Essential role of the NV protein of Novirhabdovirus for pathogenicity in rainbow trout

Novirhabdovirus, infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV) are fish rhabdoviruses that, in comparison to the other rhabdoviruses, contain an additional gene coding for a small nonvirion (NV) protein of unassigned function. A recombinant IHNV with the NV gene deleted but expressing the green fluorescent protein (rIHNV-Delta NV) has previously been shown to be efficiently recovered by reverse genetics (S. Biacchesi et al., J. Virol. 74:11247-11253, 2000). However, preliminary experiments suggested that the growth in cell culture of rIHNV-Delta NV was affected by the NV deletion. In the present study, we show that the growth in cell culture of rIHNV-Delta NV is indeed severely impaired but that a normal growth of rIHNV-Delta NV can be restored when NV is provided in trans by using fish cell clones constitutively expressing the NV protein. These results indicate that NV is a protein that has a crucial biological role for optimal replication of IHNV in cell culture. Although IHNV and VHSV NV proteins do not share any significant identity, we show here that both NV proteins play a similar role since a recombinant IHNV virus, rIHNV-NV(VHSV), in which the IHNV NV open reading frame has been replaced by that of VHSV, was shown to replicate as well as the wild-type (wt) IHNV into fish cells. Finally, data provided by experimental fish infections with the various recombinant viruses strongly suggest an essential role of the NV protein for the pathogenicity of IHNV. Furthermore, we show that juvenile trout immunized with NV-knockout IHNV were protected against challenge with wt IHNV. That opens a new perspective for the development of IHNV attenuated live vaccines.     

The cell-rounding activity of the vesicular stomatitis virus matrix protein is due to the induction of cell death

The matrix (M) protein of vesicular stomatitis virus (VSV) expressed in the absence of other viral components causes many of the cytopathic effects of VSV, including an inhibition of host gene expression and the induction of cell rounding. It was recently shown that M protein also induces apoptosis in the absence of other viral components. This raises the possibility that the activation of apoptotic pathways causes the inhibition of host gene expression and cell rounding by M protein. To test this hypothesis, host gene expression and cell rounding were analyzed after the transfection of M mRNA into HeLa cells stably overexpressing Bcl-2 (HeLa-Bcl-2 cells). We have shown previously that Bcl-2 inhibits M-protein-induced apoptosis. Here, we show that activation of the apoptotic pathways downstream of Bcl-2 is not required for the inhibition of host gene expression by M protein. In contrast, overexpression of Bcl-2 inhibited cell rounding induced by M protein, indicating that apoptotic pathways downstream of Bcl-2 are required for the cell-rounding activities of M protein.     

Effects of Influenza A Virus NS1 Protein on Protein Expression: the NS1 Protein Enhances Translation and Is Not Required for Shutoff of Host Protein Synthesis

The influenza A virus NS1 protein, a virus-encoded alpha/beta interferon (IFN-alpha/beta) antagonist, appears to be a key regulator of protein expression in infected cells. We now show that NS1 protein expression results in enhancement of reporter gene activity from transfected plasmids. This effect appears to be mediated at the translational level, and it is reminiscent of the activity of the adenoviral virus-associated I (VAI) RNA, a known inhibitor of the antiviral, IFN-induced, PKR protein. To study the effects of the NS1 protein on viral and cellular protein synthesis during influenza A virus infection, we used recombinant influenza viruses lacking the NS1 gene (delNS1) or expressing truncated NS1 proteins. Our results demonstrate that the NS1 protein is required for efficient viral protein synthesis in COS-7 cells. This activity maps to the amino-terminal domain of the NS1 protein, since cells infected with wild-type virus or with a mutant virus expressing a truncated NS1 protein-lacking approximately half of its carboxy-terminal end-showed similar kinetics of viral and cellular protein expression. Interestingly, no major differences in host cell protein synthesis shutoff or in viral protein expression were found among NS1 mutant viruses in Vero cells. Thus, another viral component(s) different from the NS1 protein is responsible for the inhibition of host protein synthesis during viral infection. In contrast to the earlier proposal suggesting that the NS1 protein regulates the levels of spliced M2 mRNA, no effects on M2 protein accumulation were seen in Vero cells infected with delNS1 virus.     

Nonvirion protein of novirhabdovirus suppresses apoptosis at the early stage of virus infection

Viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) are members of the genus Novirhabdovirus within the Rhabdoviridae family, which can cause severe hemorrhagic disease in fresh- and saltwater fish worldwide. These viruses carry an additional nonvirion (NV) gene, which codes for the nonstructural NV protein that has been implicated to play a role in viral pathogenesis. To determine the precise biological function of this NV gene and its gene product, we generated NV-deficient and NV knockout recombinant VHSVs, using reverse genetics. Comparisons of the replication kinetics and markers for virus-induced apoptosis indicated that the NV-deficient and NV knockout mutant viruses induce apoptosis earlier in cell culture than the wild-type recombinant VHSV. These results suggest that the NV protein has an antiapoptotic function at the early stage of virus infection. Furthermore, we created a chimeric VHSV, in which the NV gene of VHSV was replaced by the IHNV NV gene, which was capable of suppressing apoptosis in cell culture. These results show that the NV protein of other members of Novirhabdovirus can restore the NV protein function. In this study, we also investigated the kinetics of VHSV replication during a single round of viral replication and examined the mechanism of VHSV-induced apoptosis. Our results show that VHSV infection induced caspases 3, 8 and 9 in cell culture.     

A nuclear localization of the infectious haematopoietic necrosis virus NV protein is necessary for optimal viral growth

The nonvirion (NV) protein of infectious hematopoietic necrosis virus (IHNV) has been previously reported to be essential for efficient growth and pathogenicity of IHNV. However, little is known about the mechanism by which the NV supports the viral growth. In this study, cellular localization of NV and its role in IHNV growth in host cells was investigated. Through transient transfection in RTG-2 cells of NV fused to green fluorescent protein (GFP), a nuclear localization of NV was demonstrated. Deletion analyses showed that the (32)EGDL(35) residues were essential for nuclear localization of NV protein, and fusion of these 4 amino acids to GFP directed its transport to the nucleus. We generated a recombinant IHNV, rIHNV-NV-ΔEGDL in which the (32)EGDL(35) was deleted from the NV. rIHNVs with wild-type NV (rIHNV-NV) or with the NV gene replaced with GFP (rIHNV-ΔNV-GFP) were used as controls. RTG-2 cells infected with rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL yielded 12- and 5-fold less infectious virion, respectively, than wild type rIHNV-infected cells at 48 h post-infection (p.i.). While treatment with poly I∶C at 24 h p.i. did not inhibit replication of wild-type rIHNVs, replication rates of rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL were inhibited by poly I∶C. In addition, both rIHNV-ΔNV and rIHNV-NV-ΔEGDL induced higher levels of expressions of both IFN1 and Mx1 than wild-type rIHNV. These data suggest that the IHNV NV may support the growth of IHNV through inhibition of the INF system and the amino acid residues of (32)EGDL(35) responsible for nuclear localization are important for the inhibitory activity of NV.     

Effects of NV gene knock-out recombinant viral hemorrhagic septicemia virus (VHSV) on Mx gene expression in Epithelioma papulosum cyprini (EPC) cells and olive flounder (Paralichthys olivaceus)

To determine whether the NV gene of viral hemorrhagic septicemia virus (VHSV) is related to the type I interferon response of hosts, expression of Mx gene in Epithelioma papulosum cyprini (EPC) cells and in olive flounder (Paralichthys olivaceus) in response to infection with either wild-type VHSV or recombinant VHSVs (rVHSV-ΔNV-EGFP and rVHSV-wild) was investigated. A reporter vector was constructed for measuring Mx gene expression using olive flounder Mx promoter, in which the reporter Metridia luciferase was designed to be excreted to culture medium to facilitate measurement. The highest increase of luciferase activity was detected from supernatant of cells infected with rVHSV-ΔNV-EGFP. In contrast cells infected with wild-type VHSV showed a slight increase of the luciferase activity. Interestingly, cells infected with rVHSV-wild that has artificially changed nucleotides just before and after the NV gene ORF, also showed highly increased luciferase activity, but the increased amplitude was lower than that by rVHSV-ΔNV-EGFP. These results strongly suggest that the NV protein of VHSV plays an important role in suppressing interferon response in host cells, which provides a condition for the viruses to efficiently proliferate in host cells. In an in vivo experiment, the Mx gene expression in olive flounder challenged with the rVHSV-ΔNV-EGFP was clearly higher than fish challenged with rVHSV-wild or wild-type VHSV, suggesting that lacking of the NV gene in the genome of rVHSV-ΔNV-EGFP brought to strong interferon response that subsequently inhibit viral replication in fish.     

Matrix Protein and Another Viral Component Contribute to Induction of Apoptosis in Cells Infected with Vesicular Stomatitis Virus

The induction of apoptosis in host cells is a prominent cytopathic effect of vesicular stomatitis virus (VSV) infection. The viral matrix (M) protein is responsible for several important cytopathic effects, including the inhibition of host gene expression and the induction of cell rounding in VSV-infected cells. This raises the question of whether M protein is also involved in the induction of apoptosis. HeLa or BHK cells were transfected with M mRNA to determine whether M protein induces apoptosis when expressed in the absence of other viral components. Expression of M protein induced apoptotic morphological changes and activated caspase-3 in both cell types, indicating that M protein induces apoptosis in the absence of other viral components. An M protein containing a point mutation that renders it defective in the inhibition of host gene expression (M51R mutation) activated little, if any, caspase-3, while a deletion mutant lacking amino acids 4 to 21 that is defective in the virus assembly function but fully functional in the inhibition of host gene expression was as effective as wild-type (wt) M protein in activating caspase-3. To determine whether M protein influences the induction of apoptosis in the context of a virus infection, the M51R M protein mutation was incorporated onto a wt background by using a recombinant infectious cDNA clone (rM51R-M virus). The timing of the induction of apoptosis by rM51R-M virus was compared to that by the corresponding recombinant wt (rwt) virus and to that by tsO82 virus, the mutant virus in which the M51R mutation was originally identified. In HeLa cells, rwt virus induced apoptosis faster than did rM51R-M virus, demonstrating a role for M protein in the induction of apoptosis. In contrast to the results obtained with HeLa cells, rwt virus induced apoptosis more slowly than did rM51R-M virus in BHK cells. This indicates that a viral component other than M protein contributes to induction of apoptosis in BHK cells and that wt M protein acts to delay induction of apoptosis by the other viral component. tsO82 virus induced apoptosis more rapidly than did rM51R-M virus in both HeLa and BHK cells. These two viruses contain the same point mutation in their M proteins, suggesting that sequence differences in genes other than that for M protein affect their rates of induction of apoptosis.     

Vesicular stomatitis viruses expressing wild-type or mutant M proteins activate apoptosis through distinct pathways

Vesicular stomatitis virus (VSV) induces apoptosis by at least two mechanisms. The viral matrix (M) protein induces apoptosis via the mitochondrial pathway due to the inhibition of host gene expression. However, in some cell types, the inhibition of host gene expression by VSV expressing wild-type (wt) M protein delays VSV-induced apoptosis, indicating that another mechanism is involved. In support of this, the recombinant M51R-M (rM51R-M) virus, expressing a mutant M protein that is defective in its ability to inhibit host gene expression, induces apoptosis much more rapidly in L929 cells than do viruses expressing wt M protein. Here, we determine the caspase pathways by which the rM51R-M virus induces apoptosis. An analysis of caspase activity, using fluorometric caspase assays and Western blots, indicated that each of the main initiator caspases, caspase-8, caspase-9, and caspase-12, were activated during infection with the rM51R-M virus. The overexpression of Bcl-2, an inhibitor of the mitochondrial pathway, or MAGE-3, an inhibitor of caspase-12 activation, did not delay apoptosis induction in rM51R-M virus-infected L929 cells. However, an inhibitor of caspase-8 activity significantly delayed apoptosis induction. Furthermore, the inhibition of caspase-8 activity prevented the activation of caspase-9, suggesting that caspase-9 is activated by cross talk with caspase-8. These data indicate that VSV expressing the mutant M protein induces apoptosis via the death receptor apoptotic pathway, a mechanism distinct from that induced by VSV expressing the wt M protein.     

Coxsackievirus preferentially replicates and induces cytopathic effects in undifferentiated neural progenitor cells

Enteroviruses, including coxsackieviruses, exhibit significant tropism for the central nervous system, and these viruses are commonly associated with viral meningitis and encephalitis. Previously, we described the ability of coxsackievirus B3 (CVB3) to infect proliferating neuronal progenitor cells located in the neonatal subventricular zone and persist in the adult murine central nervous system (CNS). Here, we demonstrate that cultured murine neurospheres, which comprise neural stem cells and their progeny at different stages of development, were highly susceptible to CVB3 infection. Neurospheres, or neural progenitor and stem cells (NPSCs), isolated from neonatal C57BL/6 mice, supported high levels of infectious virus production and high viral protein expression levels following infection with a recombinant CVB3 expressing enhanced green fluorescent protein (eGFP) protein. Similarly, NPSCs isolated from neonatal actin-promoter-GFP transgenic mice (actin-GFP NPSCs) were highly susceptible to infection with a recombinant CVB3 expressing DsRed (Discosoma sp. red fluorescent protein). Both nestin-positive and NG2(+) progenitor cells within neurospheres were shown to preferentially express high levels of viral protein as soon as 24 h postinfection (p.i.). By day 3 p.i., viral protein expression and viral titers increased dramatically in NPSCs with resultant cytopathic effects (CPE) and eventual cell death. In contrast, reduced viral replication, lower levels of CPE, and diminished viral protein expression levels were observed in NPSCs differentiated for 5 or 16 days in the presence of fetal bovine serum (FBS). Despite the presence of CPE and high levels of cell death following early CVB3 infection, surviving neurospheres were readily observed and continued to express detectable levels of viral protein as long as 37 days after initial infection. Also, CVB3 infection of actin-GFP NPSCs increased the percentage of cells expressing neuronal class III β-tubulin following their differentiation in the presence of FBS. These results suggest that neural stem cells may be preferentially targeted by CVB3 and that neurogenic regions of the CNS may support persistent viral replication in the surviving host. In addition, normal progenitor cell differentiation may be altered in the host following infection.     

Identification of two additional translation products from the matrix (M) gene that contribute to vesicular stomatitis virus cytopathology

The matrix (M) protein of vesicular stomatitis virus (VSV) is a multifunctional protein that is responsible for condensation of the ribonucleocapsid core during virus assembly and also plays a critical role in virus budding. The M protein is also responsible for most of the cytopathic effects (CPE) observed in infected cells. VSV CPE include inhibition of host gene expression, disablement of nucleocytoplasmic transport, and disruption of the host cytoskeleton, which results in rounding of infected cells. In this report, we show that the VSV M gene codes for two additional polypeptides, which we have named M2 and M3. These proteins are synthesized from downstream methionines in the same open reading frame as the M protein (which we refer to here as M1) and lack the first 32 (M2) or 50 (M3) amino acids of M1. Infection of cells with a recombinant virus that does not express M2 and M3 (M33,51A) resulted in a delay in cell rounding, but virus yield was not affected. Transient expression of M2 and M3 alone caused cell rounding similar to that with the full-length M1 protein, suggesting that the cell-rounding function of the M protein does not require the N-terminal 50 amino acids. To determine if M2 and M3 were sufficient for VSV-mediated CPE, both M2 and M3 were expressed from a separate cistron in a VSV mutant background that readily establishes persistent infections and that normally lacks CPE. Infection of cells with the recombinant virus that expressed M2 and M3 resulted in cell rounding indistinguishable from that with the wild-type recombinant virus. These results suggest that M2 and M3 are important for cell rounding and may play an important role in viral cytopathogenesis. To our knowledge, this is first report of the multiple coding capacities of a rhabdovirus matrix gene.     

HIV LTR-dependent expression of Bax selectively induces apoptosis in Tat-positive cells

HIV integrates into the host cell genome where it persists for the life of the cell. One approach to reducing viral burden is to selectively eliminate cells containing integrated provirus early following infection. We have used the HIV LTR promoter to selectively express transgenes in human cells positive for the HIV transactivator protein Tat. Transient transfection of Jurkat cells, or Jurkat cells stably expressing Tat (Jurkat-Tat), with a LTR construct containing luciferase reporter gene resulted in a 37-fold increase in gene expression when Tat was present. We have demonstrated that when pro-apoptotic Bax was used as the transgene, cytotoxicity was seen only in the Jurkat-Tat cells. Annexin-V staining indicated that Bax induced cell death by apoptosis. In mixed populations of Jurkat and Jurkat-Tat cells, the LTR-Bax construct was selectively cytotoxic to the Tat-positive cells. These results suggest that Bax under the control of the HIV LTR can be used to destroy cells harbouring HIV without affecting uninfected cells.     

Clathrin-mediated endocytosis in living host cells visualized through quantum dot labeling of infectious hematopoietic necrosis virus

Infectious hematopoietic necrosis virus (IHNV) is an important fish pathogen that infects both wild and cultured salmonids. As a species of the genus Novirhabdovirus, IHNV is a valuable model system for exploring the host entry mechanisms of rhabdoviruses. In this study, quantum dots (QDs) were used as fluorescent labels for sensitive, long-term tracking of IHNV entry. Using live-cell fluorescence microscopy, we found that IHNV is internalized through clathrin-coated pits after the virus binds to host cell membranes. Pretreatment of host cells with chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and clathrin light chain (LCa) depletion using RNA interference both resulted in a marked reduction in viral entry. We also visualized transport of the virus via the cytoskeleton (i.e., actin filaments and microtubules) in real time. Actin polymerization is involved in the transport of endocytic vesicles into the cytosol, whereas microtubules are required for the trafficking of clathrin-coated vesicles to early endosomes, late endosomes, and lysosomes. Disrupting the host cell cytoskeleton with cytochalasin D or nocodazole significantly impaired IHNV infectivity. Furthermore, infection was significantly affected by pretreating the host cells with bafilomycin A1, a compound that inhibits the acidification of endosomes and lysosomes. Strong colocalizations of IHNV with endosomes indicated that the virus is internalized into these membrane-bound compartments. This is the first report in which QD labeling is used to visualize the dynamic interactions between viruses and endocytic structures; the results presented demonstrate that IHNV enters host cells via clathrin-mediated endocytic, cytoskeleton-dependent, and low-pH-dependent pathways.     

Human cytomegalovirus UL38 protein blocks apoptosis

Apoptosis is an innate cellular defense response to viral infection. The slow-replicating human cytomegalovirus (HCMV) blocks premature death of host cells prior to completion of the infection cycle. In this study, we report that the HCMV UL38 gene encodes a cell death inhibitory protein. A mutant virus lacking the pUL38 coding sequence, ADdlUL38, grew poorly in human fibroblasts, failed to accumulate viral DNA to wild-type levels, and induced excessive death of infected cells. Cells expressing pUL38 were resistant to cell death upon infection and effectively supported the growth of ADdlUL38. Cells infected with the pUL38-deficient virus showed morphological changes characteristic of apoptosis, including cell shrinkage, membrane blebbing, vesicle release, and chromatin condensation and fragmentation. The proteolytic cleavage of two key enzymes involved in apoptosis, namely, caspase 3 and poly(ADP-ribose) polymerase, was activated upon ADdlUL38 infection, and the cleavage was blocked in cells expressing pUL38. The pan-caspase inhibitor Z-VAD-FMK largely restored the growth of ADdlUL38 in normal fibroblasts, indicating that the defective growth of the mutant virus mainly resulted from premature death of host cells. Furthermore, cells expressing pUL38 were resistant to cell death induced by a mutant adenovirus lacking the antiapoptotic E1B-19K protein or by thapsigargin, which disrupts calcium homeostasis in the endoplasmic reticulum. Taken together, these results indicate that the HCMV protein pUL38 suppresses apoptosis, blocking premature death of host cells to facilitate efficient virus replication.     

Evaluation of the protective immunogenicity of the N, P, M, NV and G proteins of infectious hematopoietic necrosis virus in rainbow trout oncorhynchus mykiss using DNA vaccines.

The protective immunogenicity of the nucleoprotein (N), phosphoprotein (P), matrix protein (M), non-virion protein (NV) and glycoprotein (G) of the rhabdovirus infectious hematopoietic necrosis virus (IHNV) was assessed in rainbow trout using DNA vaccine technology. DNA vaccines were produced by amplifying and cloning the viral genes in the plasmid pCDNA 3.1. The protective immunity elicited by each vaccine was evaluated through survival of immunized fry after challenge with live virus. Neutralizing antibody titers were also determined in vaccinated rainbow trout Oncorhynchus mykiss fry (mean weight 2 g) and 150 g sockeye salmon Oncorhynchus nerka. The serum from the 150 g fish was also used in passive immunization studies with naive fry. Our results showed that neither the internal structural proteins (N, P and M) nor the NV protein of IHNV induced protective immunity in fry or neutralizing antibodies in fry and 150 g fish when expressed by a DNA vaccine construct. The G protein, however, did confer significant protection in fry up to 80 d post-immunization and induced protective neutralizing antibodies. We are currently investigating the role of different arms of the fish immune system that contribute to the high level of protection against IHNV seen in vaccinated fish.     

Ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host RNA and protein synthesis

The vesicular stomatitis virus (VSV) matrix (M) protein plays a major role in the virus-induced inhibition of host gene expression. It has been proposed that the inhibition of host gene expression by M protein is responsible for suppressing activation of host interferon gene expression. Most wild-type (wt) strains of VSV induce little if any interferon gene expression. Interferon-inducing mutants of VSV have been isolated previously, many of which contain mutations in their M proteins. However, it was not known whether these M protein mutations were responsible for the interferon-inducing phenotype of these viruses. Alternatively, mutations in other genes besides the M gene may enhance the ability of VSV to induce interferons. These hypotheses were tested by transfecting cells with mRNA expressing wt and mutant M proteins in the absence of other viral components and determining their ability to inhibit interferon gene expression. The M protein mutations were the M51R mutation originally found in the tsO82 and T1026R1 mutant viruses, the double substitution V221F and S226R found in the TP3 mutant virus, and the triple substitution E213A, V221F, and S226R found in the TP2 mutant virus. wt M proteins suppressed expression of luciferase from the simian virus 40 promoter and from the beta interferon (IFN-beta) promoter, while M proteins of interferon-inducing viruses were unable to inhibit luciferase expression from either promoter. The M genes of the interferon-inducing mutants of VSV were incorporated into the wt background of a recombinant VSV infectious cDNA clone. The resulting recombinant viruses were tested for their ability to activate interferon gene expression and for their ability to inhibit host RNA and protein synthesis. Each of the recombinant viruses containing M protein mutations induced expression of a luciferase reporter gene driven by the IFN-beta promoter and induced production of interferon bioactivity more effectively than viruses containing wt M proteins. Furthermore, the M protein mutant viruses were defective in their ability to inhibit both host RNA synthesis and host protein synthesis. These data support the idea that wt M protein suppresses interferon gene expression through the general inhibition of host RNA and protein synthesis.     

Noncytotoxic inhibition of cytomegalovirus replication through NK cell protease granzyme M-mediated cleavage of viral phosphoprotein 71

Granzyme M (GrM) is highly expressed in cytotoxic granules of NK cells, which provide the first line of defense against viral pathogens. GrM knockout mice show increased susceptibility toward murine CMV infection. Although GrM is a potent inducer of cell death, the mechanism by which GrM eliminates viruses remains elusive. In this paper, we show that purified human GrM in combination with the perforin-analog streptolysin O (SLO) strongly inhibited human CMV (HCMV) replication in fibroblasts in the absence of host cell death. In a proteomic approach, GrM was highly specific toward the HCMV proteome and most efficiently cleaved phosphoprotein 71 (pp71), an HCMV tegument protein that is critical for viral replication. Cleavage of pp71 occurred when viral lysates were incubated with purified GrM, when intact cells expressing recombinant pp71 were challenged with living cytotoxic effector cells, and when HCMV-infected fibroblasts were incubated with SLO and purified GrM. GrM directly cleaved pp71 after Leu(439), which coincided with aberrant cellular localization of both pp71 cleavage fragments as determined by confocal immunofluorescence. In a luciferase reporter assay, cleavage of pp71 after Leu(439) by GrM completely abolished the ability of pp71 to transactivate the HCMV major immediate-early promoter, which is indispensable for effective HCMV replication. Finally, GrM decreased immediate-early 1 protein expression in HCMV-infected fibroblasts. These results indicate that the NK cell protease GrM mediates cell death-independent antiviral activity by direct cleavage of a viral substrate.     

The role of viral hemorrhagic septicemia virus (VHSV) NV gene in TNF-α- and VHSV infection-mediated NF-κB activation

The role of viral hemorrhagic septicemia virus (VHSV) NV gene in nuclear factor-κB (NF-κB) activation was investigated. Epithelioma papulosum cyprini (EPC) cells pre-treated with tumor necrosis factor (TNF)-α showed a strong resistance against VHSV infection, but cells treated with TNF-α after VHSV infection showed no resistance, suggesting that immediate early TNF-α-mediated responses inhibit VHSV replication. Activation of NF-κB is a key step in TNF-α-mediated immunomodulatory pathways. In this study, activation of NF-κB by TNF-α exposure was inhibited in EPC cells harboring NV gene expressing vectors, indicating that the NV gene of VHSV can suppress TNF-α-mediated NF-κB activation. Furthermore, the NV gene knock-out recombinant VHSV (rVHSV-ΔNV-EGFP) induced significantly higher NF-κB activity in EPC cells than wild-type VHSV, suggesting that VHSV adopted a strategy to suppress early activation of NF-κB in host cells through and NV gene.     

Effective inhibition of mRNA accumulation and protein expression of H5N1 avian influenza virus NS1 gene in vitro by small interfering RNAs

Avian influenza has emerged as a devastating disease and may cross species barrier and adapt to a new host, causing enormous economic loss and great public health threats, and non-structural protein 1 (NS1) is a multifunctional non-structural protein of avian influenza virus (AIV) that counters cellular antiviral activities and is a virulence factor. RNA interference (RNAi) provides a powerful promising approach to inhibit viral infection specifically. To explore the possibility of using RNAi as a strategy against AIV infection, after the fusion protein expression plasmids pNS1-enhanced green fluorescent protein (EGFP), which contain the EGFP reporter gene and AIV NS1 as silencing target, were constructed and NS1-EGFP fusion protein expressing HEK293 cell lines were established, four small interfering RNAs (siRNAs) targeting NS1 gene were designed, synthesized, and used to transfect the stable cell lines. Flow cytometry, real-time quantitative polymerase chain reaction, and Western blot were performed to assess the expression level of NS1. The results suggested that sequence-dependent specific siRNAs effectively inhibited mRNA accumulation and protein expression of AIV NS1 in vitro. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for AIV infection.