Delay of vaccinia virus-induced apoptosis in nonpermissive Chinese hamster ovary cells by the cowpox virus CHOhr and adenovirus E1B 19K genes.

The infection of vaccinia virus in Chinese hamster ovary (CHO) cells produces a rapid shutdown in protein synthesis, and the infection is abortive (R.R. Drillien, D. Spehner, and A. Kirn, Virology 111:488-499, 1978; D.E. Hruby, D.L. Lynn, R. Condit, and J.R. Kates, J. Gen. Virol. 47:485-488, 1980). Cowpox virus, which can productively infect CHO cells, had previously been shown to contain a host range gene, CHOhr, which confers on vaccinia virus the ability to replicate in CHO cells (D. Spehner, S. Gillard, R. Drillien, and A. Kirn, J. Virol. 62:1297-1304, 1988). We found that CHO cells underwent apoptosis when infected with vaccinia virus. The expression of the CHOhr gene in vaccinia virus allowed for the expression of late virus genes. CHOhr also delayed or prevented vaccinia virus-induced apoptosis in CHO cells such that there was sufficient time for replication of the virus before the cell died. The E1B 19K gene from adenovirus also delayed vaccinia virus-induced apoptosis; however, there was no detectable expression of late virus genes. Furthermore, E1B 19K also delayed cell death in CHO cells which had been productively infected with vaccinia virus. This study identifies a new antiapoptotic gene from cowpox virus, CHOhr, for which the protein contains an ankyrin-like repeat and shows no significant homology to other proteins. This work also indicates that an antiapoptotic gene from one virus family can delay cell death in an infection of a virus from a different family.     

Rapid inhibition of processing and assembly of small nuclear ribonucleoproteins after infection with vesicular stomatitis virus.

After infection of baby hamster kidney cells with vesicular stomatitis virus (VSV), processing and assembly of small nuclear ribonucleoproteins (snRNP) were rapidly inhibited. The U1 and U2 snRNAs accumulated as precursor species approximately 3 and 10 nucleotides longer, respectively, than the mature RNAs. Alteration in snRNP assembly was noted because the precursor snRNAs were not associated with the U-series RNA-core protein complex in infected cells. However, antibodies specific for the U2 RNA-binding protein, A', were able to precipitate pre-U2 RNAs from VSV-infected cells. These results indicated that precursors to U2 RNA were bound to A' and remained bound during virus infection. Analysis of the synthesis of proteins normally associated with U1 and U2 RNAs indicated that synthesis was unaffected at times when snRNP assembly with core proteins was blocked by the VSV. These findings suggested that the core proteins associate with one another in the absence of the snRNAs in VSV-infected cells. They further suggest a correlation between the inability of the core complex to bind the U-series snRNPs and the failure to process the 3' ends of U1 and U2 RNAs in VSV-infected cells. These effects of VSV on snRNP assembly may be related to the shutoff of host-cell macromolecular synthesis.     

Down-regulation of heme oxygenase-1 by SVCV infection

Heme oxygenase (HO-1) is a cytoprotective enzyme that plays a critical role in defending the body against oxidant-induced injury during inflammatory processes. In mammalian systems, viral infection or antigen expression can down-regulate the expression of HO-1. In turn, the induction of HO-1 or overexpression of HO-1 results in potent and direct antiviral activity that targets the replication of several mammalian viruses. In this study, the HO-1 gene of Cyprinus carpio was cloned, and the expression profile of HO-1 was investigated during spring viremia of carp virus (SVCV) infection. The results demonstrate that the expression of HO-1 was down-regulated during SVCV infection in the EPC cells and in common carp. These results indicated that SVCV infection could induce host oxidative stress, which may contribute to tissue injury in affect fish.     

The vaccinia virus F1L protein interacts with the proapoptotic protein Bak and inhibits Bak activation

Many viruses have evolved strategies to counteract cellular immune responses, including apoptosis. Vaccinia virus, a member of the poxvirus family, encodes an antiapoptotic protein, F1L. F1L localizes to mitochondria and inhibits apoptosis by preventing the release of cytochrome c by an undetermined mechanism (S. T. Wasilenko, T. L. Stewart, A. F. Meyers, and M. Barry, Proc. Natl. Acad. Sci. USA 100:14345-14350, 2003; T. L. Stewart, S. T. Wasilenko, and M. Barry, J. Virol. 79:1084-1098, 2005). Here, we show that in the absence of an apoptotic stimulus, F1L associates with Bak, a proapoptotic member of the Bcl-2 family that plays a pivotal role in the release of cytochrome c. Cells infected with vaccinia virus were resistant to Bak oligomerization and the initial N-terminal exposure of Bak following the induction of apoptosis with staurosporine. A mutant vaccinia virus missing F1L was no longer able to inhibit apoptosis or Bak activation. In addition, the expression of F1L was essential to inhibit tBid-induced cytochrome c release in both wild-type murine embryonic fibroblasts (MEFs) and Bax-deficient MEFs, indicating that F1L could inhibit apoptosis in the presence and absence of Bax. tBid-induced Bak oligomerization and N-terminal exposure of Bak in Bax-deficient MEFs were inhibited during virus infection, as assessed by cross-linking and limited trypsin proteolysis. Infection with the F1L deletion virus no longer provided protection from tBid-induced Bak activation and apoptosis. Additionally, infection of Jurkat cells with the F1L deletion virus resulted in cellular apoptosis, as measured by loss of the inner mitochondrial membrane potential, caspase 3 activation, and cytochrome c release, indicating that the presence of F1L was pivotal for inhibiting vaccinia virus-induced apoptosis. Our data indicate that F1L expression during infection inhibits apoptosis and interferes with the activation of Bak.     

Open reading frame 5 of porcine reproductive and respiratory syndrome virus as a cause of virus-induced apoptosis.

The gene product of open reading frame 5 (p25) of porcine reproductive and respiratory syndrome (PRRS) virus has been expressed by coinfection of culture cells with vaccinia virus expressing the T7 RNA polymerase and a recombinant vaccinia virus encoding the open reading frame 5 gene under the T7 promoter and the encephalomyocarditis virus internal ribosome entry site. In spite of the reported efficiency of the expression system, very poor accumulation of p25 protein was observed and a strong cytotoxicity was produced in the doubly infected cells. This cell toxicity was shown to occur by induction of apoptosis, as indicated by nucleosome ladder formation, chromatin condensation, and rRNA degradation. Apoptosis induction was also observed after infection of cultured cells with an adapted PRRS virus strain and after infection of swine macrophage cells with a PRRS virus field strain. Contrary to the observations made for other cases of virus-induced apoptosis, we could not prevent p25 protein-induced apoptosis by using a cell line permanently expressing Bcl-2 protein.     

Inhibition of agonist-induced activation of phospholipase C following poxvirus infection.

Recent studies indicate that viruses may influence polyphosphoinositide levels. This study has examined the effects of vaccinia virus infection on phospholipase C activity. Infection of BS-C-1 cells, an African Green Monkey kidney cell line, or A431 cells, a human carcinoma cell line, with vaccinia virus inhibits receptor-mediated phospholipase C activation. As a consequence, agonist-mediated Ca2+ mobilization in BS-C-1 cells also was inhibited by vaccinia virus infection. Alleviation of the inhibition of phospholipase C activation was observed in vaccinia virus-infected cells treated with cycloheximide without influencing uninfected cells. Treatment of infected cells with alpha-amanitin, an inhibitor of host mRNA synthesis but not virus mRNA synthesis, failed to alleviate the inhibition of phospholipase C activation. Together these results suggest that a virus-encoded gene product mediates the inhibition of phospholipase C activation without the need of a virus-induced host factor. Analysis of the processes involved in the formation of inositol (1,4,5)-trisphosphate and mobilization of intracellular Ca2+ indicate that the vaccinia virus gene product exerts its inhibitory effects at the level of phospholipase C activity. This may occur by either directly reducing the amount of phospholipase C, reducing the specific activity of phospholipase C, or by inhibiting the association of phospholipase C with its substrate, phosphatidylinositol 4,5-bisphosphate.     

An orally bioavailable antipoxvirus compound (ST-246) inhibits extracellular virus formation and protects mice from lethal orthopoxvirus Challenge

ST-246 is a low-molecular-weight compound (molecular weight = 376), that is potent (concentration that inhibited virus replication by 50% = 0.010 microM), selective (concentration of compound that inhibited cell viability by 50% = >40 microM), and active against multiple orthopoxviruses, including vaccinia, monkeypox, camelpox, cowpox, ectromelia (mousepox), and variola viruses. Cowpox virus variants selected in cell culture for resistance to ST-246 were found to have a single amino acid change in the V061 gene. Reengineering this change back into the wild-type cowpox virus genome conferred resistance to ST-246, suggesting that V061 is the target of ST-246 antiviral activity. The cowpox virus V061 gene is homologous to vaccinia virus F13L, which encodes a major envelope protein (p37) required for production of extracellular virus. In cell culture, ST-246 inhibited plaque formation and virus-induced cytopathic effects. In single-cycle growth assays, ST-246 reduced extracellular virus formation by 10 fold relative to untreated controls, while having little effect on the production of intracellular virus. In vivo oral administration of ST-246 protected BALB/c mice from lethal infection, following intranasal inoculation with 10x 50% lethal dose (LD(50)) of vaccinia virus strain IHD-J. ST-246-treated mice that survived infection acquired protective immunity and were resistant to subsequent challenge with a lethal dose (10x LD(50)) of vaccinia virus. Orally administered ST-246 also protected A/NCr mice from lethal infection, following intranasal inoculation with 40,000x LD(50) of ectromelia virus. Infectious virus titers at day 8 postinfection in liver, spleen, and lung from ST-246-treated animals were below the limits of detection (<10 PFU/ml). In contrast, mean virus titers in liver, spleen, and lung tissues from placebo-treated mice were 6.2 x 10(7), 5.2 x 10(7), and 1.8 x 10(5) PFU/ml, respectively. Finally, oral administration of ST-246 inhibited vaccinia virus-induced tail lesions in Naval Medical Research Institute mice inoculated via the tail vein. Taken together, these results validate F13L as an antiviral target and demonstrate that an inhibitor of extracellular virus formation can protect mice from orthopoxvirus-induced disease.     

Control of thymidine kinase synthesis in IHD vaccinia virus-infected thymidine kinase-deficient LM cells.

The synthesis of vaccinia virus-induced thymidine kinase is normally arrested several hours after infection. In thymidine kinase-deficient LM cells infected with IHD strain of vaccinia virus, arrest occurs whether or not viral DNA synthesis is inhibited. With virus inactivated by UV irradiation, enzyme synthesis takes place, but arrest is abolished. It is suggested that an early viral genetic function is responsible for the cessation of thymidine kinase synthesis.     

Binding between elongation factor 1A and the 3ʹ-UTR of Chinese wheat mosaic virus is crucial for virus infection

The Chinese wheat mosaic virus (CWMV) genome consists of two positive-strand RNAs that are required for CWMV replication and translation. The eukaryotic translation elongation factor (eEF1A) is crucial for the elongation of protein translation in eukaryotes. Here, we show that silencing eEF1A expression in Nicotiana benthamiana plants by performing virus-induced gene silencing can greatly reduce the accumulation of CWMV genomic RNAs, whereas overexpression of eEF1A in plants increases the accumulation of CWMV genomic RNAs. In vivo and in vitro assays showed that eEF1A does not interact with CWMV RNA-dependent RNA polymerase. Electrophoretic mobility shift assays revealed that eEF1A can specifically bind to the 3ʹ-untranslated region (UTR) of CWMV genomic RNAs. By performing mutational analyses, we determined that the conserved region in the 3ʹ-UTR of CWMV genomic RNAs is necessary for CWMV replication and translation, and that the sixth stem-loop (SL-6) in the 3ʹ-UTR of CWMV genomic RNAs plays a key role in CWMV infection. We conclude that eEF1A is an essential host factor for CWMV infection. This finding should help us to develop new strategies for managing CWMV infections in host plants.     

Host range restriction of vaccinia virus in Chinese hamster ovary cells: relationship to shutoff of protein synthesis.

Chinese hamster ovary cells were found to be nonpermissive for vaccinia virus. Although early virus-induced events occurred in these cells (RNA and polypeptide synthesis), subsequent events appeared to be prevented by a very rapid and nonselective shutoff of protein synthesis. Within less than 2 h after infection, both host and viral protein syntheses were arrested. At low multiplicities of infection, inhibition of RNA synthesis with cordycepin resulted in failure of the virus to block protein synthesis. Moreover, infection of the cells in the presence of cycloheximide prevented the immediate onset of shutoff after reversal of cycloheximide. Inactivation of virus particles by UV irradiation also impaired the capacity of the virus to inhibit protein synthesis. These results suggested that an early vaccinia virus-coded product was implicated in the shutoff of protein synthesis. Either the nonpermissive Chinese hamster ovary cells were more sensitive to this inhibition than permissive cells, or a regulatory control of the vaccinia shutoff function was defective.     

Soluble Antigens of Vaccinia-infected Mammalian Cells II. Time Course of Synthesis of Soluble Antigens and Virus Structural Proteins

Virus-induced soluble antigens produced in mammalian cells after infection with vaccinia virus can be divided into two classes on the basis of molecular weight. Synthesis of the low molecular weight antigens begins early in the course of infection (1 to 2 hr), and is switched-off rather abruptly 4 to 5 hr after infection in a manner similar to that reported for the early enzymes characteristic of this same system. It was demonstrated, however, that these antigens do not include virus-induced thymidine kinase, a major virus-induced enzyme, nor is it likely that the low molecular weight antigens described here share identity with any of the virus-induced enzymes. A portion of the low molecular weight antigens appear to be incorporated into the structure of newly synthesized virus, probably as internal proteins. In contrast, synthesis of the high molecular weight antigen class is initiated later in the course of infection (4 to 5 hr), just prior to the appearance of newly synthesized virus. Antiserum directed specifically against virus structural proteins forms precipitin bands with all of the high molecular weight antigens recognizable by immunoelectrophoresis. This evidence, coupled with the observation that the high molecular weight antigen fraction elicits production of specific virus-neutralizing antibody, strongly suggests that this antigen class represents virus structural subunits produced in excess.     

Sequence analysis, expression, and deletion of a vaccinia virus gene encoding a homolog of profilin, a eukaryotic actin-binding protein.

A 4,500-bp BamHI fragment, located within the HindIII A segment of the vaccinia virus genome, was found to contain eight potential coding regions for polypeptides of 78 to 346 amino acids. The open reading frames with 133, 346, and 125 codons were homologous to profilin (an actin-binding protein), 3-beta-hydroxysteroid dehydrogenase, and Cu-Zn superoxide dismutase, respectively. Sequence alignments indicated that the vaccinia virus and mammalian profilins were more closely related to each other than to known profilins of other eukaryotes. The expression and possible role of the profilin homolog in the virus replicative cycle were therefore investigated. Antibody raised to Escherichia coli expressed vaccinia virus profilin was used to demonstrate the synthesis of the 15-kDa polypeptide at late times after vaccinia virus infection of mammalian cells. The protein accumulated in the cytoplasm, but only trace amounts remained associated with highly purified virions. The isolation of vaccinia virus mutants (in strains WR and IHD-J), with nearly the entire profilin gene replaced by the E. coli gpt gene, indicated that the protein is not essential for infectivity. The characteristic vaccinia virus-induced changes in actin fibers, seen by fluorescence microscopy, occurred in cells infected with the mutant. Moreover, the virus-encoded profilin homolog was not required for actin-associated events, including intracellular virus movement to the periphery of the cell, formation of specialized microvilli, or release of mature virions, as shown by electron microscopy and yields of infectious intra- and extracellular virus.     

A 14,000-Mr envelope protein of vaccinia virus is involved in cell fusion and forms covalently linked trimers.

A monoclonal antibody, MAbC3, that reacts with a 14,000-molecular-weight envelope protein (14K protein) of vaccinia virus completely inhibited virus-induced cell fusion during infection. Immunoblot and immunofluorescence studies revealed that the 14K protein was synthesized at about 6 to 7 h postinfection and transported from the cytoplasm to the cell surface. Synthesis and transport of the 14K protein during infection occurred in the presence of rifampin, an inhibitor of virus maturation. One- and two-dimensional gel electrophoretic analyses demonstrated that the 14K protein forms largely trimers (42K) that are covalently linked by disulfide bonds. The facts that MAbC3 prevents virus uncoating and blocks virus-induced cell fusion but does not prevent virus attachment to cells and the 14K envelope protein forms trimers all suggest that this protein plays major role in virus penetration.     

Further characterization of the rabies virus glycoproteins produced by virus-infected and G cDNA-transfected cells using a monoclonal antibody, #1-30-44, which recognizes an acid-sensitive epitope

Expression of rabies virus glycoprotein (G) by G cDNA-transfected mammalian cells resulted in the production of only a fusion-negative form. Low pH-dependent fusion activity, however, was seen when the expression was done under control of the T7 promoter with the help of recombinant vaccinia virus (RVV-T7) that provided T7 RNA polymerase. Fusion-inactive G proteins were transported to the cell surface as being detected by a conformational epitope-specific monoclonal antibody (mAb; #1-46-12). The fusion-inactive G proteins were recognized by most of our 13 conformation-specific mAbs, except for one mAb, #1-30-44, that recognized the low pH-sensitive conformational epitope. When the G gene expression was done with the help of RVV-T7, although most G proteins remained in the epitope-negative form, a small fraction of G gene products were 1-30-44 epitope-positive, and cell fusion activity could be seen when cells were exposed to low pH conditions. From these results, we conclude that acquisition of low pH-dependent fusion activity is closely related to structural maturation of the G protein to form the low pH-sensitive 1-30-44 epitope. Such maturation seems to be dependent on certain rabies virus-induced cellular conditions or functions, which might also be provided in part by the vaccinia virus infection. We further assume that expression of G cDNA alone mostly results in the production of mis-folded and/or differently folded forms of G protein, and only a small fraction is correctly folded even under RVV-T7-mediated expression conditions.     

Inhibition of HIV-1 multiplication by antisense U7 snRNAs and siRNAs targeting cyclophilin A

Human immunodeficiency virus 1 (HIV-1) multiplication depends on a cellular protein, cyclophilin A (CyPA), that gets integrated into viral particles. Because CyPA is not required for cell viability, we attempted to block its synthesis in order to inhibit HIV-1 replication. For this purpose, we used antisense U7 small nuclear RNAs (snRNAs) that disturb CyPA pre-mRNA splicing and short interfering RNAs (siRNAs) that target CyPA mRNA for degradation. With dual-specificity U7 snRNAs targeting the 3′ and 5′ splice sites of CyPA exons 3 or 4, we obtained an efficient skipping of these exons and a strong reduction of CyPA protein. Furthermore, short interfering RNAs targeting two segments of the CyPA coding region strongly reduced CyPA mRNA and protein levels. Upon lentiviral vector-mediated transduction, prolonged antisense effects were obtained for both types of antisense RNAs in the human T-cell line CEM-SS. These transduced CEM-SS cells showed a delayed, and for the siRNAs also reduced, HIV-1 multiplication. Since the two types of antisense RNAs function by different mechanisms, combining the two approaches may result in a synergistic effect.     

In vivo antiviral effect of interleukin 18 in a mouse model of vaccinia virus infection.

Interleukin-18 (IL-18), originally called interferon-gamma (IFN-gamma)-inducing factor is a novel cytokine which exhibits pleiotropic immunomodulatory activities such as the activation of natural killer (NK) cells and cytotoxic T lymphocytes (CTL). In this study, the efficacy of IL-18 on viral infection in mice was investigated. IL-18 treatment significantly suppressed pock formation on the tails of BALB/c mice inoculated intravenously with vaccinia virus when the cytokine was administered intraperitoneally on days 0, 2 and 4 after infection. Sequentially, NK and CTL activity of the infected mice were significantly augmented by IL-18 injection. The in vivo anti-vaccinia virus activity of IL-18 was only partially inhibited by treating the infected mice with anti-asialo GM1 antibody. When infected mice were injected with anti-IFN-gamma antibody only, severe deterioration of health and significant body weight loss were observed, suggesting that IFN-gamma is very important in protecting mice against vaccinia virus infection. Interestingly, IL-18 injection visibly improved the severe vaccinia virus-induced symptoms in mice treated with anti-IFN-gamma antibody, even though a pivotal involvement of IFN-gamma in IL-18-mediated anti-vaccinia virus effect is not yet determined. Taken together, these results indicate that the IL-18-elicited anti-vaccinia virus effect in the acute phase of infection would be raised by the sum of various host defence mechanisms including NK cells and CTL, and not from a specific immunocompetent cell population or effector molecule.     

F11-Mediated Inhibition of RhoA Signalling Enhances the Spread of Vaccinia Virus In Vitro and In Vivo in an Intranasal Mouse Model of Infection

The cortical actin cytoskeleton beneath the plasma membrane represents a physical barrier that vaccinia virus has to overcome during its exit from an infected cell. Previous observations using overexpression and pharmacological approaches suggest that vaccinia enhances its release by modulating the cortical actin cytoskeleton by inhibiting RhoA signalling using the viral protein F11. We have now examined the role of F11 and its ability to interact with RhoA to inhibit its downstream signalling in the spread of vaccinia infection both in vitro and in vivo. Live cell imaging over 48 hours reveals that loss of F11 or its ability to bind RhoA dramatically reduces the rate of cell-to-cell spread of the virus in a cell monolayer. Cells infected with the ΔF11L virus also maintained their cell-to-cell contacts, and did not undergo virus-induced motility as observed during wild-type infections. The ΔF11L virus is also attenuated in intranasal mouse models of infection, as it is impaired in its ability to spread from the initial sites of infection to the lungs and spleen. Loss of the ability of F11 to bind RhoA also reduces viral spread in vivo. Our results clearly establish that viral-mediated inibition of RhoA signalling can enhance the spread of infection not only in cell monolayers, but also in vivo.     

HO-1与HCV的相关性研究

血红素加氧酶-1（hemeoxygenase-1,HO-1）在肝脏和脾脏高表达,可以被包括某些病毒感染在内的多种因素诱导表达,具有抗氧化、抗炎、抗凋亡等保护作用。丙型肝炎病毒（hepatitisCvirus,HCV）感染可以造成慢型肝炎、肝硬化和肝癌等疾病,对人类健康造成很大威胁。研究发现HO-1通过影响HCV的复制发挥其保护作用,同时HCV也可以反向调控HO-1的表达。尽管HO-1与HCV相互作用的分子机制还不明确,但H0—1与HCV感染相关性研究不断取得重大进展,将为HCV感染的治疗提供一种新方法。     

Inhibition of antigen-induced and interleukin-2-induced proliferation of bovine peripheral blood leukocytes by inactivated bovine herpes virus 1.

The mechanism by which bovine herpesvirus 1 (BHV-1) predisposes cattle to bacterial pneumonia was investigated by using an in vitro system to demonstrate immunosuppression. At a multiplicity of infection of 0.001, live or inactivated BHV-1 induced a 50% inhibition of the proliferative response of peripheral blood mononuclear leukocytes to antigen (vaccinia virus in vaccinia virus-immunized cattle which were BHV-1 negative) or interleukin-2. At this same multiplicity of infection, the mitogen-induced proliferation of peripheral blood mononuclear leukocytes was unaffected. This inhibition of antigen and interleukin-2-induced proliferative responses could not be reversed by the addition of excess amounts of interleukin-2 and could not be prevented by the addition of indomethacin to block prostaglandin production. Antibodies to BHV-1, especially those specific for glycoproteins gI and gIV, were able to block the inhibitory effect of BHV-1 in these in vitro assays. These results showed that antibody to BHV-1 blocks the immunosuppressive effect of the virus in vitro and suggested that an appropriate antibody response to BHV-1 could protect cattle from virus-induced immunosuppression leading to secondary bacterial pneumonia.