitis virus (MHV) nonstructural protein 2 (ns2) is a 2',5'-phosphodiesterase that inhibits activation of the interferon-induced oligoadenylate synthetase (OAS)-RNase L pathway. Enzymatically active ns2 is required for efficient MHV replication in macrophages, as well as for the induction of hepatitis in C57BL/6 mice. In contrast, following intranasal or intracranial inoculation, efficient replication of MHV in the brain is not dependent on an enzymatically active ns2. The replication of wild-type MHV strain A59 (A59) and a mutant with an inactive phosphodiesterase (ns2-H126R) was assessed in primary hepatocytes and primary central nervous system (CNS) cell types-neurons, astrocytes, and oligodendrocytes. A59 and ns2-H126R replicated with similar kinetics in all cell types tested, except macrophages and microglia. RNase L activity, as assessed by rRNA cleavage, was induced by ns2-H126R, but not by A59, and only in macrophages and microglia. Activation of RNase L correlated with the induction of type I interferon and the consequent high levels of OAS mRNA induced in these cell types. Pretreatment of nonmyeloid cells with interferon restricted A59 and ns2-H126R to the same extent and failed to activate RNase L following infection, despite induction of OAS expression. However, rRNA degradation was induced by treatment of astrocytes or oligodendrocytes with poly(I·C). Thus, RNase L activation during MHV infection is cell type specific and correlates with relatively high levels of expression of OAS genes, which are necessary but not sufficient for induction of an effective RNase L antiviral response.

Cell-type-specific type I interferon antagonism influences organ tropism of murine coronavirus.

Previous studies have demonstrated that mouse hepatitis virus (MHV) hepatotropism is determined largely by postentry events rather than by availability of the viral receptor. In addition, mutation of MHV nonstructural protein 2 (ns2) abrogates the ability of the virus to replicate in the liver and induce hepatitis but does not affect replication in the central nervous system (CNS). Here we show that replication of ns2 mutant viruses is attenuated in bone marrow-derived macrophages (BMM) generated from wild-type (wt) mice but not in L2 fibroblasts, primary astrocytes, or BMM generated from type I interferon receptor-deficient (IFNAR(-/-)) mice. In addition, ns2 mutants are more sensitive than wt virus to pretreatment of BMM, but not L2 fibroblasts or primary astrocytes, with alpha/beta interferon (IFN-α/ß). The ns2 mutants induced similar levels of IFN-α/ß in wt and IFNAR(-/-) BMM, indicating that ns2 expression has no effect on the induction of IFN but rather that it antagonizes a later step in IFN signaling. Consistent with these in vitro data, the virulence of ns2 mutants increased to near that of wt virus after depletion of macrophages in vivo. These data imply that the ability of MHV to replicate in macrophages is a prerequisite for replication in the liver and induction of hepatitis but not for replication or disease in the CNS, underscoring the importance of IFN signaling in macrophages in vivo for protection of the host from hepatitis. Our results further support the notion that viral tissue tropism is determined in part by postentry events, including the early type I interferon response.

Murine coronavirus delays expression of a subset of interferon-stimulated genes.

The importance of the type I interferon (IFN-I) system in limiting coronavirus replication and dissemination has been unequivocally demonstrated by rapid lethality following infection of mice lacking the alpha/beta IFN (IFN-alpha/beta) receptor with mouse hepatitis virus (MHV), a murine coronavirus. Interestingly, MHV has a cell-type-dependent ability to resist the antiviral effects of IFN-alpha/beta. In primary bone-marrow-derived macrophages and mouse embryonic fibroblasts, MHV replication was significantly reduced by the IFN-alpha/beta-induced antiviral state, whereas IFN treatment of cell lines (L2 and 293T) has only minor effects on replication (K. M. Rose and S. R. Weiss, Viruses 1:689-712, 2009). Replication of other RNA viruses, including Theiler's murine encephalitis virus (TMEV), vesicular stomatitis virus (VSV), Sindbis virus, Newcastle disease virus (NDV), and Sendai virus (SeV), was significantly inhibited in L2 cells treated with IFN-alpha/beta, and MHV had the ability to rescue only SeV replication. We present evidence that MHV infection can delay interferon-stimulated gene (ISG) induction mediated by both SeV and IFN-beta but only when MHV infection precedes SeV or IFN-beta exposure. Curiously, we observed no block in the well-defined IFN-beta signaling pathway that leads to STAT1-STAT2 phosphorylation and translocation to the nucleus in cultures infected with MHV. This observation suggests that MHV must inhibit an alternative IFN-induced pathway that is essential for early induction of ISGs. The ability of MHV to delay SeV-mediated ISG production may partially involve limiting the ability of IFN regulatory factor 3 (IRF-3) to function as a transcription factor. Transcription from an IRF-3-responsive promoter was partially inhibited by MHV; however, IRF-3 was transported to the nucleus and bound DNA in MHV-infected cells superinfected with SeV.

HIV-1 Vif protein binds the editing enzyme APOBEC3G and induces its degradation.

The viral infectivity factor (Vif) encoded by HIV-1 neutralizes a potent antiviral pathway that occurs in human T lymphocytes and several leukemic T-cell lines termed nonpermissive, but not in other cells termed permissive. In the absence of Vif, this antiviral pathway efficiently inactivates HIV-1. It was recently reported that APOBEC3G (also known as CEM-15), a cytidine deaminase nucleic acid-editing enzyme, confers this antiviral phenotype on permissive cells. Here we describe evidence that Vif binds APOBEC3G and induces its rapid degradation, thus eliminating it from cells and preventing its incorporation into HIV-1 virions. Studies of Vif mutants imply that it contains two domains, one that binds APOBEC3G and another with a conserved SLQ(Y/F)LA motif that mediates APOBEC3G degradation by a proteasome-dependent pathway. These results provide promising approaches for drug discovery.

Human immunodeficiency virus type 1 Vif functionally interacts with diverse APOBEC3 cytidine deaminases and moves with them between cytoplasmic sites of mRNA metabolism.

Vif(IIIB), which has been a standard model for the viral infectivity factor of human immunodeficiency virus type 1 (HIV-1), binds the cytidine deaminase APOBEC3G (A3G) and induces its degradation, thereby precluding its lethal incorporation into assembling virions. Additionally, Vif(IIIB) less efficiently degrades A3F, another potent anti-HIV-1 cytidine deaminase. Although the APOBEC3 paralogs A3A, A3B, and A3C have weaker anti-HIV-1 activities and are only partially degraded by Vif(IIIB), we found that Vif(IIIB) induces their emigration from the nucleus to the cytosol and thereby causes net increases in the cytosolic concentrations and anti-HIV-1 activities of A3A and A3B. In contrast, some other Vifs, exemplified by Vif(HXB2) and Vif(ELI-1), much more efficiently degrade and thereby neutralize all APOBEC3s. Studies focused mainly on A3F imply that it occurs associated with mRNA-PABP1 in translationally active polysomes and to a lesser extent in mRNA processing bodies (P-bodies). A3F appears to stabilize the P-bodies with which it is associated. A correspondingly small proportion of Vif(IIIB) also localizes in P-bodies in an A3F-dependent manner. Stress causes A3A, A3B, A3C, and A3F to colocalize efficiently with Vif(IIIB) and mRNA-PABP1 complexes in stress granules in a manner that is prevented by cycloheximide, an inhibitor of translational elongation. Coimmunoprecipitation studies suggest that Vifs from different HIV-1 isolates associate with all tested APOBEC3s. Thus, Vifs interact closely with structurally diverse APOBEC3s, with effects on their subcellular localization, degradation rates, and antiviral activities. Cytosolic APOBEC3-Vif complexes are predominantly bound to mRNAs that dynamically move between translationally active and storage or processing pools.

The viral infectivity factor (Vif) of HIV-1 unveiled.

The viral infectivity factor (Vif) of HIV type-1 (HIV-1) is essential for efficient viral replication, yet was, until recently, enigmatic. This resulted from the complexity and cellular specificity of its function and the correspondingly complex systems that are required for its investigation. These limitations have been overcome and Vif function has been rapidly elucidated, with implications for the development of drugs to block its activity. These studies have revealed a novel component of the innate immune system, APOBEC3G, that lethally hypermutates retroviruses, including HIV-1. For HIV-1, the competition between the virus and APOBEC3G is tipped in favor of the invader by Vif, which binds to APOBEC3G and triggers its polyubiquitination and rapid degradation, thereby preventing its entry into progeny virions.

Regulated production and anti-HIV type 1 activities of cytidine deaminases APOBEC3B, 3F, and 3G.

APOBEC3G and 3F (A3G and A3F) cytidine deaminases incorporate into retroviral cores where they lethally hypermutate nascent DNA reverse transcripts. As substantiated here, the viral infectivity factor (Vif) encoded by human immunodeficiency virus type-1 (HIV-1) binds A3G and A3F and induces their degradation, thereby precluding their incorporation into viral progeny. Previous evidence suggested that A3G is expressed in H9 and other nonpermissive cells that contain this antiviral defense but not in several permissive cells, and that overexpression of A3G or A3F makes permissive cells nonpermissive. Using a broader panel of cell lines, we confirmed a correlation between A3G and cellular abilities to inactivate HIV-1(Deltavif). However, there was a quantitative discrepancy because several cells with weak antiviral activities had similar amounts of wild-type A3G mRNA and protein compared to H9 cells. Antiviral activity of H9 cells was also attenuated in some conditions. These quantitative discrepancies could not be explained by the presence of A3F or other A3G paralogs in some of the cell lines. Thus, A3A, A3B, and A3C had weak but significant anti-HIV-1 activities and did not dominantly interfere with A3G or A3F antiviral functions. Control of A3G synthesis by the protein kinase C/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway was also similar in permissive and nonpermissive cells. A3G in highly permissive cells is degraded by Vif, suggesting that it is not in a sequestered site, and is specifically incorporated in low amounts into HIV-1(Deltavif). Although A3G and/or A3F inactivate HIV-1(Deltavif) and are neutralized by Vif, the antiviral properties of cell lines are also influenced by other cellular and viral factors.

Murine Coronavirus Cell Type Dependent Interaction with the Type I Interferon Response.

Coronaviruses infect many species of animal including humans, causing acute and chronic diseases of many organ systems. Murine coronavirus, mouse hepatitis virus (MHV) infection of the mouse, provides animal models for the study of central nervous system disease, including encephalitis and demyelinating diseases such as Multiple Sclerosis and for hepatitis. While there are many studies of the adaptive immune response to MHV, there has until recently been scant information on the type I interferon (IFN) response to MHV. The relationship between MHV and the IFN-alpha/beta response is paradoxical. While the type I IFN response is a crucial aspect of host defense against MHV in its natural host, there is little if any induction of IFN following infection of mouse fibroblast cell lines in vitro. Furthermore, MHV is relatively resistant to the antiviral effects of IFN-alpha/beta in mouse fibroblast cell lines and in human 293T cells. MHV can, under some circumstances, compromise the antiviral effects of IFN signaling. The nucleocapsid protein as well as the nsp1 and nsp3 proteins of MHV has been reported to have IFN antagonist activity. However, in primary cell types such as plasmacytoid dendritic cells (pDC) and macrophages, IFN is induced by MHV infection and an antiviral state is established. Other primary cell types such as neurons, astrocytes and hepatocytes fail to produce IFN following infection and, in vivo, likely depend on IFN produced by pDCs and macrophages for protection from MHV. Thus MHV induction of IFN-alpha/beta and the ability to induce an antiviral state in response to interferon is extremely cell type dependent. IFN induced protection from MHV pathogenesis likely requires the orchestrated activities of several cell types, however, the cell types involved in limiting MHV replication may be different in the liver and in the immune privileged CNS.

The anti-HIV-1 editing enzyme APOBEC3G binds HIV-1 RNA and messenger RNAs that shuttle between polysomes and stress granules.

Deoxycytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) (members of the apolipoprotein B mRNA-editing catalytic polypeptide 3 family) have RNA-binding motifs, invade assembling human immunodeficiency virus (HIV-1), and hypermutate reverse transcripts. Antagonistically, HIV-1 viral infectivity factor degrades these enzymes. A3G is enzymatically inhibited by binding RNA within an unidentified large cytosolic ribonucleoprotein, implying that RNA degradation during reverse transcription may activate intravirion A3G at the necessary moment. We purified a biologically active tandem affinity-tagged A3G from human HEK293T cells. Mass spectrometry and coimmunoprecipitation from HEK293T and T lymphocyte extracts identified many RNA-binding proteins specifically associated with A3G and A3F, including poly(A)-binding proteins (PABPs), YB-1, Ro-La, RNA helicases, ribosomal proteins, and Staufen1. Most strikingly, nearly all A3G-associated proteins were known to bind exclusively or intermittently to translating and/or dormant mRNAs. Accordingly, A3G in HEK293T and T lymphocyte extracts was almost completely in A3G-mRNA-PABP complexes that shifted reversibly between polysomes and dormant pools in response to translational inhibitors. For example arsenite, which inhibits 5'-cap-dependent translational initiation, shifted mRNA-A3G-PABP from polysomes into stress granules in a manner that was blocked and reversed by the elongation inhibitor cycloheximide. Immunofluorescence microscopy showed A3G-mRNA-PABP stress granules only partially overlapping with Staufen1. A3G coimmunoprecipitated HIV-1 RNA and many mRNAs. Ribonuclease released nearly all A3G-associated proteins, including A3G homo-oligomers and A3G-A3F hetero-oligomers, but the viral infectivity factor remained bound. Many proteins and RNAs associated with A3G are excluded from A3G-containing virions, implying that A3G competitively partitions into virions based on affinity for HIV-1 RNA.

Transcriptional regulation of APOBEC3G, a cytidine deaminase that hypermutates human immunodeficiency virus.

Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (APOBEC3G) is an antiretroviral deoxycytidine deaminase that lethally hypermutates human immunodeficiency virus type 1 (HIV-1) but is itself neutralized by the HIV-1-encoded viral infectivity factor. Accordingly, APOBEC3G occurs specifically in human T lymphocytic cell lines that contain this antiviral defense, including H9. Since the substrate specificities of related cytidine deaminases are strongly influenced by their intracellular quantities, we analyzed the factors that control APOBEC3G expression. The levels of APOBEC3G mRNA and protein were unaffected by treatment of proliferating H9 cells with interferons or tumor necrosis factor-alpha but were enhanced up to 20-fold by phorbol myristate acetate. This induction was mediated at the transcriptional level by a pathway that required activation of the protein kinase Calpha/betaI isozyme (PKC), mitogen-activated protein kinase kinase (MEK) 1 and 2, and extracellular signal-regulated kinase (ERK). Correspondingly, induction of APOBEC3G was blocked by multiple inhibitors that act at diverse steps of this pathway. The PKCalpha/betaI/MEK/ERK pathway also controlled basal levels of APOBEC3G mRNA and protein, which consequently declined when cells were treated with these inhibitors or arrested in the G(0) state of the cell cycle by serum starvation. We conclude that expression of the antiviral APOBEC3G editing enzyme is dynamically controlled by the PKCalpha/betaI/MEK/ERK protein kinase cascade in human T lymphocytes.