The Second-Site Mutation in the Herpes Simplex Virus Recombinants Lacking the γ134.5 Genes Precludes Shutoff of Protein Synthesis by Blocking the Phosphorylation of eIF-2α

In cells infected with the herpes simplex virus 1 (HSV-1) recombinant R3616 lacking both copies of the γ₁34.5 gene, the double-stranded protein kinase R (PKR) is activated, eIF-2α is phosphorylated, and protein synthesis is shut off. Although PKR is also activated in cells infected with the wild-type virus, the product of the γ₁34.5 gene, infected-cell protein 34.5 (ICP34.5), binds protein phosphatase 1α and redirects it to dephosphorylate eIF-2α, thus enabling sustained protein synthesis. Serial passage in human cells of a mutant lacking the γ₁34.5 gene yields second-site, compensatory mutants lacking various domains of the α47 gene situated next to the U_S11 gene (I. Mohr and Y. Gluzman, EMBO J. 15:4759–4766, 1996). We report the construction of two recombinant viruses: R5103, lacking the γ₁34.5, U_S8, -9, -10, and -11, and α47 (U_S12) genes; and R5104, derived from R5103 and carrying a chimeric DNA fragment containing the U_S10 gene and the promoter of the α47 gene fused to the coding domain of the U_S11 gene. R5104 exhibited a protein synthesis profile similar to that of wild-type virus, whereas protein synthesis was shut off in cells infected with R5103 virus. Studies on the wild-type parent and mutant viruses showed the following: (i) PKR was activated in cells infected with parent or mutant virus but not in mock-infected cells, consistent with earlier studies; (ii) lysates of R3616, R5103, and R5104 virus-infected cells lacked the phosphatase activity specific for eIF-2α characteristic of wild-type virus-infected cells; and (iii) lysates of R3616 and R5103, which lacked the second-site compensatory mutation, contained an activity which phosphorylated eIF-2α in vitro, whereas lysates of mock-infected cells or cells infected with HSV-1(F) or R5104 did not phosphorylate eIF-2α. We conclude that in contrast to wild-type virus-infected cells, which preclude the shutoff of protein synthesis by causing rapid dephosphorylation of eIF-2α, in cells infected with γ₁34.5⁻ virus carrying the compensatory mutation, eIF-2α is not phosphorylated. The activity made apparent by the second-site mutation may represent a more ancient mechanism evolved to preclude the shutoff of protein synthesis.     

Rhinovirus and dsRNA Induce RIG-I-Like Receptors and Expression of Interferon β and λ1 in Human Bronchial Smooth Muscle Cells

Rhinovirus (RV) infections cause exacerbations and development of severe asthma highlighting the importance of antiviral interferon (IFN) defence by airway cells. Little is known about bronchial smooth muscle cell (BSMC) production of IFNs and whether BSMCs have dsRNA-sensing receptors besides TLR3. dsRNA is a rhinoviral replication intermediate and necrotic cell effect mimic that mediates innate immune responses in bronchial epithelial cells. We have explored dsRNA-evoked IFN-β and IFN-λ₁ production in human BSMCs and potential involvement of TLR3 and RIG-I-like receptors (RLRs). Primary BSMCs were stimulated with 0.1–10 µg/ml dsRNA, 0.1–1 µg/ml dsRNA in complex with the transfection agent LyoVec (dsRNA/LyoVec; selectively activating cytosolic RLRs) or infected with 0.05–0.5 MOI RV1B. Both dsRNA stimuli evoked early (3 h), concentration-dependent IFN-β and IFN-λ₁ mRNA expression, which with dsRNA/LyoVec was much greater, and with dsRNA was much less, after 24 h. The effects were inhibited by dexamethasone. Further, dsRNA and dsRNA/LyoVec concentration-dependently upregulated RIG-I and MDA5 mRNA and protein. dsRNA and particularly dsRNA/LyoVec caused IFN-β and IFN-λ₁ protein production (24 h). dsRNA- but not dsRNA/LyoVec-induced IFN expression was partly inhibited by chloroquine that suppresses endosomal TLR3 activation. RV1B dose-dependently increased BSMC expression of RIG-I, MDA5, IFN-β, and IFN-λ₁ mRNA. We suggest that BSMCs express functional RLRs and that both RLRs and TLR3 are involved in viral stimulus-induced BSMC expression of IFN-β and IFN-λ₁.     

The Essential,Nonredundant Roles of RIG-I and MDA5 in Detecting and Controlling West Nile Virus Infection

Virus recognition and response by the innate immune system are critical components of host defense against infection. Activation of cell-intrinsic immunity and optimal priming of adaptive immunity against West Nile virus (WNV), an emerging vector-borne virus, depend on recognition by RIG-I and MDA5, two cytosolic pattern recognition receptors (PRRs) of the RIG-I-like receptor (RLR) protein family that recognize viral RNA and activate defense programs that suppress infection. We evaluated the individual functions of RIG-I and MDA5 both in vitro and in vivo in pathogen recognition and control of WNV. Lack of RIG-I or MDA5 alone results in decreased innate immune signaling and virus control in primary cells in vitro and increased mortality in mice. We also generated RIG-I^−/− × MDA5^−/− double-knockout mice and found that a lack of both RLRs results in a complete absence of innate immune gene induction in target cells of WNV infection and a severe pathogenesis during infection in vivo, similar to findings for animals lacking MAVS, the central adaptor molecule for RLR signaling. We also found that RNA products from WNV-infected cells but not incoming virion RNA display at least two distinct pathogen-associated molecular patterns (PAMPs) containing 5′ triphosphate and double-stranded RNA that are temporally distributed and sensed by RIG-I and MDA5 during infection. Thus, RIG-I and MDA5 are essential PRRs that recognize distinct PAMPs that accumulate during WNV replication. Collectively, these experiments highlight the necessity and function of multiple related, cytoplasmic host sensors in orchestrating an effective immune response against an acute viral infection.     

In Vivo Immune Evasion Mediated by the Herpes Simplex Virus Type 1 Immunoglobulin G Fc Receptor

Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcγR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcγR activity without affecting other gE functions, we constructed a mutant virus, NS-gE₃₃₉, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcγRs. NS-gE₃₃₉ expresses gE and gI, is FcγR⁻, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcγR does not bind murine IgG; therefore, the absence of an FcγR should not affect virulence in mice. NS-gE₃₃₉ causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcγR⁻ mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcγR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE₃₃₉-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcγR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.     

Evidence for a Novel Mechanism of Influenza Virus-Induced Type I Interferon Expression by a Defective RNA-Encoded Protein

Influenza A virus (IAV) defective RNAs are generated as byproducts of error-prone viral RNA replication. They are commonly derived from the larger segments of the viral genome and harbor deletions of various sizes resulting in the generation of replication incompatible viral particles. Furthermore, small subgenomic RNAs are known to be strong inducers of pattern recognition receptor RIG-I-dependent type I interferon (IFN) responses. The present study identifies a novel IAV-induced defective RNA derived from the PB2 segment of A/Thailand/1(KAN-1)/2004 (H5N1). It encodes a 10 kDa protein (PB2_∆) sharing the N-terminal amino acid sequence of the parental PB2 protein followed by frame shift after internal deletion. PB2_∆ induces the expression of IFNβ and IFN-stimulated genes by direct interaction with the cellular adapter protein MAVS, thereby reducing viral replication of IFN-sensitive viruses such as IAV or vesicular stomatitis virus. This induction of IFN is completely independent of the defective RNA itself that usually serves as pathogen-associated pattern and thus does not require the cytoplasmic sensor RIG-I. These data suggest that not only defective RNAs, but also some defective RNA-encoded proteins can act immunostimulatory. In this particular case, the KAN-1-induced defective RNA-encoded protein PB2_∆ enhances the overwhelming immune response characteristic for highly pathogenic H5N1 viruses, leading to a more severe phenotype in vivo.     

Melanoma Differentiation-Associated Gene 5 (MDA5) Is Involved in the Innate Immune Response to Paramyxoviridae Infection In Vivo

The early host response to pathogens is mediated by several distinct pattern recognition receptors. Cytoplasmic RNA helicases including RIG-I and MDA5 have been shown to respond to viral RNA by inducing interferon (IFN) production. Previous in vitro studies have demonstrated a direct role for MDA5 in the response to members of the Picornaviridae, Flaviviridae and Caliciviridae virus families ((+) ssRNA viruses) but not to Paramyxoviridae or Orthomyxoviridae ((−) ssRNA viruses). Contrary to these findings, we now show that MDA5 responds critically to infections caused by Paramyxoviridae in vivo. Using an established model of natural Sendai virus (SeV) infection, we demonstrate that MDA5^−/− mice exhibit increased morbidity and mortality as well as severe histopathological changes in the lower airways in response to SeV. Moreover, analysis of viral propagation in the lungs of MDA5^−/− mice reveals enhanced replication and a distinct distribution involving the interstitium. Though the levels of antiviral cytokines were comparable early during SeV infection, type I, II, and III IFN mRNA expression profiles were significantly decreased in MDA5^−/− mice by day 5 post infection. Taken together, these findings indicate that MDA5 is indispensable for sustained expression of IFN in response to paramyxovirus infection and provide the first evidence of MDA5-dependent containment of in vivo infections caused by (−) sense RNA viruses.     

The Herpes Simplex Virus US11 Protein Effectively Compensates for the γ134.5 Gene if Present before Activation of Protein Kinase R by Precluding Its Phosphorylation and That of the α Subunit of Eukaryotic Translation Initiation Factor 2

In herpes simplex virus-infected cells, viral γ₁34.5 protein blocks the shutoff of protein synthesis by activated protein kinase R (PKR) by directing the protein phosphatase 1α to dephosphorylate the α subunit of eukaryotic translation initiation factor 2 (eIF-2α). The amino acid sequence of the γ₁34.5 protein which interacts with the phosphatase has high homology to a domain of the eukaryotic protein GADD34. A class of compensatory mutants characterized by a deletion which results in the juxtaposition of the α47 promoter next to U_S11, a γ₂ (late) gene in wild-type virus-infected cells, has been described. In cells infected with these mutants, protein synthesis continues even in the absence of the γ₁34.5 gene. In these cells, PKR is activated but eIF-2α is not phosphorylated, and the phosphatase is not redirected to dephosphorylate eIF-2α. We report the following: (i) in cells infected with these mutants, U_S11 protein was made early in infection; (ii) U_S11 protein bound PKR and was phosphorylated; (iii) in in vitro assays, U_S11 blocked the phosphorylation of eIF-2α by PKR activated by poly(I-C); and (iv) U_S11 was more effective if present in the reaction mixture during the activation of PKR than if added after PKR had been activated by poly(I-C). We conclude the following: (i) in cells infected with the compensatory mutants, U_S11 made early in infection binds to PKR and precludes the phosphorylation of eIF-2α, whereas U_S11 driven by its natural promoter and expressed late in infection is ineffective; and (ii) activation of PKR by double-stranded RNA is a common impediment countered by most viruses by different mechanisms. The γ₁34.5 gene is not highly conserved among herpesviruses. A likely scenario is that acquisition by a progenitor of herpes simplex virus of a portion of the cellular GADD34 gene resulted in a more potent and reliable means of curbing the effects of activated PKR. U_S11 was retained as a γ₂ gene because, like many viral proteins, it has multiple functions.The herpes simplex virus 1 (HSV-1) genome encodes two sets of functions. The first and paramount are functions related to viral gene expression, replication of viral DNA, synthesis of virion proteins, assembly, packaging, and egress of the virus from the infected cell. The second set of functions, no less important in the survival of the virus in the human population, is creation of the environment necessary to maximize the yield and spread of virus from cell to cell and from infected to uninfected individuals (reviewed in reference 38). Of these known genes, several play a significant role in abating or delaying a host response to infection. The earliest to be expressed is the U_L41 gene which encodes a protein that is introduced into the cell in virions during infection (26, 27). This protein reduces the synthesis of host proteins by causing the destruction of mRNA in a rather nonspecific manner and therefore could be expected to reduce the synthesis of cellular proteins deleterious to viral replication (26, 27, 44).A second and very different approach to blocking host defense mechanisms is exemplified by infected cell protein 47 (ICP47). Proteosomal degradation of viral proteins could be expected to produce antigenic peptides which, if presented on the cell surface, could provoke a cytotoxic cell response early in infection and thus reduce viral yield. ICP47, an α protein made immediately after infection, blocks the presentation of antigenic peptides on the surface of the infected cells (20).The focus of this laboratory has been on a third viral pathway designed to block cellular response to infection. In cells infected with most viruses, the synthesis of complementary mRNA leads to activation of double-stranded RNA-dependent protein kinase R (PKR). This enzyme phosphorylates the α subunit of eukaryotic translation initiation factor 2 (eIF-2α) (23). A consequence of this phosphorylation is total shutoff of protein synthesis. This would be an example of a noble sacrifice of the infected cell for the sake of survival of the organism were it not for the fact that viruses, while activating the PKR kinase pathway by making double-stranded RNA, also express functions which block this host defense system (2–4, 6, 7, 10, 28, 30, 34). In the case of HSV-1, more than 50% of the viral DNA is represented late in infection in the form of cRNA (21, 25), and the gene whose product blocks the consequences of activation of PKR is γ₁34.5 (7). In the absence of the gene, eIF-2α is phosphorylated and protein synthesis is impaired beginning approximately 5 h after infection (7, 9). In its presence, protein synthesis continues unabated even though PKR is activated (9). Recent studies have shown that the carboxyl terminus of the γ₁34.5 gene binds to the protein phosphatase 1α (PP1) and redirects it to dephosphorylate eIF-2α (19). The effectiveness of the γ₁34.5-PP1 complex is apparent from the observation that the rate of dephosphorylation of eIF-2α in cells infected with wild-type virus is more than 1000 times that of uninfected cells or cells infected with the γ₁34.5⁻ virus (5, 19).The studies described in this report concern another aspect of virus-induced block of the consequence of activation of PKR. Briefly, Mohr and Gluzman reported that serial passage of a γ₁34.5⁻ mutant resulted in the selection of a compensatory mutation capable of sustained protein synthesis (35). A characteristic of the compensatory mutants isolated by Mohr and Gluzman is a deletion in the α47 gene resulting in the juxtaposition of the promoter of the α47 gene next to the 5′ end of U_S11, a late (γ₂) viral gene. Preliminary studies of those mutants revealed that PKR was activated in cells infected with either the wild-type parent or the γ₁34.5⁻ virus, but protein synthesis was unaffected in cells infected with wild-type virus or the mutant carrying the compensatory mutations (5, 18).In an attempt to define the phenotype of the virus carrying the compensatory mutation, we constructed a mutant lacking the γ₁34.5 and the U_S8 to -12 genes. This mutant, designated R5103, activated PKR and caused a shutoff of protein synthesis (5). We then inserted into the R5103 genome a DNA fragment consisting of the intact U_S10 gene and the U_S11 open reading frame fused to the α47 promoter. This virus, designated R5104, activated PKR but did not induce the shutoff of protein synthesis. Consistent with the conclusion of Mohr and Gluzman (35), the mutation maps in the domain inserted into the R5104 virus (5). Further studies yielded two significant observations. First, in stark contrast to lysates of cells infected with R5103 and other γ₁34.5⁻ mutants, the lysates of R5104 virus failed to phosphorylate the α subunit of eIF-2 (5). Second, in striking contrast to lysates of wild-type virus-infected cells, the phosphatase activity of lysates of R5104 virus-infected cells specific for eIF-2α could not be differentiated from that of mock-infected cells or those of cells infected with other γ₁34.5⁻ mutants (5). These results indicated that the compensatory mutation blocks PKR from phosphorylating eIF-2α.The studies summarized in this report focused on U_S11 protein. We report that in cells infected with the R5104 recombinant the U_S11 protein is made early in infection, that U_S11 protein interacts with PKR and blocks the phosphorylation of eIF-2α by activated PKR in in vitro assays, and that the effectiveness of the U_S11 protein is greater if the protein is present in the reaction before activation of PKR than if it is after PKR has been activated by the addition of poly(I-C). We also found that U_S11 is phosphorylated in the presence of activated PKR but not in its absence. We conclude that U_S11 may have been an ancient mechanism for blocking the effects of activated PKR and that it has been supplanted by acquisition of the carboxyl-terminal domain of the γ₁34.5 protein from a cellular gene. We also note that U_S11 protein made late in infection, after PKR has been activated, is ineffective.Relevant to this report are some of the properties of the U_S11 protein. U_S11 is one of the most abundant viral proteins expressed at late times in viral infection (22, 31). It binds mRNA in a sequence- and conformation-specific fashion (39–41). In HSV-1-infected cells, U_S11 suppresses the synthesis of a truncated RNA colinear with the 5′ domain of the U_L34 mRNA (40). The protein accumulates in nucleoli, in the cytoplasm in association with the 60S ribosomal subunit, and it is also packaged in virions (31, 37, 41). In newly infected cells, the U_S11 protein has been found associated with ribosomes (41).Recently a plethora of reports suggested that U_S11 may have novel functions not readily apparent from its localization in the infected cell. Thus, U_S11 protein has been reported to have functions similar to those of human immunodeficiency Tat and Rev proteins and has also been reported to complement Rev function in a Rev⁻ human immunodeficiency virus mutant (11). The U_S11 protein has been reported to confer thermotolerance and help restore protein synthesis in HeLa cells subjected to thermal injury (12).     

Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity

Alpha/beta interferon immune defenses are essential for resistance to viruses and can be triggered through the actions of the cytoplasmic helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). Signaling by each is initiated by the recognition of viral products such as RNA and occurs through downstream interaction with the IPS-1 adaptor protein. We directly compared the innate immune signaling requirements of representative viruses of the Flaviviridae, Orthomyxoviridae, Paramyxoviridae, and Reoviridae for RIG-I, MDA5, and interferon promoter-stimulating factor 1 (IPS-1). In cultured fibroblasts, IPS-1 was essential for innate immune signaling of downstream interferon regulatory factor 3 activation and interferon-stimulated gene expression, but the requirements for RIG-I and MDA5 were variable. Each was individually dispensable for signaling triggered by reovirus and dengue virus, whereas RIG-I was essential for signaling by influenza A virus, influenza B virus, and human respiratory syncytial virus. Functional genomics analyses identified cellular genes triggered during influenza A virus infection whose expression was strictly dependent on RIG-I and which are involved in processes of innate or adaptive immunity, apoptosis, cytokine signaling, and inflammation associated with the host response to contemporary and pandemic strains of influenza virus. These results define IPS-1-dependent signaling as an essential feature of host immunity to RNA virus infection. Our observations further demonstrate differential and redundant roles for RIG-I and MDA5 in pathogen recognition and innate immune signaling that may reflect unique and shared biologic properties of RNA viruses whose differential triggering and control of gene expression may impact pathogenesis and infection.     

Origin and evolution of the RIG-I like RNA helicase gene family

Background  

The DExD/H domain containing RNA helicases such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) of invading viruses. The RIG-I and MDA5 proteins differentially recognise conserved PAMPs in double stranded or single stranded viral RNA molecules, leading to activation of the interferon system in vertebrates. They share three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. The RIG-I/MDA5 directed interferon response is negatively regulated by laboratory of genetics and physiology 2 (LGP2) and is believed to be controlled by the mitochondria antiviral signalling protein (MAVS), a CARD containing protein associated with mitochondria.