Induction and Inhibition of Type I Interferon Responses by Distinct Components of Lymphocytic Choriomeningitis Virus

Type I interferons (IFNs) play a critical role in the host defense against viruses. Lymphocytic choriomeningitis virus (LCMV) infection induces robust type I IFN production in its natural host, the mouse. However, the mechanisms underlying the induction of type I IFNs in response to LCMV infection have not yet been clearly defined. In the present study, we demonstrate that IRF7 is required for both the early phase (day 1 postinfection) and the late phase (day 2 postinfection) of the type I IFN response to LCMV, and melanoma differentiation-associated gene 5 (MDA5)/mitochondrial antiviral signaling protein (MAVS) signaling is crucial for the late phase of the type I IFN response to LCMV. We further demonstrate that LCMV genomic RNA itself (without other LCMV components) is able to induce type I IFN responses in various cell types by activation of the RNA helicases retinoic acid-inducible gene I (RIG-I) and MDA5. We also show that expression of the LCMV nucleoprotein (NP) inhibits the type I IFN response induced by LCMV RNA and other RIG-I/MDA5 ligands. These virus-host interactions may play important roles in the pathogeneses of LCMV and other human arenavirus diseases.Type I interferons (IFNs), namely, alpha interferon (IFN-α) and IFN-β, are not only essential for host innate defense against viral pathogens but also critically modulate the development of virus-specific adaptive immune responses (6, 8, 28, 30, 36, 50, 61). The importance of type I IFNs in host defense has been demonstrated by studying mice deficient in the type I IFN receptor, which are highly susceptible to most viral pathogens (2, 47, 62).Recent studies have suggested that the production of type I IFNs is controlled by different innate pattern recognition receptors (PRRs) (19, 32, 55, 60). There are three major classes of PRRs, including Toll-like receptors (TLRs) (3, 40), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) (25, 48, 51), and nucleotide oligomerization domain (NOD)-like receptors (9, 22). TLRs are a group of transmembrane proteins expressed on either cell surfaces or endosomal compartments. RLRs localize in the cytosol. Both TLRs and RLRs are involved in detecting viral pathogens and controlling the production of type I IFNs (52, 60). In particular, the endosome-localized TLRs (TLR3, TLR7/8, and TLR9) play important roles in detecting virus-derived double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), and DNA-containing unmethylated CpG motifs, respectively. In contrast, RIG-I detects virus-derived ssRNA with 5′-triphosphates (5′-PPPs) or short dsRNA (<1 kb), whereas melanoma differentiation-associated gene 5 (MDA5) is responsible for recognizing virus-derived long dsRNA as well as a synthetic mimic of viral dsRNA poly(I):poly(C) [poly(I·C)] (24, 60). Recognition of viral pathogen-associated molecular patterns (PAMPs) ultimately leads to the activation and nuclear translocation of interferon regulatory factors (IRFs) and nuclear factor κB (NF-κB), which, in turn, switches on a cascade of genes controlling the production of both type I IFNs and other proinflammatory cytokines (10, 11, 60).Lymphocytic choriomeningitis virus (LCMV) infection in its natural host, the mouse, is an excellent system to study the impact of virus-host interactions on viral pathogenesis and to address important issues related to human viral diseases (1, 45, 49, 67). LCMV infection induces type I IFNs as well as other proinflammatory chemokines and cytokines (6, 41). Our previous studies have demonstrated that TLR2, TLR6, and CD14 are involved in LCMV-induced proinflammatory chemokines and cytokines (66). The mechanism by which LCMV induces type I IFN responses, however, has not been clearly defined (7, 8, 31, 44). The role of the helicase family members RIG-I and MDA5 in virus-induced type I IFN responses has been recently established. RIG-I has been found to be critical in controlling the production of type I IFN in response to a number of RNA viruses, including influenza virus, rabies virus, Hantaan virus, vesicular stomatitis virus (VSV), Sendai virus (SeV), etc. In contrast, MDA5 is required for responses to picornaviruses (15, 25, 63).In the present study, we demonstrated that LCMV genomic RNA strongly activates type I IFNs through a RIG-I/MDA5-dependent signaling pathway. Our present study further demonstrated that the LCMV nucleoprotein (NP) blocks LCMV RNA- and other viral ligand-induced type I IFN responses.     

The 3C Protein of Enterovirus 71 Inhibits Retinoid Acid-Inducible Gene I-Mediated Interferon Regulatory Factor 3 Activation and Type I Interferon Responses

Enterovirus 71 (EV71) is a human pathogen that induces hand, foot, and mouth disease and fatal neurological diseases. Immature or impaired immunity is thought to associate with increased morbidity and mortality. In a murine model, EV71 does not facilitate the production of type I interferon (IFN) that plays a critical role in the first-line defense against viral infection. Administration of a neutralizing antibody to IFN-α/β exacerbates the virus-induced disease. However, the molecular events governing this process remain elusive. Here, we report that EV71 suppresses the induction of antiviral immunity by targeting the cytosolic receptor retinoid acid-inducible gene I (RIG-I). In infected cells, EV71 inhibits the expression of IFN-β, IFN-stimulated gene 54 (ISG54), ISG56, and tumor necrosis factor alpha. Among structural and nonstructural proteins encoded by EV71, the 3C protein is capable of inhibiting IFN-β activation by virus and RIG-I. Nevertheless, EV71 3C exhibits no inhibitory activity on MDA5. Remarkably, when expressed in mammalian cells, EV71 3C associates with RIG-I via the caspase recruitment domain. This precludes the recruitment of an adaptor IPS-1 by RIG-I and subsequent nuclear translocation of interferon regulatory factor 3. An R84Q or V154S substitution in the RNA binding motifs has no effect. An H40D substitution is detrimental, but the protease activity associated with 3C is dispensable. Together, these results suggest that inhibition of RIG-I-mediated type I IFN responses by the 3C protein may contribute to the pathogenesis of EV71 infection.Enterovirus 71 (EV71) is a single-stranded, positive-sense RNA virus belonging to the Picornaviridae family. The viral genome is approximately 7,500 nucleotides in length, with a single open reading frame that encodes a large precursor protein. Upon infection, this protein precursor is processed into four structural (VP1, VP2, VP3, and VP4) and seven nonstructural (2A, 2B, 2C, 3A, 3B, 3C, and 3D) proteins (32). EV71 infection manifests most frequently as the childhood exanthema known as hand, foot, and mouth disease (HFMD). Additionally, EV71 infection may cause neurological diseases, which include aseptic meningitis, brain stem and/or cerebellar encephalitis, and acute flaccid paralysis (32). Young children and infants are especially susceptible to EV71 infection. Since the initial recognition of EV71 in the United States, outbreaks have been reported in Southeast Asia, Europe, and Australia (1-3, 11, 14, 24, 30-32). Recently, large epidemics of HFMD occurred in the mainland of China (26, 42, 52).The mechanism of EV71 pathogenesis remains obscure. It is believed that immature or impaired immunity, upon EV71 infection, is associated with increased morbidity and mortality (7, 14, 17). In a murine infection model, lymphocyte as well as antibody responses reduce tissue viral loads and EV71 lethality (28). Notably, EV71 induces skin rash at the early stage and hind limb paralysis or death at the late stage. Oral infection leads to initial replication in the intestine and subsequent spread to various organs such as the spinal cord and the brain stem (8). Intriguingly, EV71 does not facilitate the production of type I interferon (IFN), a family of cytokines involved in first-line defense against virus infection. Indeed, administration of neutralizing antibody to IFN-α/β increases tissue viral loads and exacerbates the virus-induced disease (29).Type I IFN is produced in response to viral infections (22). For example, Toll-like receptor 3 (TLR3) in the endosome recognizes double-stranded RNA (dsRNA), where it recruits the adaptor Toll/interleukin-1 receptor (TIR) domain-containing adaptor inducing IFN-β (TRIF) (22). TRIF, together with tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3), then activates the two IKK-related kinases, TANK-binding kinase 1 (TBK1) and inducible IκB kinase (IKKi), both of which phosphorylate interferon regulatory factor 3/7 (IRF3/7) (10, 13, 36, 45). IRF3 or IRF7, in turn, stimulates the expression of target genes, such as IFN-α/β (33, 37, 39, 51). In parallel, TRIF also induces NF-κB activation via TRAF6 (18, 19). In addition, alternative mechanisms exist in host cells to detect cytosolic nucleic acids. Two RNA helicases, retinoid acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5), recognize viral RNA present in the cytoplasm and subsequently recruit the adaptor IFN promoter-stimulating factor 1 (IPS-1; also called Cardif, MAVS, and VISA) (22, 23, 54). The interaction of IPS-1, TRAF3, and TBK1/IKKi activates IRF3/IRF7 and induces the expression of IFN-α/β while the interaction of IPS-1 with the Fas-associated protein-containing death domain (FADD) leads to NF-κB activation. It has been shown that MDA5 recognizes long double-stranded RNAs, such as in cells infected with picornaviruses, whereas RIG-I senses 5′ triphosphate single-stranded RNA with poly(U/A) motifs and short dsRNA in cells infected with a variety of RNA viruses (16, 20, 40, 43).The objective of this study was to investigate the interaction of EV71 with the type I IFN system. We demonstrate that, unlike Sendai virus or double-stranded RNA, EV71 does not stimulate the expression of antiviral genes in mammalian cells. Among structural and nonstructural proteins encoded by EV71, the 3C protein is able to inhibit virus-induced activation of the IFN-β promoter. We provide evidence that when expressed in mammalian cells, the 3C protein suppresses RIG-I signaling by disruption of the RIG-I-IPS-1 complex and IRF3 nuclear translocation. While H40, KFRDI, and VGK motifs are involved, the protease and RNA binding activities are dispensable. Collectively, these results suggest that control of RIG-I by the 3C protein impairs type I IFN responses, which may contribute to the pathogenesis of EV71 infection.     

Early Innate Recognition of Herpes Simplex Virus in Human Primary Macrophages Is Mediated via the MDA5/MAVS-Dependent and MDA5/MAVS/RNA Polymerase III-Independent Pathways

Innate recognition of viruses is mediated by pattern recognition receptors (PRRs) triggering expression of antiviral interferons (IFNs) and proinflammatory cytokines. In mice, Toll-like receptor 2 (TLR2) and TLR9 as well as intracellular nucleotide-sensing pathways have been shown to recognize herpes simplex virus (HSV). Here, we describe how human primary macrophages recognize early HSV infection via intracellular pathways. A number of inflammatory cytokines, IFNs, and IFN-stimulated genes were upregulated after HSV infection. We show that early recognition of HSV and induction of IFNs and inflammatory cytokines are independent of TLR2 and TLR9, since inhibition of TLR2 using TLR2 neutralizing antibodies did not affect virus-induced responses and the macrophages were unresponsive to TLR9 stimulation. Instead, HSV recognition involves intracellular recognition systems, since induction of tumor necrosis factor alpha (TNF-α) and IFNs was dependent on virus entry and replication. Importantly, expression of IFNs was strongly inhibited by small interfering RNA (siRNA) knockdown of MAVS, but this MAVS-dependent IFN induction occurred independently of the recently discovered polymerase III (Pol III)/RIG-I DNA sensing system. In contrast, induction of TNF-α was largely independent of MAVS, suggesting that induction of inflammatory cytokines during HSV infection proceeds via a novel pathway. Transfection with ODN2006, a broad inhibitor of intracellular nucleotide recognition, revealed that nucleotide-sensing systems are employed to induce both IFNs and TNF-α. Finally, using siRNA knockdown, we found that MDA5, but not RIG-I, was the primary mediator of HSV recognition. Thus, innate recognition of HSV by human primary macrophages occurs via two distinct intracellular nucleotide-sensing pathways responsible for induction of IFNs and inflammatory cytokine expression, respectively.Virus recognition is essential for activation of innate antiviral immune defense and the subsequent induction of acquired immunity. Conserved pathogen motifs, termed pathogen-associated molecular patterns (PAMPs), are recognized by pattern recognition receptors (PRRs). Virus-recognizing PRRs include Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and a number of intracellular DNA receptors. Several TLRs have been attributed roles in the recognition of virus. TLR2 and TLR4 recognize viral surface structures (3, 6, 18, 31), TLR3 recognizes double-stranded RNA (dsRNA) (2), and TLR7/8 and TLR9 function as signaling receptors for viral single-stranded RNA (ssRNA) (8, 11, 21) and CpG DNA (12, 20), respectively.Within the cell, cytoplasmic RLRs RIG-I and MDA5 both recognize accumulation of virus-derived dsRNA; in addition, RIG-I recognizes 5′-triphosphated RNA (14, 27, 39, 40). In addition to the RLRs, a number of receptors recognize foreign DNA. Presently, three DNA receptors have been identified: Z-DNA binding protein 1 (ZBP-1, or DAI) (36) and RNA polymerase III (Pol III) (1, 4) both mediate interferon (IFN) and cytokine production, whereas the AIM2 inflammasome is involved in caspase 1 activation in response to cytoplasmic dsDNA (13).Herpes simplex virus type 1 (HSV-1) and HSV-2 are two closely related human DNA viruses associated with a number of serious diseases, including orofacial infections, encephalitis, and genital infections (34). Macrophages play an important role in the first line of defense against viral infection via production of IFNs, cytokines, and chemokines that regulate the progress of the virus infection and activate and support appropriate defense mechanisms (9, 10, 24).TLR2, TLR3, and TLR9 have been identified as mediators of proinflammatory cytokine production during HSV infections. TLR2 mediates an overzealous inflammatory cytokine response following HSV-1 infection in mice, promoting mononuclear cell infiltration of the brain and development of encephalitis (18). TLR3 mediates type I and III IFN production in human fibroblasts (41). TLR9 recognizes genomic DNA from HSV-1 and HSV-2 in murine plasmacytoid dendritic cells (DCs) (17, 20) and mediates tumor necrosis factor alpha (TNF-α) and CCL5 production in murine macrophages (22). Both TLR2 and TLR9 mediate recognition of HSV and cytokine production in murine conventional DCs (35). HSV is recognized by an RLR/MAVS-dependent mechanism in murine macrophages and mouse embryonic fibroblasts (MEFs) (5, 29, 30). Recent data suggest that RNA Pol III mediates IFN production following HSV-1 infection and transfection with HSV-1 DNA in macrophage-like RAW 264.7 cells (4). Finally, murine L929 fibroblast-like cells are moderately inhibited in their ability to produce IFN after HSV-1 infection when ZBP-1 is knocked down (19, 36). Thus, several PRRs have been reported to recognize HSV-1 in murine cells and different cell lines, but the pathways responsible for sensing this virus in human primary macrophages and their impact on cytokine expression have not previously been described.In this work, we investigate the recognition pathways underlying HSV-induced cytokine and chemokine expression in human primary macrophages. We demonstrate that HSV-1-induced IFN and cytokine expression is independent of TLR2 and TLR9 but highly dependent on virus replication and intracellular nucleotide recognition systems. Specifically, induction of IFNs is dependent on MAVS and MDA5, whereas TNF-α is induced by a novel intracellular nucleotide-sensing system.     

Heavily Isotype-Dependent Protective Activities of Human Antibodies against Vaccinia Virus Extracellular Virion Antigen B5

Antibodies against the extracellular virion (EV or EEV) form of vaccinia virus are an important component of protective immunity in animal models and likely contribute to the protection of immunized humans against poxviruses. Using fully human monoclonal antibodies (MAbs), we now have shown that the protective attributes of the human anti-B5 antibody response to the smallpox vaccine (vaccinia virus) are heavily dependent on effector functions. By switching Fc domains of a single MAb, we have definitively shown that neutralization in vitro—and protection in vivo in a mouse model—by the human anti-B5 immunoglobulin G MAbs is isotype dependent, thereby demonstrating that efficient protection by these antibodies is not simply dependent on binding an appropriate vaccinia virion antigen with high affinity but in fact requires antibody effector function. The complement components C3 and C1q, but not C5, were required for neutralization. We also have demonstrated that human MAbs against B5 can potently direct complement-dependent cytotoxicity of vaccinia virus-infected cells. Each of these results was then extended to the polyclonal human antibody response to the smallpox vaccine. A model is proposed to explain the mechanism of EV neutralization. Altogether these findings enhance our understanding of the central protective activities of smallpox vaccine-elicited antibodies in immunized humans.The smallpox vaccine, live vaccinia virus (VACV), is frequently considered the gold standard of human vaccines and has been enormously effective in preventing smallpox disease. The smallpox vaccine led to the worldwide eradication of the disease via massive vaccination campaigns in the 1960s and 1970s, one of the greatest successes of modern medicine (30). However, despite the efficacy of the smallpox vaccine, the mechanisms of protection remain unclear. Understanding those mechanisms is key for developing immunologically sound vaccinology principles that can be applied to the design of future vaccines for other infectious diseases (3, 101).Clinical studies of fatal human cases of smallpox disease (variola virus infection) have shown that neutralizing antibody titers were either low or absent in patient serum (24, 68). In contrast, neutralizing antibody titers for the VACV intracellular mature virion (MV or IMV) were correlated with protection of vaccinees against smallpox (68). VACV immune globulin (VIG) (human polyclonal antibodies) is a promising treatment against smallpox (47), since it was able to reduce the number of smallpox cases ∼80% among variola-exposed individuals in four case-controlled clinical studies (43, 47, 52, 53, 69). In animal studies, neutralizing antibodies are crucial for protecting primates and mice against pathogenic poxviruses (3, 7, 17, 21, 27, 35, 61, 66, 85).The specificities and the functions of protective antipoxvirus antibodies have been areas of intensive research, and the mechanics of poxvirus neutralization have been debated for years. There are several interesting features and problems associated with the antibody response to variola virus and related poxviruses, including the large size of the viral particles and the various abundances of many distinct surface proteins (18, 75, 91, 93). Furthermore, poxviruses have two distinct virion forms, intracellular MV and extracellular enveloped virions (EV or EEV), each with a unique biology. Most importantly, MV and EV virions share no surface proteins (18, 93), and therefore, there is no single neutralizing antibody that can neutralize both virion forms. As such, an understanding of virion structure is required to develop knowledge regarding the targets of protective antibodies.Neutralizing antibodies confer protection mainly through the recognition of antigens on the surface of a virus. A number of groups have discovered neutralizing antibody targets of poxviruses in animals and humans (3). The relative roles of antibodies against MV and EV in protective immunity still remain somewhat unclear. There are compelling data that antibodies against MV (21, 35, 39, 66, 85, 90, 91) or EV (7, 16, 17, 36, 66, 91) are sufficient for protection, and a combination of antibodies against both targets is most protective (66). It remains controversial whether antibodies to one virion form are more important than those to the other (3, 61, 66). The most abundant viral particles are MV, which accumulate in infected cells and are released as cells die (75). Neutralization of MV is relatively well characterized (3, 8, 21, 35). EV, while less abundant, are critical for viral spread and virulence in vivo (93, 108). Neutralization of EV has remained more enigmatic (3).B5R (also known as B5 or WR187), one of five known EV-specific proteins, is highly conserved among different strains of VACV and in other orthopoxviruses (28, 49). B5 was identified as a protective antigen by Galmiche et al., and the available evidence indicated that the protection was mediated by anti-B5 antibodies (36). Since then, a series of studies have examined B5 as a potential recombinant vaccine antigen or as a target of therapeutic monoclonal antibodies (MAbs) (1, 2, 7, 17, 40, 46, 66, 91, 110). It is known that humans immunized with the smallpox vaccine make antibodies against B5 (5, 22, 62, 82). It is also known that animals receiving the smallpox vaccine generate antibodies against B5 (7, 20, 27, 70). Furthermore, previous neutralization assays have indicated that antibodies generated against B5 are primarily responsible for neutralization of VACV EV (5, 83). Recently Chen at al. generated chimpanzee-human fusion MAbs against B5 and showed that the MAbs can protect mice from lethal challenge with virulent VACV (17). We recently reported, in connection with a study using murine monoclonal antibodies, that neutralization of EV is highly complement dependent and the ability of anti-B5 MAbs to protect in vivo correlated with their ability to neutralize EV in a complement-dependent manner (7).The focus of the study described here was to elucidate the mechanisms of EV neutralization, focusing on the human antibody response to B5. Our overall goal is to understand underlying immunobiological and virological parameters that determine the emergence of protective antiviral immune responses in humans.     

Previously Unrecognized Amino Acid Substitutions in the Hemagglutinin and Fusion Proteins of Measles Virus Modulate Cell-Cell Fusion,Hemadsorption, Virus Growth,and Penetration Rate

Wild-type measles virus (MV) isolated in B95a cells could be adapted to Vero cells after several blind passages. In this study, we have determined the complete nucleotide sequences of the genomes of the wild type (T11wild) and its Vero cell-adapted (T11Ve-23) MV strain and identified amino acid substitutions R516G, E271K, D439E and G464W (D439E/G464W), N481Y/H495R, and Y187H/L204F in the nucleocapsid, V, fusion (F), hemagglutinin (H), and large proteins, respectively. Expression of mutated H and F proteins from cDNA revealed that the H495R substitution, in addition to N481Y, in the H protein was necessary for the wild-type H protein to use CD46 efficiently as a receptor and that the G464W substitution in the F protein was important for enhanced cell-cell fusion. Recombinant wild-type MV strains harboring the F protein with the mutations D439E/G464W [F(D439E/G464W)] and/or H(N481Y/H495R) protein revealed that both mutated F and H proteins were required for efficient syncytium formation and virus growth in Vero cells. Interestingly, a recombinant wild-type MV strain harboring the H(N481Y/H495R) protein penetrated slowly into Vero cells, while a recombinant wild-type MV strain harboring both the F(D439E/G464W) and H(N481Y/H495R) proteins penetrated efficiently into Vero cells, indicating that the F(D439E/G464W) protein compensates for the inefficient penetration of a wild-type MV strain harboring the H(N481Y/H495R) protein. Thus, the F and H proteins synergistically function to ensure efficient wild-type MV growth in Vero cells.Measles virus (MV), which belongs to the genus Morbillivirus in the family Paramyxoviridae, is an enveloped virus with a nonsegmented negative-strand RNA genome. The MV genome encodes six structural proteins: the nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin (H), and large (L) proteins. The P gene also encodes two other accessory proteins, the C and V proteins. The C protein is translated from an alternative translational initiation site leading a different reading frame, and the V protein is synthesized from an edited mRNA. MV has two envelope glycoproteins, the F and H proteins. The former is responsible for envelope fusion, and the latter is responsible for receptor binding (12).Wild-type MV strains isolated in B95a cells and laboratory-adapted MV strains have distinct phenotypes (18). Wild-type MV strains can grow in B95a cells but not in Vero cells, while laboratory-adapted MV strains can grow in both B95a and Vero cells. Wild-type MV strains do not cause hemadsorption (HAd) in African green monkey red blood cells (AGM-RBC), while most of laboratory-adapted MV strains cause HAd. Importantly, wild-type MV strains are pathogenic and induce clinical signs that resemble human measles in experimentally infected monkeys while laboratory-adapted MV strains do not.One approach to identify amino acid substitutions responsible for these phenotypic differences is the comparison of a wild-type MV strain with a standard laboratory-adapted MV strain such as the Edmonston strain. With regard to the H protein, amino acid substitutions important for HAd activity and cell-cell fusion in tissue culture cells were identified by expressing the H proteins in mammalian cells (15, 21). Recently, Tahara et al. revealed that the M, H, and L proteins are responsible for efficient growth in Vero cells by constructing a series of recombinant viruses in which part of the genome of the wild-type MV was replaced with the corresponding sequences of the Edmonston strain (45, 46, 47).Another approach is the comparison of wild-type MV strains with their Vero cell-adapted MV strains. It was reported that Vero cell-adapted MV strains could be obtained by successive blind passages of wild-type MV strains in Vero cells (18, 24, 30, 43). Interestingly, in vivo and in vitro phenotypes of Vero cell-adapted MV strains were similar to those of laboratory-adapted standard MV strains (18, 19, 24, 30, 43). Comparison of the complete nucleotide sequences of the genomes of wild-type MV strains with those of Vero cell-adapted wild-type MV strains revealed amino acid substitutions in the P, C, V, M, H, and L proteins (27, 42, 48, 53).At present, these phenotypic differences are explained mainly by the receptor usage of MV. Wild-type MV strains can use signaling lymphocyte activation molecule (SLAM; also called CD150) but not CD46 as a cellular receptor, whereas laboratory-adapted MV strains can use both SLAM and CD46 as cellular receptors (7, 10, 16, 29, 56, 60).However, receptor usage per se cannot explain all of the phenotypic differences (20, 25, 48, 53). For example, recombinant Edmonston strains expressing wild-type H proteins can grow in Vero cells to some extent (17, 54). Several reports suggested the presence of the third MV receptor on Vero cells (14, 44, 54, 60). Other reports indicated the contribution of the M protein on cell-cell fusion and growth of MV in Vero cells (4, 27, 47). Recently, the unidentified epithelial cell receptor for MV was predicted in primary culture of human cells (1, 55) and several epithelial cell lines (23, 51). However, the identity of the third receptor on Vero cells and the unidentified epithelial cell receptor is not clear yet. Thus, the mechanism of Vero cell adaptation of wild-type MV is not completely understood.In order to understand the molecular mechanism of these phenotypic changes of wild-type MV strains during adaptation in Vero cells, we determined the complete nucleotide sequences of the genomes of the wild-type (T11wild) and its Vero cell-adapted (T11Ve-23) MV strains (43) and examined the effect of individual amino acid substitutions using a mammalian cell expression system and reverse genetics. We show here that previously unrecognized new amino acid substitutions in the H and F proteins are important for MV adaptation and HAd activity.     

The Interferon Stimulator Mitochondrial Antiviral Signaling Protein Facilitates Cell Death by Disrupting the Mitochondrial Membrane Potential and by Activating Caspases

Interferon (IFN) signaling is initiated by the recognition of viral components by host pattern recognition receptors. Dengue virus (DEN) triggers IFN-β induction through a molecular mechanism involving the cellular RIG-I/MAVS signaling pathway. Here we report that the MAVS protein level is reduced in DEN-infected cells and that caspase-1 and caspase-3 cleave MAVS at residue D429. In addition to its well-known function in IFN induction, MAVS is also a proapoptotic molecule that triggers disruption of the mitochondrial membrane potential and activation of caspases. Although different domains are required for the induction of cytotoxicity and IFN, caspase cleavage at residue 429 abolished both functions of MAVS. The apoptotic role of MAVS in viral infection and double-stranded RNA (dsRNA) stimulation was demonstrated in cells with reduced endogenous MAVS expression induced by RNA interference. Even though IFN-β promoter activation was largely suppressed, DEN production was not affected greatly in MAVS knockdown cells. Instead, DEN- and dsRNA-induced cell death and caspase activation were delayed and attenuated in the cells with reduced levels of MAVS. These results reveal a new role of MAVS in the regulation of cell death beyond its well-known function of IFN induction in antiviral innate immunity.In the battle of hosts and microbes, the innate immune system uses pathogen recognition receptors (PRRs) to sense pathogen-associated molecular patterns (23). There are several functionally distinct classes of PRRs, such as the transmembrane (TM) Toll-like receptors (TLRs) and the intracellular retinoic acid-inducible gene I (RIG-I)-like helicase (RLH) receptors (15, 23, 25, 38). RLHs, including RIG-I and melanoma differentiation-associated gene 5 (MDA5), comprise an N-terminal caspase recruitment domain (CARD), a middle DEXD/H box RNA helicase domain, and a C-terminal domain. RLHs sense intracellular viral RNA and initiate an antiviral interferon (IFN) response (1, 43). RIG-I binding to viral RNA triggers conformational changes that expose the CARD for subsequent signaling (42). The adaptor molecule providing a link between RIG-I and downstream events was identified independently by four research groups as a mitochondrial CARD-containing protein, which was named mitochondrial antiviral signaling protein (MAVS) (34), IFN-β promoter stimulator 1 (IPS-1) (12), virus-induced signaling adaptor (VISA) (40), and CARD adaptor-inducing IFN-β (Cardif) (24). We refer to this adaptor as MAVS in this paper. MAVS transduces signals from RIG-I through CARD-CARD interactions, which then lead to interferon regulatory factor 3 (IRF-3) and NF-κB activation of IFN-β induction through a signaling cascade involving IKKα/β/γ, IKKɛ, and TBK1 (15). Recently, a protein termed STING (11) or MITA (47) was identified as a mediator that acts downstream of RIG-I and MAVS and upstream of TBK1.MAVS protein contains an N-terminal CARD required for signaling, a proline-rich domain that interacts with TRAF3, and a C-terminal TM region that targets MAVS to the mitochondrial outer membrane (29). Several cellular and viral proteins target MAVS in the attenuation of the IFN induction pathway. Cleavage of MAVS by hepatitis C virus (HCV) and hepatitis A virus (HAV) proteases, at residues C508 (18, 24) and Q428 (41), respectively, results in the loss of MAVS mitochondrial localization, thereby disrupting its function in IFN induction. Another mitochondrial outer membrane protein, NLRX1, can sequester MAVS from its association with RIG-I and act as a negative regulator of the IFN pathway (28). MAVS was recently found to be cleaved and inactivated by caspases during apoptosis (31, 33).The caspases are a well-known family of cysteinyl aspartate-specific proteases. The diverse roles of caspases in the cell cycle, proliferation, differentiation, cytokine production, innate immune regulation, and microbial infection suggest various functions of caspases beyond apoptosis (13, 14). The caspases can be separated into two subfamilies, namely, the cell death and inflammation subfamilies. In response to apoptotic stimuli, the initiators caspase-2, -8, -9, and -10 and effectors caspase-3, -6, and -7 mediate cell death events. Caspase-1, -4, -5, and -12 are known as the inflammatory caspases. Caspase-1 is involved in the cleavage and maturation of cytokines (8, 17). Caspase-8 and -10 were discovered as essential components that mediate antiviral signaling (37). Caspase-1 and -3 are activated in innate immune signaling (32). These findings indicate that caspases are involved in the regulation of innate immunity, in addition to their well-known apoptotic role. However, the details of how caspases are activated, the role of caspase activation, and how caspases manipulate the signaling pathways in innate immunity are still obscure.The family Flaviviridae contains three genera: Hepacivirus, Flavivirus, and Pestivirus. Infections with flaviviruses, such as dengue virus (DEN), Japanese encephalitis virus, and West Nile virus, are emerging worldwide. DEN triggers IFN-β through a molecular mechanism involving the RIG-I/MAVS signaling pathway (5, 20). In this study, we found that MAVS is cleaved during DEN serotype 2 (DEN-2) infection, in a caspase-dependent manner; this contrasts with viral protease-dependent cleavage of MAVS during infection with HCV and HAV. In a cell-free caspase assay system, MAVS was cleaved at residue D429 by caspase-1 and caspase-3. Cleaved MAVS failed to induce IFN production and caspase activation, and overexpression of MAVS also triggered caspase activation, which then negatively regulated its own function. Importantly, the role of MAVS in viral infection was verified by knockdown of MAVS expression. We discuss the possible regulatory mechanisms of MAVS and the biological significance of this cleavage event by caspases in the context of understanding how these apoptosis-related proteases might achieve cross talk with the innate immune pathway during viral infection.