Activation and evasion of innate antiviral immunity by herpes simplex virus

Herpes simplex virus (HSV), a human pathogenic virus, has evolved several strategies to evade the production and function of interferons (IFNs) and cytokines generated by the innate immune system to restrict the virus. Equilibrium exists between the virus and the immune response, and a shift in this delicate balance either restricts the virus or enhances virus spread and tissue damage. Therefore, understanding of the cytokine response generated after HSV infection and the underlying virus-cell interactions is essential to improve our understanding of viral pathogenesis. This review summarizes the current knowledge on induction and evasion of the innate immune response by HSV.     

Hepatitis C virus triggers mitochondrial fission and attenuates apoptosis to promote viral persistence

Mitochondrial dynamics is crucial for the regulation of cell homeostasis. Our recent findings suggest that hepatitis C virus (HCV) promotes Parkin-mediated elimination of damaged mitochondria (mitophagy). Here we show that HCV perturbs mitochondrial dynamics by promoting mitochondrial fission followed by mitophagy, which attenuates HCV-induced apoptosis. HCV infection stimulated expression of dynamin-related protein 1 (Drp1) and its mitochondrial receptor, mitochondrial fission factor. HCV further induced the phosphorylation of Drp1 (Ser616) and caused its subsequent translocation to the mitochondria, followed by mitophagy. Interference of HCV-induced mitochondrial fission and mitophagy by Drp1 silencing suppressed HCV secretion, with a concomitant decrease in cellular glycolysis and ATP levels, as well as enhanced innate immune signaling. More importantly, silencing Drp1 or Parkin caused significant increase in apoptotic signaling, evidenced by increased cytochrome C release from mitochondria, caspase 3 activity, and cleavage of poly(ADP-ribose) polymerase. These results suggest that HCV-induced mitochondrial fission and mitophagy serve to attenuate apoptosis and may contribute to persistent HCV infection.Hepatitis C virus (HCV) infection often leads to chronic hepatitis that can progress to fibrosis, cirrhosis, and hepatocellular carcinoma (1). HCV is a hepatotropic, noncytopathic (2, 3), single-stranded, positive-sense RNA virus that replicates its RNA genome on the endoplasmic reticulum (ER)-derived membranous structures (4, 5). HCV stimulates lipogenesis, leading to the accumulation of lipid droplets that facilitate virion assembly and maturation (5–8). HCV infection also induces mitochondrial dysfunction via ER and oxidative stress that results in mitochondrial Ca²⁺ overload, collapse of mitochondrial transmembrane potential (ΔΨm), elevated levels of reactive oxygen species, and disruption of mitochondrial respiration (9–15). Liver tissues of patients with chronic hepatitis C frequently exhibit traits of mitochondrial injury such as swollen, ruptured, and empty mitochondria (16).Mitochondria are dynamic organelles that constantly undergo fission, fusion, and mitophagy to facilitate mitochondrial quality control, which is crucial for maintaining cell viability and bioenergetics (17). Aberrant mitochondrial dynamics are associated with the pathogenesis of several genetic and neurological disorders, cardiac dysfunctions, cancer, and metabolic diseases such as diabetes and obesity (18). Depending on their physiological and cellular context, the balance between mitochondrial fission and fusion processes modulates the mitochondrial morphology (17). Mitochondrial fission/fragmentation is mediated by recruitment of cytosolic Drp1 to the mitochondria, forming spirals that constrict both the inner and outer mitochondrial membranes (19). The mitochondrial fission is modulated by mitochondrial outer membrane proteins, which include mitochondrial fission 1 (Fis1), mitochondria fission factor (Mff), and mitochondrial dynamics proteins of 49 and 51 kDa. These proteins coordinate to recruit Drp1 to mitochondria (20, 21). Mitochondrial fusion involves mitofusin 1 and 2 proteins and the inner mitochondrial membrane protein optic atrophy 1 (19, 21). More specifically, Drp1 recruitment to mitochondria is regulated by phosphorylation and dephosphorylation of respective serine residues by putative kinases and phosphatases (19). Mitochondrial dynamics is tightly regulated in response to alterations in cellular physiology such as stress, infections, and nutrient supply, and is also shown to play a critical role in apoptosis (18, 22).In this study, we investigated the HCV-induced modulation of mitochondrial dynamics, which plays a crucial role in attenuating apoptosis of infected cells resulting from mitochondrial injury associated with infection. We show that HCV stimulates the gene expression of Drp1 and Mff and promotes Drp1 recruitment to mitochondria by stimulating the phosphorylation of Drp1 (Ser616), leading to mitochondrial fission analyzed by confocal and electron microscopy. By using a dual-fluorescence mito-monomeric red fluorescent protein (mRFP)-EGFP reporter for monitoring complete mitophagy, we demonstrate that HCV-induced Drp1-mediated mitochondrial fission was followed by mitophagy. Interference of HCV-induced mitochondrial fission by silencing either Drp1 or Mff led to the accumulation of swollen mitochondria that resisted mitophagic degradation. Interestingly, interference of mitochondrial fission also suppressed viral secretion and glycolysis paralleled by a concomitant decline in cellular ATP levels and increased IFN synthesis in the HCV-infected cells. More importantly, inhibition of HCV-induced aberrant mitochondrial fission and mitophagy triggered robust apoptosis evidenced by a marked increase in cytochrome C release, caspase 3 activity, and cleavage of poly(ADP-ribose) polymerase. These observations unambiguously implicate the functional relevance of mitochondrial dynamics in the maintenance of persistent HCV infection.     

Structural basis for the sequence-specific recognition of human ISG15 by the NS1 protein of influenza B virus

Interferon-induced ISG15 conjugation plays an important antiviral role against several viruses, including influenza viruses. The NS1 protein of influenza B virus (NS1B) specifically binds only human and nonhuman primate ISG15s and inhibits their conjugation. To elucidate the structural basis for the sequence-specific recognition of human ISG15, we determined the crystal structure of the complex formed between human ISG15 and the N-terminal region of NS1B (NS1B-NTR). The NS1B-NTR homodimer interacts with two ISG15 molecules in the crystal and also in solution. The two ISG15-binding sites on the NS1B-NTR dimer are composed of residues from both chains, namely residues in the RNA-binding domain (RBD) from one chain, and residues in the linker between the RBD and the effector domain from the other chain. The primary contact region of NS1B-NTR on ISG15 is composed of residues at the junction of the N-terminal ubiquitin-like (Ubl) domain and the short linker region between the two Ubl domains, explaining why the sequence of the short linker in human and nonhuman primate ISG15s is essential for the species-specific binding of these ISG15s. In addition, the crystal structure identifies NS1B-NTR binding sites in the N-terminal Ubl domain of ISG15, and shows that there are essentially no contacts with the C-terminal Ubl domain of ISG15. Consequently, NS1B-NTR binding to ISG15 would not occlude access of the C-terminal Ubl domain of ISG15 to its conjugating enzymes. Nonetheless, transfection assays show that NS1B-NTR binding of ISG15 is responsible for the inhibition of interferon-induced ISG15 conjugation in cells.     

US3 protein kinase of HSV-1 cycles between the cytoplasm and nucleus and interacts with programmed cell death protein 4 (PDCD4) to block apoptosis

The U(S)3 protein kinase of herpes simplex virus 1 plays a key role in blocking apoptosis induced by viral gene products or exogenous agents. The U(S)3 protein kinase is similar to protein kinase A with respect to substrate range and specificity. We report that in the yeast two-hybrid system a domain of U(S)3 essential for antiapoptotic activity reacted with programmed cell death protein 4 (PDCD4). We report that U(S)3 interacts with PDCD4, that PDCD4 is posttranslationally modified in infected cells both in a U(S)3-dependent and -independent fashion, and that depletion of PDCD4 by siRNA blocked apoptosis induced by a Δα4 mutant virus. In infected cells, PDCD4 accumulates in the nucleus, whereas U(S)3 accumulates in the cytoplasm. Studies designed to elucidate the convergence of these proteins led to the discovery that U(S)3 protein kinase cycles between the nucleus and cytoplasm and that U(S)3 retains PDCD4 in infected cell nuclei.     

Inhibition of RIG-I and MDA5-dependent antiviral response by gC1qR at mitochondria

gC1qR is one of the C1q receptors implicated in the regulation of innate and adaptive immunity. We found that gC1qR inhibits RIG-I and MDA5-dependent antiviral signaling. Double stranded RNA and virus trigger the translocation of gC1qR to the mitochondrial outer membrane leading to the interaction of gC1qR with the RIG-I and MDA5 adaptor, VISA/MAVS/IPS-1/Cardif. The interaction of gC1qR with VISA/MAVS/IPS-1/Cardif at mitochondria results in the disruption of RIG-I and MDA5 signaling and the promotion of virus replication. Knockdown of endogenous gC1qR enhances RIG-I-dependent antiviral signaling, and augments the inhibition of virus proliferation. Therefore, gC1qR is a physiological inhibitor of the RIG-I and MDA5-mediated antiviral signaling pathway. These data uncover a new viral mechanism used to negatively control antiviral signaling in host cells.     

Interleukin-1 receptor antagonist prevents murine bronchopulmonary dysplasia induced by perinatal inflammation and hyperoxia

Bronchopulmonary dysplasia (BPD) is a common lung disease of premature infants, with devastating short- and long-term consequences. The pathogenesis of BPD is multifactorial, but all triggers cause pulmonary inflammation. No therapy exists; therefore, we investigated whether the anti-inflammatory interleukin-1 receptor antagonist (IL-1Ra) prevents murine BPD. We precipitated BPD by perinatal inflammation (lipopolysaccharide injection to pregnant dams) and rearing pups in hyperoxia (65% or 85% O₂). Pups were treated daily with IL-1Ra or vehicle for up to 28 d. Vehicle-injected animals in both levels of hyperoxia developed a severe BPD-like lung disease (alveolar number and gas exchange area decreased by up to 60%, alveolar size increased up to fourfold). IL-1Ra prevented this structural disintegration at 65%, but not 85% O₂. Hyperoxia depleted pulmonary immune cells by 67%; however, extant macrophages and dendritic cells were hyperactivated, with CD11b and GR1 (Ly6G/C) highly expressed. IL-1Ra partially rescued the immune cell population in hyperoxia (doubling the viable cells), reduced the percentage that were activated by up to 63%, and abolished the unexpected persistence of IL-1α and IL-1β on day 28 in hyperoxia/vehicle-treated lungs. On day 3, perinatal inflammation and hyperoxia each triggered a distinct pulmonary immune response, with some proinflammatory mediators increasing up to 20-fold and some amenable to partial or complete reversal with IL-1Ra. In summary, our analysis reveals a pivotal role for IL-1α/β in murine BPD and an involvement for MIP (macrophage inflammatory protein)-1α and TREM (triggering receptor expressed on myeloid cells)-1. Because it effectively shields newborn mice from BPD, IL-1Ra emerges as a promising treatment for a currently irremediable disease that may potentially brighten the prognosis of the tiny preterm patients.     

Phospholipase A2 inhibitor and LY6/PLAUR domain-containing protein PINLYP regulates type I interferon innate immunity

Type I interferons (IFNs) are the first frontline of the host innate immune response against invading pathogens. Herein, we characterized an unknown protein encoded by phospholipase A2 inhibitor and LY6/PLAUR domain-containing (PINLYP) gene that interacted with TBK1 and induced type I IFN in a TBK1- and IRF3-dependent manner. Loss of PINLYP impaired the activation of IRF3 and production of IFN-β induced by DNA virus, RNA virus, and various Toll-like receptor ligands in multiple cell types. Because PINLYP deficiency in mice engendered an early embryonic lethality in mice, we generated a conditional mouse in which PINLYP was depleted in dendritic cells. Mice lacking PINLYP in dendritic cells were defective in type I IFN induction and more susceptible to lethal virus infection. Thus, PINLYP is a positive regulator of type I IFN innate immunity and important for effective host defense against viral infection.

Interferon (IFN)-mediated antiviral responses serve as the first line of the host innate immune defense against viral infection. IFNs are divided into three families based on sequence homology: type I, type II, and type III (1, 2). The type I IFN family encodes 13 subtypes of IFN-α in humans (14 in mice), a single IFN-β subtype, and several poorly defined subtypes (3, 4). Type I IFNs were originally identified based on their ability to interfere with viral replication, restrain virus dissemination, and activate adaptive immune responses (5–7). They can be induced in most cell types by microbial pathogen-associated and damage-associated molecular patterns recognized by pattern recognition receptors (PRRs) (3). By inducing the expression of IFN-stimulated genes (ISGs), type I IFNs elicit antiviral innate immunity and mediate adaptive immune responses (8, 9).The induction of antiviral type I IFN response is elicited in response to the stimulation of PRRs that detect pathogen-associated molecular patterns, such as viral nucleic acids, viral replicative intermediates, and surface glycoproteins (10, 11). There are four major subfamilies of PRRs: the Toll-like receptors (TLRs), nucleotide-binding oligomerization domain/leucine-rich repeat-containing receptors, RIG-1-like receptors (RLRs), and the C-type lectin receptors, which are located at the cell surface, in the cytosol, or endosomal compartments (11–14). Among the TLR family members, TLR3, TLR7, TLR8, and TLR9 are involved in the recognition of viral nucleotides. Viral DNA enriched in CpG-DNA motifs is recognized by TLR9, single-stranded RNA is recognized by TLR7 and TLR8, and double-stranded RNA and its synthetic analog polyinosinic-polycytidylic acid (poly I:C) are recognized by TLR3 (15, 16). Some viral envelope proteins can be recognized by TLR4 or TLR2 (16, 17).Following viral infection, cytosolic DNA can be sensed by cyclic guanosine monophosphate (GMP)–adenosine monophosphate (AMP) synthase (cGAS) that induces the production of cyclic GMP-AMP (cGAMP) (18, 19). cGAMP functions as a second messenger that binds and activates the endoplasmic reticulum (ER) adaptor STING (19–22). Translocation of activated STING from the ER to the Golgi apparatus leads to the activation of kinase TBK1, which subsequently phosphorylates IRF3 and triggers the production of type I IFN (22–24). Cytosolic RNA can be recognized by the RLRs like RIG-1 and MDA5, which signal via mitochondrial antiviral signaling protein (MAVS; also known as CARDIF, IPS1, and VISA) and subsequently activate TBK1 and IRF3–IRF7, leading to the induction of type I IFNs and other antiviral genes (25–27).The lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) superfamily is characterized by the LU domain and a domain containing 10 cysteines that form distinct disulfide bridges, which create the three-fingered structural motif. The Ly6/uPAR family members regulate a wide range of functions in various cell types (28). Here, we uncovered the previously uncharacterized role of the Ly6/uPAR family member PINLYP in the induction of type I IFNs in response to DNA virus, RNA virus, and other TLR ligands. This study further defined the pivotal function of PINLYP in the effective host defense against virus infection.     

Viral PB1-F2 and host IFN-γ guide ILC2 and T cell activity during influenza virus infection

Functional plasticity of innate lymphoid cells (ILCs) and T cells is regulated by host environmental cues, but the influence of pathogen-derived virulence factors has not been described. We now report the interplay between host interferon (IFN)-γ and viral PB1-F2 virulence protein in regulating the functions of ILC2s and T cells that lead to recovery from influenza virus infection of mice. In the absence of IFN-γ, lung ILC2s from mice challenged with the A/California/04/2009 (CA04) H1N1 virus, containing nonfunctional viral PB1-F2, initiated a robust IL-5 response, which also led to improved tissue integrity and increased survival. Conversely, challenge with Puerto Rico/8/1934 (PR8) H1N1 virus expressing fully functional PB1-F2, suppressed IL-5⁺ ILC2 responses, and induced a dominant IL-13⁺ CD8 T cell response, regardless of host IFN-γ expression. IFN-γ–deficient mice had increased survival and improved tissue integrity following challenge with lethal doses of CA04, but not PR8 virus, and increased resistance was dependent on the presence of IFN-γR⁺ ILC2s. Reverse-engineered influenza viruses differing in functional PB1-F2 activity induced ILC2 and T cell phenotypes similar to the PB1-F2 donor strains, demonstrating the potent role of viral PB1-F2 in host resistance. These results show the ability of a pathogen virulence factor together with host IFN-γ to regulate protective pulmonary immunity during influenza infection.

Innate lymphoid cells (ILCs) and T cells represent critical populations of cells that have diverse roles in inflammation and protection (1, 2). Both cell populations consist of subsets that differ in cytokine expression and function. While T cells are important for viral clearance, they can also exacerbate lung immunopathology (1, 3–5) Among ILC subsets, ILC2s play a critical role in pulmonary immunity, particularly in maintaining the lung barrier surface (6–9). During infection, ILC2s respond to the epithelial cell–derived cytokines IL-25, IL-33, and thymic stromal lymphopoietin, and produce the type 2 cytokine IL-5 (10–12). This, in turn, can lead to increased eosinophil recruitment and airway hyperreactivity (AHR) (8, 13–15). Like T cells, ILC2s can play both beneficial and detrimental roles during viral lung infection (6–8).It is known that host cytokines can regulate the activity of ILC and T cell subsets. For example, we previously found that interferon (IFN)-γ deficiency results in enhanced ILC2 activity and increased survival from challenge with the 2009 pandemic strain A/California/04/2009 (CA04) influenza A virus (8). However, our current studies have shown no effect of IFN-γ following challenge with the Puerto Rico/8/1934 (PR8) influenza A virus, a strain that is a commonly used model for the highly virulent 1918 pandemic influenza virus. Although both strains are H1N1 influenza A viruses, they have striking differences in expression of functional PB1-F2, a viral proapoptotic protein that is associated with immunopathology and mortality (16). While the PR8 viral strain expresses full-length PB1-F2, the PB1-F2 gene in the CA04 strain is truncated and nonfunctional (16–20). As a result, the PR8 virus exhibits significantly increased virulence compared to the CA04 viral strain. However, the impact of PB1-F2 on the lymphocyte function that is critical for protection during influenza is not known. A better understanding of the role of pathogen virulence factors in regulating immune cell activity during influenza may aid in designing future therapies for human use.We hypothesized that the PB1-F2 virulence protein can differentially regulate ILC2 and T cell activity in conjunction with host IFN-γ signaling. To test this hypothesis, we have investigated pulmonary immunity in wild-type (WT) and IFN-γ–deficient BALB/c mice infected with PB1-F2 gene reassortant PR8 and CA04 viruses. Our findings demonstrate that viral virulence genes, together with host factors, play critical roles in regulating both ILC2 and T cell responses during influenza, and this, in turn, determines host survival.     

Natural killer cell activation enhances immune pathology and promotes chronic infection by limiting CD8+ T-cell immunity

Infections with HIV, hepatitis B virus, and hepatitis C virus can turn into chronic infections, which currently affect more than 500 million patients worldwide. It is generally thought that virus-mediated T-cell exhaustion limits T-cell function, thus promoting chronic disease. Here we demonstrate that natural killer (NK) cells have a negative impact on the development of T-cell immunity by using the murine lymphocytic choriomeningitis virus. NK cell-deficient (Nfil3(-/-), E4BP4(-/-)) mice exhibited a higher virus-specific T-cell response. In addition, NK cell depletion caused enhanced T-cell immunity in WT mice, which led to rapid virus control and prevented chronic infection in lymphocytic choriomeningitis virus clone 13- and reduced viral load in DOCILE-infected animals. Further experiments showed that NKG2D triggered regulatory NK cell functions, which were mediated by perforin, and limited T-cell responses. Therefore, we identified an important role of regulatory NK cells in limiting T-cell immunity during virus infection.     

Ca2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca2+ channels

A family of peptide signaling molecules (AtPeps) and their plasma membrane receptor AtPepR1 are known to act in pathogen-defense signaling cascades in plants. Little is currently known about the molecular mechanisms that link these signaling peptides and their receptor, a leucine-rich repeat receptor-like kinase, to downstream pathogen-defense responses. We identify some cellular activities of these molecules that provide the context for a model for their action in signaling cascades. AtPeps activate plasma membrane inwardly conducting Ca(2+) permeable channels in mesophyll cells, resulting in cytosolic Ca(2+) elevation. This activity is dependent on their receptor as well as a cyclic nucleotide-gated channel (CNGC2). We also show that the leucine-rich repeat receptor-like kinase receptor AtPepR1 has guanylyl cyclase activity, generating cGMP from GTP, and that cGMP can activate CNGC2-dependent cytosolic Ca(2+) elevation. AtPep-dependent expression of pathogen-defense genes (PDF1.2, MPK3, and WRKY33) is mediated by the Ca(2+) signaling pathway associated with AtPep peptides and their receptor. The work presented here indicates that extracellular AtPeps, which can act as danger-associated molecular patterns, signal by interaction with their receptor, AtPepR1, a plasma membrane protein that can generate cGMP. Downstream from AtPep and AtPepR1 in a signaling cascade, the cGMP-activated channel CNGC2 is involved in AtPep- and AtPepR1-dependent inward Ca(2+) conductance and resulting cytosolic Ca(2+) elevation. The signaling cascade initiated by AtPeps leads to expression of pathogen-defense genes in a Ca(2+)-dependent manner.     

Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection

The plant immune system is activated by microbial patterns that are detected as nonself molecules. Such patterns are recognized by immune receptors that are cytoplasmic or localized at the plasma membrane. Cell surface receptors are represented by receptor-like kinases (RLKs) that frequently contain extracellular leucine-rich repeats and an intracellular kinase domain for activation of downstream signaling, as well as receptor-like proteins (RLPs) that lack this signaling domain. It is therefore hypothesized that RLKs are required for RLPs to activate downstream signaling. The RLPs Cf-4 and Ve1 of tomato (Solanum lycopersicum) mediate resistance to the fungal pathogens Cladosporium fulvum and Verticillium dahliae, respectively. Despite their importance, the mechanism by which these immune receptors mediate downstream signaling upon recognition of their matching ligand, Avr4 and Ave1, remained enigmatic. Here we show that the tomato ortholog of the Arabidopsis thaliana RLK Suppressor Of BIR1-1/Evershed (SOBIR1/EVR) and its close homolog S. lycopersicum (Sl)SOBIR1-like interact in planta with both Cf-4 and Ve1 and are required for the Cf-4– and Ve1-mediated hypersensitive response and immunity. Tomato SOBIR1/EVR interacts with most of the tested RLPs, but not with the RLKs FLS2, SERK1, SERK3a, BAK1, and CLV1. SOBIR1/EVR is required for stability of the Cf-4 and Ve1 receptors, supporting our observation that these RLPs are present in a complex with SOBIR1/EVR in planta. We show that SOBIR1/EVR is essential for RLP-mediated immunity and propose that the protein functions as a regulatory RLK of this type of cell-surface receptors.     

Cytokine response in pediatric patients with pandemic influenza H1N1 2009 virus infection and pneumonia: comparison with pediatric pneumonia without H1N1 2009 infection

Objectives

We investigated serum cytokine levels in pediatric patients with pandemic influenza H1N1 2009 virus (H1N1) infection‐pneumonia and in pediatric patients with pneumonia but without H1N1 infection, and examined correlations between cytokine levels and clinical/laboratory findings.

Methods

Fifty‐seven cases of infection by H1N1 were confirmed by RT‐PCR and enrolled. Of these 57 cases, 26 had a severe H1N1 infection (group 1), and 31 had a mild H1N1 infection (group 2). Sera from 18 cases with pneumonia without H1N1 infection (group 3) were used as controls. The serum levels of 10 cytokines were determined by multiplex assay.

Results

The serum levels of IFN‐α, IL‐6, and IP‐10 were significantly higher in H1N1 infected cases than in group 3, and levels of IL‐6 and IP‐10 were significantly higher in group 1 than in group 2. The level of IL‐10 was significantly higher in groups 1 and 3 than in group 2. However, levels of IFN‐γ and IL‐17 were not significantly different between the three groups. IL‐1β, IL‐4, and MIP‐1α were not detectable in most patients. IP‐10 and IL‐6 levels were found to show negative correlations with lymphocyte count and oxygen saturation.

Conclusions

We found higher levels of cytokines (IFN‐α, IL‐6, IP‐10) of innate immunity than those of acquired immunity in pediatric H1N1 infection. Of the cytokines found to be increased in cases with H1N1 infection, IP‐10 and IL‐6 were found to be correlated with disease severity (lymphopenia and hypoxia). IP‐10 and IL‐6 may be important markers in pediatric H1N1 infection. Pediatr Pulmonol. 2011; 46: 1233–1239. © 2011 Wiley Periodicals, Inc.

     

Tobacco calmodulin-like protein provides secondary defense by binding to and directing degradation of virus RNA silencing suppressors

RNA silencing (RNAi) induced by virus-derived double-stranded RNA (dsRNA), which is in a sense regarded as a pathogen-associated molecular pattern (PAMP) of viruses, is a general plant defense mechanism. To counteract this defense, plant viruses express RNA silencing suppressors (RSSs), many of which bind to dsRNA and attenuate RNAi. We showed that the tobacco calmodulin-like protein, rgs-CaM, counterattacked viral RSSs by binding to their dsRNA-binding domains and sequestering them from inhibiting RNAi. Autophagy-like protein degradation seemed to operate to degrade RSSs with the sacrifice of rgs-CaM. These RSSs could thus be regarded as secondary viral PAMPs. This study uncovered a unique defense system in which an rgs-CaM-mediated countermeasure against viral RSSs enhanced host antiviral RNAi in tobacco.     

Host immunity influences disease progression and antiviral efficacy in humans infected with hepatitis B virus

Hepatitis B virus (HBV) infection can lead to several severe liver diseases, including hepatitis, cirrhosis and hepatocellular carcinoma, although the underlying mechanisms responsible for the clinical outcome have not been well characterized. In this review, we retrospectively examine the history of immunological responses to HBV infection and summarize the current understanding of innate and adaptive immunity in the context of HBV-associated liver disease. Recent data indicate that the interaction between HBV and the host immune response not only substantially drives disease progression, but also significantly influences antiviral efficacy in HBV-infected individuals. Advances in the field have provided insight into the immunopathology of HBV infection. Based on the characteristics of host immune responses in patients with HBV infection, a ‘climbing slope hypothesis’ is proposed to suggest that therapeutic strategies aimed at modulating the immune activity of the host may represent a complementary approach to antiviral drug treatment for the management of chronically HBV-infected patients.     

HSV-1 degrades,stabilizes, requires,or is stung by STING depending on ICP0, the US3 protein kinase,and cell derivation

STING (stimulator of IFN genes) activates the IFN pathway in response to cytosolic DNA. Knockout of STING in mice was reported to exacerbate the pathogenicity of herpes simplex virus 1 (HSV-1). Here we report the following: (i) STING is stable in cancer-derived HEp-2 or HeLa cells infected with wild-type HSV-1 but is degraded in cells infected with mutants lacking the genes encoding functional infected cell protein 0 (ICP0), ICP4, or the US3 protein kinase (US3-PK). In HEp-2 cells, depletion of STING by shRNA results in a decrease in the yields of wild-type or ΔICP0 viruses. (ii) STING is stable throughout infection with either wild-type or ICP0 mutant viruses in human embryonic lung cells (HEL) or HEK293T cells derived from normal tissues. In these cells, depletion of STING results in higher yields of both wild-type and ΔICP0 viruses. (iii) The US3-PK is also required for stabilization of IFI16, a nuclear DNA sensor. However, the stability of IFI16 does not correlate positively or negatively with that of STING. IFI16 is stable in STING-depleted HEL cells infected with wild-type virus. In contrast to HEL cells, IFI16 was undetectable in STING-depleted HEp-2 cells, and hence the role of HSV-1 in maintaining IFI16 could not be ascertained. The results indicate that in HSV-1–infected cells the stability of IFI16 and the function and stability of STING are dependent on cell derivation, the functional integrity of ICP0, and US3-PK, an indication that in wild-type virus-infected cells both proteins are actively stabilized. In HEp-2 cells, the stability of IFI16 requires STING.The studies described in this report stem from two observations. First, a voluminous literature singles out the infected cell protein 0 (ICP0) as the major herpes simplex virus 1 (HSV-1) protein dedicated to defeating host responses to infection (1). Many of the functions of ICP0 designed to defeat host responses are executed by direct interaction between ICP0 and host proteins (2–8). In some instances, a plausible connection is apparent but no physical contact between ICP0 and the host effector protein is demonstrable (9, 10). Thus, ICP0 is associated with blocking the activation of IRF3 although a physical interaction between ICP0 and IRF3 has not been reported (10). One hypothesis that could explain such observations is that ICP0 interacts with the partners of the targeted protein rather than the target itself.Second, ICP0 accumulates during the first phase of the replicative cycle in the nucleus in which it performs multiple functions to enable efficient replication. Sometime between 5 and 9 h after exposure of cells to virus and depending on cell line, functional integrity of ICP0, and the amount of foreign DNA introduced into the cell, etc., ICP0 disappears from the nucleus and accumulates in the cytoplasm (11, 12). Several functions of ICP0 linked to physical interactions with cytoplasmic proteins have been described. Thus, ICP0 interacts with EF-1δ and enhances translation efficiency and with CIN85 to recruit Cbl and deplete receptors from the cell surface (5, 8). An intriguing observation is that the appearance of ICP0 in the cytoplasm correlates with inhibition of IRF3-dependent activation of interferon-stimulated genes (ISGs) even though no evidence has emerged that ICP0 and IRF3 interact (10). Recent studies suggest that IFN synthesis via the IRF3/NF-κB pathway is activated by STING (stimulator of IFN genes also known as TMEM173, MPYS, MITA, and ERIS) (13–16). STING is a cytoplasmic sensor of ligand-bound or free DNA (13–16). Activated STING binds the protein kinase TBK1 and directs it to phosphorylate IRF3 to induce IFNβ production (13–16). One of the ligands of STING is IFI16, a nuclear DNA sensor (13, 15, 16). It has been proposed that IF116 presents DNA to STING (13, 15, 16). Moreover, it has been proposed that up-regulation of IFI16 down-regulates STING, and, conversely, down-regulation of IF116 up-regulates STING (17). The interest in STING stems from the reports that it plays a critical role in innate immune responses to viral, bacterial, and eukaryotic pathogens (13–16). STING knockout mice were significantly more susceptible to HSV-1 than the normal siblings (18). In essence, STING mediates the activation of the IFN pathway that is effectively blocked by a large number of functions encoded by HSV proteins. The question that led to the initiation of the studies reported here is the nature of the interaction between STING and viral gene products. In light of the reports of the interplay between STING and IFI16, a nuclear DNA sensor (13, 15, 16), and the evidence that STING is actively stabilized in infected cells, we also examined the fate of this protein in infected cells.In this report we show that the end result of the interplay between STING, IFI16, and HSV-1 is determined by the genotype of the infected cells and the functional integrity of ICP0 and of US3-PK. In essence, we report that both proteins appear to be actively stabilized by viral gene products and that STING is both essential and inimical to viral replication depending on the genotype of the cells. We also report that the patterns of stabilization of IFI16 and STING are not interdependent or covariant.     

Prior infection with classical swine H1N1 influenza viruses is associated with protective immunity to the 2009 pandemic H1N1 virus

Please cite this paper as: Kash et al. (2010) Prior infection with classical swine H1N1 influenza viruses is associated with protective immunity to the 2009 pandemic H1N1 virus. Influenza and Other Respiratory Viruses 4(3), 121–127. Background  The 2009 H1N1 pandemic emerged even though seasonal H1N1 viruses have circulated for decades. Epidemio‐logical evidence suggested that the current seasonal vaccine did not offer significant protection from the novel pandemic, and that people over the age of 50 might were less susceptible to infection. Objectives  In a mouse challenge study with the 2009 pandemic H1N1 virus, we evaluated protective immune responses elicited by prior infection with human and swine influenza A viruses. Results  Mice infected with A/Mexico/4108/2009 (Mex09) showed significant weight loss and 40% mortality. Prior infection with a 1976 classical swine H1N1 virus resulted in complete protection from Mex09 challenge. Prior infection with either a 2009 or a 1940 seasonal H1N1 influenza virus provided partial protection and a >100‐fold reduction in viral lung titers at day 4 post‐infection. Conclusions  These findings indicate that in experimental animals recently induced immunity to 1918‐derived H1N1 seasonal influenza viruses, and to a 1976 swine influenza virus, afford a degree of protection against the 2009 pandemic virus. Implications of these findings are discussed in the context of accumulating data suggesting partial protection of older persons during the 2009 pandemic.     

Inhibition of mitochondrial permeability transition prevents mitochondrial dysfunction,cytochrome c release and apoptosis induced by heart ischemia

Ischemia/reperfusion of heart causes contractile dysfunction, necrosis and/or apoptosis and is a major cause of human death, but the molecular mechanisms are unclear. We show that ischemia alone (without reperfusion) is sufficient to induce apoptosis and mitochondrial dysfunction, and we have investigated the mechanism responsible; 30 and 60 min stop-flow ischemia in Langendorff-perfused rat hearts induced progressive (a). release of cytochrome c from mitochondria to cytosol, (b). inhibition of the mitochondrial respiratory functions, (c). activation of caspase-3-like protease activity and (d). DNA strand breaks (however, only 2% of myocyte nuclei were TUNEL positive at 60 min). Fifteen minutes pre-perfusion of hearts with cyclosporin A, an inhibitor of mitochondrial-permeability transition (MPT), largely prevented all these ischemic changes. Pre-perfusion of hearts with FK506, an inhibitor of calcineurin, caused no protection. Pre-perfusion with DEVD-CHO, an inhibitor of caspase-3-like proteases, completely prevented ischemia-induced DNA strand breaks, but only partially blocked cytochrome c release and mitochondrial respiratory inhibition. Reperfusion of hearts after 30 min ischemia further stimulated caspase activity and nuclear apoptosis. We conclude that ischemia-induced MPT causes release of cytochrome c, which then activates the caspases that execute apoptosis and feedback to cause further cytochrome c release. The MPT-induced cytochrome c release is also largely responsible for the ischemic respiratory inhibition, which might contribute to contractile dysfunction or necrosis at reperfusion.