Influenza virus RNA structure: unique and common features

The influenza A virus genome consists of eight negative-sense RNA segments. Here we review the currently available data on structure-function relationships in influenza virus RNAs. Various ideas and hypotheses about the roles of influenza virus RNA folding in the virus replication are also discussed in relation to other viruses.     

Induction of potent cellular immune response in mice by hepatitis C virus NS3 protein with double-stranded RNA

Double-stranded RNA is produced during virus replication and, together with the viral antigen, is responsible for inducing host antivirus immunity. The hepatitis C virus (HCV) non-structural protein-3 (NS3) has been implicated in the immune evasion of HCV, and is one of the prime targets for inducing immunity against HCV infection. Mice were immunized with recombinant NS3 protein (rNS3) and poly (I:C) emulsified in Montanide ISA 720 (M720). Cytokine production was assayed by enzyme-linked immunospot assay, and CD4(+) IFN-gamma(+) T helper (Th) cells or CD8(+) IFN-gamma(+) cytotoxic T lymphocytes were detected by flow cytometry. Anti-NS3 titre and immunoglobulin G2a (IgG2a) and IgG1 levels were monitored by enzyme-linked immunosorbent assay. Administration of rNS3 formulated in poly (I:C) and M720 induced anti-NS3 titres with a predominantly IgG2a isotype comparable to those induced by rNS3 in CpG-ODN and M720. The cytokine profiles showed that this formulation induced a Th1-biased immune response with several-fold more interferon-gamma (IFN-gamma)-producing cells than interleukin-4-producing cells. In contrast, rNS3 in M720 induced a Th2-biased immune response. The frequency of IFN-gamma-producing CD4(+) and CD8(+) cells induced by rNS3 in poly (I:C) and M720 was significantly higher than that induced by rNS3, rNS3 in M720, or rNS3 in poly (I:C), and was comparable to that induced by rNS3 in CpG-ODN with M720. The antigen-specific CD8(+) T-cell immune response persisted for up to 7 months after immunization. In conclusion, poly (I:C) with rNS3 in M720 can elicit a strong and persistent Th1-biased immune response and a cytotoxic T-lymphocyte response through cross-priming in mice. This study highlighted a promising formulation for inducing an efficient cellular immune response against HCV that has potential for HCV vaccine development.     

Induction of the human protein P56 by interferon, double-stranded RNA, or virus infection

P56 is the most abundant protein induced by interferon (IFN) treatment of human cells. To facilitate studies on its induction pattern and cellular functions, we expressed recombinant P56 as a hexahistidine-tagged protein in Escherichia coli and purified it to apparent homogeneity using affinity chromatography. A polyclonal antibody raised against this recombinant protein was used to show that P56 is primarily a cytoplasmic protein. Cellular expression of P56 by transfection did not inhibit the replication of vesicular stomatitis virus and encephalomyocarditis virus. P56 synthesis was rapidly induced by IFN-beta, and the protein had a half-life of 6 h. IFN-gamma or poly(A)(+) could not induce the protein, but poly(I)-poly(C) or an 85-bp synthetic double-stranded RNA efficiently induced it. Similarly, infection of GRE cells, which are devoid of type I IFN genes, by vesicular stomatitis virus, encephalomyocarditis virus, or Sendai virus caused P56 induction. Surprisingly, Sendai virus could also induce P56 in the mutant cell line P2.1, which cannot respond to either IFN-alpha/beta or double-stranded RNA. Induction of P56 in the P2.1 cells and the parental U4C cells by virus infection was preceded by activation of IRF-3 as judged by its translocation to the nucleus from the cytoplasm.     

Atlantic salmon ISG15: Expression and conjugation to cellular proteins in response to interferon, double-stranded RNA and virus infections

ISG15 is one of the earliest and most predominant proteins to be induced in mammals following IFN-alpha/beta stimulation, which suggests that it has an important function in the interferon system. Similar to ubiquitin, ISG15 forms covalent conjugates with its target proteins, but free ISG15 is released from human lymphocytes and monocytes during IFN-alpha/beta stimulation. In this work we describe a 17.3 kDa ISG15 orthologue in Atlantic salmon (AsISG15) with characteristic features of ISG15 proteins including tandem ubiquitin-homology domains and a conserved carboxy-terminal conjugating motif (LRLRGG). Furthermore, Northern blot analysis revealed strong induction by polyinosinic polycytidylic acid (poly I:C) and by viral infections, while Western blot analysis using a specific antibody generated against AsISG15 confirmed induction mediated by recombinant Atlantic salmon IFN-alpha/beta and demonstrated conjugation of AsISG15 to cellular proteins. Interestingly, the pattern of AsISG15 modified target proteins differed during ISAV infection compared to direct IFN-alpha/beta stimulation. Immunoprecipitation experiments demonstrated extracellular, free AsISG15 in supernatants of leucocytes stimulated with poly I:C. Moreover, immunoprecipitation of an about 65 kDa ISAV protein from infected TO cells using anti-AsISG15 antiserum suggests that binding between the AsISG15 and the ISAV protein occurred. Taken together, the results suggest that AsISG15 has a role in the antiviral interferon response of Atlantic salmon.     

Biology and host response to Cyprinid herpesvirus 3 infection in common carp

Viruses from the family Alloherpesviridae form an aquatic clade of herpesviruses infecting fish and amphibia. Diseases caused by these herpesviruses are of increasing importance because of the high morbidity and mortality associated with the infection, and the difficulties in diagnosing latently infected carriers. Cyprinid herpesvirus 3 (CyHV-3) induces a severe disease and mortality in common carp and thus greatly affects carp aquaculture and trade. This review summarises advancements in the understanding of the infection process and the current knowledge on immune responses of carp to CyHV-3. A focus is laid on host genetics and immunity responsible for resistance/survival from the disease and on the viral mechanisms accountable for evasion of carp immune responses. As current knowledge of immune responses to CyHV-3 is still limited, perspectives for future studies are outlined. Analysing CyHV-3 fish-host interactions will be useful and thought-provoking for a basic understanding of fish immune responses.     

Immunofluorescent demonstration of double-stranded RNA and virus antigen in RNA virus-infected cells

The indirect immunofluorescence procedure has been used for demonstration of double-stranded RNA in cells infected with reovirus, poliomyelitis, and tick-borne encephalitis (TBE) viruses. Rabbit sera against poly(A)-poly(U) and poly(I)-poly(C) react specifically with double-stranded RNA. Double-stranded RNA is found in the cytoplasm of the cells infected with high multiplicities of poliomyelitis and tick-borne encephalitis viruses 3 hr postinoculation. In parallel, preparations were stained with sera against viral proteins. During one cycle of reproduction the dynamics of accumulation of double-stranded RNA and virus protein was synchronous both for poliomyelitis and tick-borne encephalitis viruses. When poliovirus-infected cells degenerated, the number of cells containing TBE virus double-stranded RNA decreased markedly while the proportion of cells containing virus protein remained high.     

Dendritic cells respond to influenza virus through TLR7- and PKR-independent pathways

Natural interferon-producing cells (IPC) secrete type I IFN (IFN-alpha and -beta) in response to influenza virus. This process is independent of viral replication and is mediated by Toll-like receptor 7 (TLR7), which recognizes single-stranded RNA (ssRNA). DC also express TLR7 but its function in DC response to influenza virus is unknown. To address this, we compared the DC and IPC responses to influenza virus and ssRNA oligoribonucleotides (ORN) that activate TLR7. When stimulated by ORN in vitro and in vivo, DC matured and produced inflammatory cytokines but not IFN-alpha. DC did secrete IFN-alpha in response to influenza virus. However, this response was independent of TLR7 signaling and required viral replication but not dsRNA-activated protein kinase (PKR). We conclude that DC and IPC are hard-wired to secrete IFN-alpha via different pathways, reflecting their complementary but distinct roles in anti-viral immunity.     

Mechanisms of beta-cell death in response to double-stranded (ds) RNA and interferon-gamma: dsRNA-dependent protein kinase apoptosis and nitric oxide-dependent necrosis.

Viral infection is one environmental factor that has been implicated as a precipitating event that may initiate beta-cell damage during the development of diabetes. This study examines the mechanisms by which the viral replicative intermediate, double-stranded (ds) RNA impairs beta-cell function and induces beta-cell death. The synthetic dsRNA molecule polyinosinic-polycytidylic acid (poly IC) stimulates beta-cell DNA damage and apoptosis without impairing islet secretory function. In contrast, the combination of poly IC and interferon (IFN)-gamma stimulates DNA damage, apoptosis, and necrosis of islet cells, and this damage is associated with the inhibition of glucose-stimulated insulin secretion. Nitric oxide mediates the inhibitory and destructive actions of poly IC + IFN-gamma on insulin secretion and islet cell necrosis. Inhibitors of nitric oxide synthase, aminoguanidine, and N(G)-monomethyl-L-arginine, attenuate poly IC + IFN-gamma-induced DNA damage to levels observed in response to poly IC alone, prevent islet cell necrosis, and prevent the inhibitory actions on glucose-stimulated insulin secretion. N(G)-monomethyl-L-arginine fails to prevent poly IC- and poly IC + IFN-gamma-induced islet cell apoptosis. PKR, the dsRNA-dependent protein kinase that mediates the antiviral response in infected cells, is required for poly IC- and poly IC + IFN-gamma-induced islet cell apoptosis, but not nitric oxide-mediated islet cell necrosis. Alone, poly IC fails to stimulate DNA damage in islets isolated from PKR-deficient mice; however, nitric oxide-dependent DNA damage induced by the combination of poly IC + IFN-gamma is not attenuated by the genetic absence of PKR. These findings indicate that dsRNA stimulates PKR-dependent islet cell apoptosis, an event that is associated with normal islet secretory function. In contrast, poly IC + IFN-gamma-induced inhibition of glucose-stimulated insulin secretion and islet cell necrosis are events that are mediated by islet production of nitric oxide. These findings suggest that at least one IFN-gamma-induced antiviral response (islet cell necrosis) is mediated through a PKR-independent pathway.     

Induction and distribution of amylolytic activity in Cucumis sativus L. in response to virus infection

Inoculation of cucumber (Cucumis sativus L. cv. Laura) cotyledons with tobacco necrosis virus (TNV) causes both qualitative and quantitative changes in the total and fractionated protein extracts as well as in amylolytic activity. Using a specific test it was demonstrated that the virus infection strongly enhances a major band (Rf 0.0645) of amylolytic activity, predominantly located in apoplast space. The accumulation of this extracellular amylolytic activity is regulated by a time-dependent manner and is correlated with the development of necrotic lesions. The amylolytic activity may be related to degradation of starch shown to be accumulated in the immediate vicinity of necrotic lesions associated with the hypersensitive response (HR). The possible biological function of the identified amylolytic activity in the term of ,,pathosmosis" is also discussed.     

RIG-I-like receptors: Sensing and responding to RNA virus infection

Viral and microbial pathogens contain specific motifs or pathogen-associated molecular patterns (PAMPs) that are recognized by cell surface- and endosome-associated Toll-like receptors (TLRs). RNA virus infection is also detected through TLR-independent mechanisms. Early viral replicative intermediates are detected by two recently characterized cystolic viral RNA receptors—RIG-I and MDA-5. Both are DExDH/box RNA helicases, and RIG-I specifically recognizes 5′-triphosphate containing viral RNA and transmits signals that induce type I interferon-mediated host immunity against virus infection. In this review, we will focus on RIG-I-like receptor (RLR) signal transduction and the regulatory mechanisms – ubiquitination, deubiquitination, ISGylation – underlying this important host response.     

Morphoproteomic profile of mTOR, Ras/Raf kinase/ERK, and NF-kappaB pathways in human gastric adenocarcinoma

Preclinical studies using human gastric adenocarcinoma (GAC) cell lines have shown that the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, can inhibit tumor growth and that the extracellular signal-regulated kinase (ERK) of the Ras/Raf kinase/ERK pathway is related to chemoresistance and apoptosis. We examined the state of activation of components of mTOR, Ras/Raf kinase/ERK, and nuclear factor (NF)-kappaB signal transduction pathways, as well as cell cycle protein analyte correlates in GAC cases. Formalin-fixed paraffin-embedded tissue microarray blocks containing samples from 210 cases of GAC were examined. Immunohistochemistry was utilized to detect the following antigens: S100P, upstream stimulator of ERK, and NF-kappaB pathways; phosphorylated (p)-mTOR (Ser 2448), p-ERK-1/2 (Thr 202/Tyr 204), and one of their common down-stream effectors, p-p70S6K(Thr 389); p-NF-kappaBp65(Ser 536); and cell cycle associated proteins, Ki-67, and S phase kinase-associated protein (Skp)2. Immunoreactivity (0 to 4+) of protein expression and compartmentalization were assessed by bright-field microscopy. The majority of cases showed positive (1+ to 4+) cytoplasmic/plasmalemmal p-mTOR (88%), and moderate-strong (2+ to 4+) nuclear p-p70S6K (93%) and nuclear S100P (81%) expression. A subset of cases exhibited moderate-strong nuclear p-ERK-1/2 (15%) and p-NF-kappaBp65 (36%) expression. The majority of cases showed concomitant moderate-strong (2+ to 4+) nuclear Ki-67 (71%) and Skp2 (68%). Nuclear expression levels of p-ERK-1/2 and p-NF-kappaBp65, of p-p70S6K and p-NF-kappaB, and of Ki-67 and Skp2, respectively, showed significant linear correlations in GAC (p <0.001). Additionally, there were statistically significant differences in the mean expression levels of p-ERK-1/2 and p-NF-kappaBp65 in diffuse vs intestinal types of GAC, with higher levels of both in the diffuse type ( p = 0.001 and p <0.0001, respectively). In summary, morphoproteomic analysis reveals constitutive activation of mTOR and to some extent, Ras/Raf kinase and NF-kappaB pathways in GAC, as evidenced by increased cytoplasmic p-mTOR, nuclear translocation of p-p70S6K and p-ERK-1/2 phosphorylated at putative sites of activation (Ser 2448, Thr 389, and Thr 202/Tyr 204, respectively), as well as correlative expression of cell cycle analytes, Ki-67, and Skp2. These results suggest that a prospective study is warranted to evaluate the use of morphoproteomic profiling of individual patients with GAC in order to design combinatorial treatment strategies that target the mTOR, Ras/Raf kinase/ERK, and/or NF-kappaB pathways.     

C and V proteins of Sendai virus target signaling pathways leading to IRF-3 activation for the negative regulation of interferon-beta production

We here report a molecular basis for downregulation of interferon (IFN)-beta production by V and C proteins of Sendai virus (SeV). The infection of HeLa cells with SeV poorly induced IFN-beta even if the expression of C/C' was disrupted. In contrast, when the expression of C/C'/Y1/Y2 or V/W was disrupted, SeV infection strongly induced IFN-beta production and significantly activated the interferon regulatory factor (IRF)-3 pathway. The independent expression of C or V inhibited the double-stranded (ds) RNA- or Newcastle disease virus (NDV)-induced activation of IRF-3 and NF-kappa B, as well as the IFN-beta promoter. This inhibitory effect was also observed when Y1, Y2, or a C-terminal half fragment (aa 85-204) of C was independently expressed. Phosphorylation and homodimer formation of IRF-3 were suppressed not only in cells infected with SeV capable of expressing both C/C'/Y1/Y2 (or Y1/Y2) and V/W, but also in HeLa cells constitutively expressing Y1. These results suggest that C, Y1, Y2, and V block signaling pathways leading to IRF-3 activation to downregulate IFN-beta production.     

Human oviductal epithelial cells express Toll-like receptor 3 and respond to double-stranded RNA: Fallopian tube-specific mucosal immunity against viral infection

BACKGROUND: The aim of this study was to evaluate the site-specific immunoregulatory mechanisms against viral infection in human Fallopian tubes. METHODS: We therefore investigated the effects of double-stranded RNA (dsRNA) on the production of interleukin (IL)-6, IL-8 and granulocyte chemotactic protein-2 (GCP-2) by cultured oviductal epithelial cells (OECs) using enzyme-linked immunosorbent assays. Phosphorylation of inhibitor kappaB-alpha (IkappaB-alpha) protein after dsRNA stimulation and the expression of Toll-like receptor (TLR) 3 in these cells were also evaluated by western blot analysis. RESULTS: Polyriboinosinic:polyribocytidylic acid (poly I:C), a synthetic dsRNA that antagonizes TLR3, stimulated the secretion of IL-6, IL-8 and GCP-2 by OECs. Poly I:C-induced production of these cytokines by OECs was inhibited by the pretreatment of these cells with anti-TLR3 antibody. The phosphorylation of IkappaB-alpha protein was detected in OECs after stimulation by poly I:C. The expression of TLR3 was also detected in OECs. CONCLUSION: These results suggest that the epithelial cells of the human Fallopian tube have evolved a unique, site-specific mechanism for recognizing viral infection. TLR3-mediated production of proinflammatory cytokines and chemokines in OECs in response to viral dsRNA may be important for antiviral immunity in the human female reproductive tract.     

Roles of NF-κB and MAPK signaling pathways in morphological and cytoskeletal responses of microglia to double-stranded RNA

Following virus infection of the central nervous system, microglia become activated and undergo morphological as well as functional transformations, thereby initiating effective antiviral actions. Herein, we have examined the contribution of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways to cell shape determination and cytoskeletal organization in microglia upon stimulation with double-stranded RNA (dsRNA), a conserved molecular pattern of virus infection. Under non-proliferative condition, microglial MG6-1 cells displayed a distinctive morphology with spinescent processes and small somata. Following dsRNA stimulation, the process-bearing microglial cells exhibited swift and drastic changes in cell morphology, filamentous actin (F-actin) structure, and intracellular signaling. In the dsRNA-stimulated microglial cells, the activation of c-Jun N-terminal kinase (JNK) pathway was involved in morphological alteration into an ameboid state. We also found that p38 signaling pathway negatively regulates the formation of cytoplasmic vacuoles in microglial cells. Furthermore, the dsRNA-induced accumulation of F-actin was partly mediated by NF-κB, JNK, and p38 pathways. These results indicate that NF-κB and MAPK signaling pathways mediate morphological and cytoskeletal changes during dsRNA-induced microglial activation.     

Adeno-associated virus and adenovirus coinfection induces a cellular DNA damage and repair response via redundant phosphatidylinositol 3-like kinase pathways

During adeno-associated virus and adenovirus (AAV/Ad) coinfection, accumulation of viral genomes and proteins can alter cellular stress responses. To determine how AAV/Ad coinfection affects the host we screened over 60 cellular proteins for their responses. AAV/Ad coinfections induce a robust DNA damage response (DDR) that is distinct from that induced by Ad infection alone. Using chemical inhibitors, deficient cell lines and siRNA knockdowns of the DDR kinases, ATM, ATR and DNA-PK, we determined that DNA-PK and ATM kinases are the initial transducers of this response. AAV/Ad coinfection induces ATM- and DNA-PK mediated phosphorylation of RPA2, NBS1, H2AX and the checkpoint kinases CHK1/2. Inhibition of one or more of the DDR kinases reduces the level of phosphorylation of downstream targets but does not dramatically reduce Ad or AAV protein expression. However, AAV DNA levels are moderately affected by kinase inhibition. These experiments provide new insights into the cellular responses to AAV/Ad coinfections.     

Down-regulation of Sclerotinia sclerotiorum gene expression in response to infection with Sclerotinia sclerotiorum debilitation-associated RNA virus

We have previously presented convincing evidence in support of a viral etiology for the debilitation phenotype exhibited by strain Ep-1PN of Sclerotinia sclerotiorum. To explore the possible mechanisms underlying fungal pathogenicity and hyphal growth, potential genes whose expression was down-regulated in Ep-1PN were identified from a cDNA library of the virus-free strain Ep-1PNAa, which is a single ascospore derivative of strain Ep-1PN, using reverse northern blot analysis. A total of 1116 cDNA clones were targeted and, following PCR re-amplification, 210 cDNA clones were selected as candidates, of which 16 cDNA clones were subjected to northern blot analysis for further confirmation. The results showed that 12 clones represented genes that were differentially expressed in the virus-free strain compared to the virus-infected one. Of the 210 clones that were sequenced, 150 had non-redundant sequences and of these 92% (138 clones) had significant homology to fungal genes in the databases examined. The remaining 12 clones did not have any matches. The differentially expressed genes represented a broad spectrum of biological functions including carbon and energy metabolism, protein synthesis and transport, signal transduction and stress response. This study provides the first insight into genes differentially expressed between the virus-free strain Ep-1PNAa and the virus-infected strain Ep-1PN. The possible relationships between mycovirus-mediated changes in cellular gene expression and observed phenotypes are discussed.