Viral Cell Proteins Research Articles

The mitochondrial carrier homolog 2 is involved in down-regulation of influenza A virus replication.

The mitochondrial carrier homolog 2 (MTCH2) is a mitochondrial outer membrane protein regulating mitochondrial metabolism and functions in lipid homeostasis and apoptosis. Experimental data on the interaction of MTCH2 with viral proteins in virus-infected cells are very limited. Here, the interaction of MTCH2 with PA subunit of influenza A virus RdRp and its effects on viral replication was investigated. The human MTCH2 protein was identified as the influenza A virus PA-related cellular factor with the Y2H assay. The interaction between GST.MTCH2 and PA protein co-expressed in transfected HEK293 cells was evaluated by GST-pull down. The effect of MTCH2 on virus replication was determined by quantification of viral transcript and/or viral proteins in the cells transfected with MTCH2-encoding plasmid or MTCH2-siRNA. An interaction model of MTCH2 and PA was predicted with protein modeling/docking algorithms. It was observed that PA and GST.MTCH2 proteins expressed in HEK293 cells were co-precipitated by glutathione-agarose beads. The influenza A virus replication was stimulated in HeLa cells whose MTCH2 expression was suppressed with specific siRNA, whereas the increase of MTCH2 in transiently transfected HEK293 cells inhibited viral RdRp activity. The results of a Y2H assay and protein-protein docking analysis suggested that the amino terminal part of the viral PA (nPA) can bind to the cytoplasmic domain comprising amino acid residues 253 to 282 of the MTCH2. It is suggested that the host mitochondrial MTCH2 protein is probably involved in the interaction with the viral polymerase protein PA to cause negative regulatory effect on influenza A virus replication in infected cells.

Quantitative microspectroscopic imaging reveals viral and cellular RNA helicase interactions in live cells

Human cells detect RNA viruses through a set of helicases called RIG-I-like receptors (RLRs) that initiate the interferon response via a mitochondrial signaling complex. Many RNA viruses also encode helicases, which are sometimes covalently linked to proteases that cleave signaling proteins. One unresolved question is how RLRs interact with each other and with viral proteins in cells. This study examined the interactions among the hepatitis C virus (HCV) helicase and RLR helicases in live cells with quantitative microspectroscopic imaging (Q-MSI), a technique that determines FRET efficiency and subcellular donor and acceptor concentrations. HEK293T cells were transfected with various vector combinations to express cyan fluorescent protein (CFP) or YFP fused to either biologically active HCV helicase or one RLR (i.e. RIG-I, MDA5, or LGP2), expressed in the presence or absence of polyinosinic-polycytidylic acid (poly(I:C)), which elicits RLR accumulation at mitochondria. Q-MSI confirmed previously reported RLR interactions and revealed an interaction between HCV helicase and LGP2. Mitochondria in CFP-RIG-I:YFP-RIG-I cells, CFP-MDA5:YFP-MDA5 cells, and CFP-MDA5:YFP-LGP2 cells had higher FRET efficiencies in the presence of poly(I:C), indicating that RNA causes these proteins to accumulate at mitochondria in higher-order complexes than those formed in the absence of poly(I:C). However, mitochondria in CFP-LGP2:YFP-LGP2 cells had lower FRET signal in the presence of poly(I:C), suggesting that LGP2 oligomers disperse so that LGP2 can bind MDA5. Data support a new model where an LGP2-MDA5 oligomer shuttles NS3 to the mitochondria to block antiviral signaling.

In Vitro Reconstitution of Membrane Budding by Influenza A Virus Matrix Protein 1

Influenza A is an enveloped virus comprised of eight RNA segments that encode the virus's ten major structural and non-structural proteins. In the final stage of its replication cycle, influenza virus's genetic and structural components assemble at the plasma membrane of the infected cell, where they form a bud that extrudes and eventually separates from the host. Although the production of infectious virus particles requires the convergence of all viral components at the site of bud formation, the mechanism by which assembly is coordinated remains poorly understood. One candidate that appears to play a central role is the matrix protein M1. M1 forms an oligomeric layer immediately adjacent to the viral membrane, shaping the virus and providing a link between the envelope proteins (HA, NA, and M2) and the internal ribonucleoprotein (RNP) complex that packages the viral genome. Despite evidence for its importance in the budding process, efforts to characterize the role of M1 and other viral proteins in cells has been challenging due to the complexity of the cellular environment. In order to study influenza A M1 in a well-defined setting, we have developed a minimal in vitro system that reconstitutes M1 assembly and budding on giant unilamellar vesicles. With this system, we have found that M1 oligomerization shapes the membrane into structures that geometrically and topologically resemble the budding virus. These structures are capable of incorporating cargoes with dimensions comparable to the RNP complex, and their assembly drives sorting of both lipids and membrane-bound proteins. By varying the mechanical and biochemical environment in which M1 assembles, we have identified a key mechanistic role for M1 in coordinating influenza A virus bud assembly.

Distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens in nervous and non-nervous organs of European seabass (Dicentrarchus labrax) during the course of an experimental challenge

The distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens was examined by immunohistochemistry in the nervous and non-nervous organs of juvenile European seabass (Dicentrarchus labrax) during the course of an intramuscular infection. Histological changes resulting from the infection were evaluated from 3 days to 2 months post-infection. The specific antibody response was also studied 2 months post-challenge. Viral proteins were present throughout the experimental period in the retina (inner nuclear layer, ganglion layer, outer limiting membrane, and outer plexiform layer), brain (cerebellum and tectum opticum), and liver (hepatocytes and endothelial cells). These proteins were also observed in the renal tubular cells, white pulp of spleen, and in fibroblasts and cartilage of caudal fin. This is the first report of RGNNV proteins appearing in these organs, where the immunostaining was only detected at certain sampling times after the onset of mortality. Brain and retina of virus-exposed fish showed high levels of vacuolation, while accumulation of fat vacuoles was observed in the liver. RGNNV infection also induced a specific antibody response as measured by an ELISA. In summary, this is the first study demonstrating the presence of viral proteins in cells of caudal fin, kidney and spleen of European seabass.

An inactivated West Nile Virus vaccine derived from a chemically synthesized cDNA system

A cDNA comprising the complete genome of West Nile Virus (WNV) was generated by chemical synthesis using published sequence data, independent of any preformed viral components. The synthetic WNV, produced by transfection of in vitro transcribed RNA into cell culture, exhibited undistinguishable biological properties compared to the corresponding animal-derived wild-type virus. No differences were found concerning viral growth in mammalian and insect cell lines and concerning expression of viral proteins in cells. There were also no significant differences in virulence in mice following intranasal challenge. After immunizations of mice with experimental vaccines derived from the synthetic and wild-type viruses, protection from lethal challenge was achieved with similar amounts of antigen. Both vaccine preparations also induced comparable levels of neutralizing antibodies in mice. In addition, the synthetic approach turned out to be very accurate, since the rescued WNV genome contained no undesired mutations. Thus, the first flavivirus based on chemical gene synthesis was indistinguishable from the parent virus. This demonstrates that virus isolates from animal sources are dispensable to derive seed viruses for vaccine production or research.

Trypsin is associated with the rotavirus capsid and is activated by solubilization of outer capsid proteins

The rotavirus capsid is made up of three concentric protein layers. The outer layer, consisting of VP7 and VP4, is lost during virus entry into the host cell. Rotavirus field isolates can be adapted to high-titre growth in tissue culture by treatment with trypsin and by supplementing the culture medium with trypsin, which cleaves VP4 into two fragments, VP8* and VP5*. It is known that protease inhibitors reduce the replication of rotavirus in vitro and in vivo and also diminish disease symptoms in a mouse model. To clarify the molecular basis of these observations, a series of assays were conducted on purified rotavirus particles grown in the presence of trypsin. Results of HPLC and mass spectrometry followed by N-terminal sequencing showed that viral particles contain molecules of trypsin. When associated with triple-layer particles (TLPs), trypsin is inactive and not accessible to protease inhibitors, such as aprotinin. When the outer layer is solubilized by calcium-chelating agents, VP5*, VP8* and VP7 are released and the associated trypsin is activated, allowing cleavage of the viral capsid proteins, as well as other exogenous proteins. It is shown that addition of trypsin inhibitors significantly reduces synthesis of viral mRNA and viral proteins in cells and has a major inhibitory effect if present when virus enters the cell. These data indicate that incorporation of trypsin into rotavirus particles may enhance its infectivity.

The Influence of Potato Virus Y Infection on the Ultrastructure of Pssu‐ipt Transgenic Tobacco

We studied the effect of Potato virus YNTN (PVY) on the cell ultrastructure of control (rooted control plants [C] and control plants grafted onto control rootstock [C/C]) and transgenic Pssu‐ipt plants overproducing cytokinins (CK; rooted [T] or grafted [T/C]). The PVY infection caused visible symptoms, i.e., reduction of leaf surface and plant growth, acceleration of leaf senescence, veinal necrosis, and leaf distortion in all infected C plants; in T and C/C plants, plant age affected whether symptoms of infection occurred. Even though DAS‐ELISA proved the presence of virus coat protein in all studied plants, the symptoms of infection were never observed in T/C plants. Relative content of virus proteins in cells correlated with the stage of symptom development in infected plants. A massive accumulation of virus proteins was found in the cytoplasm of infected plants, and it was proved by immunocytochemical methods. The most prominent effect of viral infection was a decrease of volume density of starch, an...

Herpes simplex virus 1 gene expression is accelerated by inhibitors of histone deacetylases in rabbit skin cells infected with a mutant carrying a cDNA copy of the infected-cell protein no. 0.

An earlier report showed that the expression of viral genes by a herpes simplex virus 1 mutant [HSV-1(vCPc0)] in which the wild-type, spliced gene encoding infected-cell protein no. 0 (ICP0) was replaced by a cDNA copy is dependent on both the cell type and multiplicity of infection. At low multiplicities of infection, viral gene expression in rabbit skin cells was delayed by many hours, although ultimately virus yield was comparable to that of the wild-type virus. This defect was rescued by replacement of the cDNA copy with the wild-type gene. To test the hypothesis that the delay reflected a dysfunction of ICP0 in altering the structure of host protein-viral DNA complexes, we examined the state of histone deacetylases (HDACs) (HDAC1, HDAC2, and HDAC3). We report the following. (i) HDAC1 and HDAC2, but not HDAC3, were modified in infected cells. The modification was mediated by the viral protein kinase U(S)3 and occurred between 3 and 6 h after infection with wild-type virus but was delayed in rabbit skin cells infected with HSV-1(vCPc0) mutant, concordant with a delay in the expression of viral genes. (ii) Pretreatment of rabbit skin cells with inhibitors of HDAC activity (e.g., sodium butyrate, Helminthosporium carbonum toxin, or trichostatin A) accelerated the expression of HSV-1(vCPc0) but not that of wild-type virus. We conclude the following. (i) In the interval in which HSV-1(vCPc0) DNA is silent, its DNA is in chromatin-like structures amenable to modification by inhibitors of histone deacetylases. (ii) Expression of wild-type virus genes in these cells precluded the formation of DNA-protein structures that would be affected by either the HDACs or their inhibitors. (iii) Since the defect in HSV-1(vCPc0) maps to ICP0, the results suggest that this protein initiates the process of divestiture of viral DNA from tight chromatin structures but could be replaced by other viral proteins in cells infected with a large number of virions.

Localization of viral proteins in cells infected with bovine viral diarrhoea virus.

Bovine viral diarrhoea virus (BVDV) is a member of the genus Pestivirus within the family Flaviviridae. In this report, protein localization studies were performed to assess the mechanism for the release of mature virus particles from infected cells. Since BVDV is an enveloped virus, budding from either intra- or extracellular membranes is feasible. A prerequisite for the latter mechanism is the integration of viral glycoproteins into the host cell membrane. Using monoclonal antibodies (MAbs) directed against the viral envelope glycoproteins E2 and E(RNS), no specific signals were detected on the surface of BVDV-infected cells by indirect fluorescence, confocal microscopy or fluorescence-activated cell sorter analyses. Furthermore, biotin-labelled cell surface proteins of virus-infected and non-infected cells were not detected by immunoprecipitation using MAbs directed against E(RNS) and E2 or the non-structural protein NS2-3. None of these proteins was detected on the cell surface. In addition, to analyse the intracellular localization of the two viral glycoproteins E(RNS) and E2 and the non-structural proteins NS2-3 and NS3, subcellular fractionation of virus-infected cells followed by radioimmunoprecipitation with the MAbs were performed. These results led to the conclusion that the BVDV envelope glycoproteins E(RNS) and E2 as well as the non-structural proteins NS2-3 and NS3 were almost quantitatively associated with intracellular membranes. These findings indicate that BVDV is released by budding into the cisternae of the endoplasmic reticulum and that there seems to be no correlation between the location and function of the analysed proteins.

Liquid chromatography/tandem mass spectrometry of synthesis products associated with the viral protein US11.

US11 is a small basic protein composed of 161 amino acid residues, and is among the most abundant viral proteins in cells infected with herpes simplex viruses HSV1 and HSV2. The amino acid sequence [91-121] is considered essential for the binding of this protein with RNA. Automated solid phase synthesis of this fragment resulted in a crude reaction mixture containing the desired sequence as well as a number of unknown side products. On-line liquid chromatography/electrospray mass spectrometry (LC/ES-MS) and LC/ES tandem mass spectrometry (MS/MS) allowed the identification of the separated components and furnished relevant sequencing information. The unusual sequences of the monitored components, which consist of a tandemly repeated three-amino-acid motif with the sequence Arg-X-Pro, where X is an acidic or uncharged polar amino acid residue, yielded product ion spectra lacking substantial sequence information and rich in fragment ions manifesting the neutral losses 17, 42 and 60 u.

T cells chronically infected with HIV do not contain sufficient Nef to promote CD4 downmodulation in the absence of envelope-mediated effects.

Among HIV viral proteins, envelope glycoproteins and Nef have been both suggested to participate in CD4 downregulation during the course of HIV infection. In a previous study, we provided evidence that a mutant form of CD4 that does not bind gp120 was never downregulated in chronically HIV-1- and HIV-2-infected CEM cells. To further investigate the relative effects of Nef or glycoproteins in CD4 downregulation, recombinant vaccinia virus (VV) vectors were used to express high levels of HIV-1 viral proteins in cells expressing both wild-type and mutant CD4. It was demonstrated that during HIV infection, overexpression of Nef, achieved through the VV expression system, was necessary to induce CD4 downregulation in the mutant CD4-expressing cell model. These results are consistent with the hypothesis that Nef-mediated CD4 downregulation depends on the cellular levels of Nef expression. We concluded that during the late stage of viral replication, CD4 downregulation is mostly due to gp120 and not to Nef because of a low level of Nef expression.

Rapamycin stimulates viral protein synthesis and augments the shutoff of host protein synthesis upon picornavirus infection.

The immunosuppressant drug rapamycin blocks progression of the cell cycle at G1 in mammalian cells and yeast. We recently showed that rapamycin inhibits both in vitro and in vivo cap-dependent, but not cap-independent, translation. This inhibition is causally related to reduced phosphorylation and consequent activation of 4E-BP1, a repressor of the function of the cap-binding protein, eIF4E. Two members of the picornavirus family, encephalomyocarditis virus and poliovirus, inhibit phosphorylation of 4E-BP1. Since translation of picornavirus mRNAs is cap independent, inhibition of phosphorylation of 4E-BP1 could contribute to the shutoff of host protein synthesis. Here, we show that rapamycin augments both the shutoff of host protein synthesis and the initial rate of synthesis of viral proteins in cells infected with encephalomyocarditis virus and poliovirus.

Characterization of temperature-sensitive mutants of Pichinde virus.

The synthesis of viral proteins and S RNAs in cells infected with 12 temperature-sensitive (ts) mutants of Pichinde virus was characterized. The mutants could be divided into five groups on the basis of the patterns of radiolabeled proteins immunoprecipitated from infected-cell lysates. Markedly reduced nucleoprotein levels and undetectable amounts of glycoprotein precursor and L protein were synthesized at the nonpermissive temperature in cells infected with five of the mutants. Reduced but detectable amounts of the viral proteins were synthesized at the nonpermissive temperature in cells infected with a single mutant. Two mutants were associated with the intracellular accumulation of glycoprotein precursor, which was apparently not transported across the cell membrane in cells incubated at the nonpermissive temperature. The synthesis of viral proteins in cells infected with two mutants was indistinguishable from those produced by wild-type virus. Two additional mutants were associated with markedly reduced amounts of immunoprecipitable proteins in infected cells incubated at both the permissive and nonpermissive temperatures. Analysis of viral RNA with radiolabeled single-stranded cDNA probes representing complementary and genomic-sense sequences corresponding to the 3' region of S RNA revealed two basic patterns of viral RNA synthesis. At the nonpermissive temperature, the synthesis of complementary- and genomic-sense sequences and mRNA of the S RNA segment was markedly reduced in cells infected with representative members of these mutant groups, suggesting the presence of mutations altering transcriptase activity. Viral-complementary- and genomic-sense sequence and RNA synthesis, as well as nucleoprotein mRNA in cells, was detected in reduced amounts for mutants associated with reduced levels of proteins at both temperatures. Interestingly, RNA species larger than the S RNA segment were detected in cells infected with some of the mutants, especially those with putative transcriptase lesions. These molecules suggest a possible oligomeric intermediate in the synthesis of S RNA of Pichinde virus.

Maturation of viral proteins in cells infected with temperature-sensitive mutants of vesicular stomatitis virus

Maturation of viral proteins in cells infected with mutants of vesicular stomatitis virus was studied by surface iodination and cell fractionation. The movement of G, M, and N proteins to the virion bud appeared to be interdependent. Mutations thought to be in G protein prevented its migration to the cell surface, allowed neither M nor N protein to become membrane bound, and blocked formation of viral particles. Mutant G protein appeared not to leave the endoplasmic reticulum at the nonpermissive temperature, but this defect was partially reversible. In cells infected with mutants that caused N protein to be degraded rapidly or prevented its assembly into nucleocapsids, M protein did not bind to membranes and G protein matured to the cell surface, but never entered structures with the density of virions. Mutations causing M protein to be degraded prevented virion formation, and G protein behaved as in cells infected by mutants in N protein. These results are consistent with a model of virion formation involving coalescence of soluble nucleocapsid and soluble M protein with G protein already in the plasma membrane.

Expression of viral proteins in mammalian cells transformed by avian sarcoma viruses

The expression of viral proteins in nine lines of hamster and rat cells transformed by avian sarcoma viruses (ASV) was studied by indirect immunofluorescence with monospecific antisera to purified gp85 and p27 of AMV-B and a polyvalent antiserum to all the p proteins of this same virus. The lines of ASV-transformed cells were either low virus producers (VP) or inducible or non-inducible non producers (NP). Cytoplasmic expression of p proteins was observed in all the cell lines except the least inducible NP cell line, and cytoplasmic expression of gp85 in all the cell lines. The degree of expression varied widely with the lines and was not related to the class of permissiveness or inducibility. However, in the inducible NP class, the expression of p proteins and gp85 was higher in the most inducible cell lines. The data also suggest that the expression of the p proteins must be uncoordinate in at least some cell lines and must also be uncoordinate with the expression of gp85. In the VP cell lines and the most inducible NP lines, g85 and some p proteins other than p27 were also expressed on the cell membrane. The membrane expression of gp85 and the p proteins which were expressed appeared to be coordinate and to parallel the degree of cytoplasmic expression. In contrast, no, or a negligible expression of viral proteins was observed on the membrane of the least inducible and the non-inducible cell lines. These results suggest that there may exist translational and/or post-translational controls of the expression of viral proteins in the ASV-transformed mammalian cells and that the permissiveness and the inducibility of the cells may depend on the insertion of viral proteins in the cell membrane. The failure of p27 to insert in the cell membrane could account for the low permissiveness or the non-permissiveness of the cells.