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PubMed1990

Antibody response to viral proteins U (vpu) and R (vpr) in HIV-1-infected individuals.

Antibodies to E. coli-produced HIV-1 vpr and vpu were determined by enzyme immunoassay in serial sets of sera from 72 men seroconverting for antibodies to HIV-1 structural proteins, and from 196 initially symptom-free men who were positive for such antibodies at study entry. First detection of vpr- and vpu-specific antibodies always was within 12 months of seroconversion for antibodies to structural proteins. In the combined cohort of 268 men, vpr- and vpu-specific antibodies were found persistently in 26 and 43% of men, respectively. Vpr- and vpu-specific antibodies were transiently detected in 3 and 7%, respectively, and intermittently detected in 18 and 13% of men, respectively. No association was found between the patterns of vpr- or vpu-specific antibody response and clinical outcome. In subjects with different patterns of vpr- and vpu-specific antibody response, no clear temporal relationship existed between the appearance or disappearance of antibodies and the onset of HIV-1-related disease.

Cross-Sectional Studies↗
PubMed2026

Metaproteomic profiling reveals viral proteins and associated host proteomic alterations in glioblastoma.

Glioblastoma (GB) is a WHO grade 4 brain cancer with dismal prognosis, yet its aetiology remains poorly defined. Although viral involvement has been proposed, findings across studies remain inconsistent, reflecting inherent limitations of individual technologies and cohort size. Here we applied metaproteomic profiling to a publicly available GB proteome dataset (12 control, 21 adjacent, 159 tumour) and an independent cohort of 81 samples (37 control, 44 tumour) to detect viral proteins in tumour and controls tissues. Across cohorts, we detected viral proteins from diverse species, with human herpesviruses (HHV-1, 2, and 8) more frequently detected in GB tumours compared with control tissues. Analysis of the host tumour proteome revealed differential abundance of proteins related to transcriptional regulation, RNA processing, protein translation, immune responses, and mitochondrial-associated metabolism. Correlation analysis identified associations between viral and human proteins, with several linked to biological processes previously implicated in DNA virus-host interactions. Further stratification of tumour by HHV-1 status showed consistent alterations in proteins associated with mitochondrial-associated metabolism, protein turnover, and cell adhesion/signalling.In summary, this study demonstrates the feasibility of metaproteomics for detecting viral components in archival GB tissues. Using this approach, we observed differences in viral protein landscape across cohorts and identified associations between viral presence and host proteomic features, providing a protein-level framework for future studies of virus-host interactions in GB.

Humans↗
PubMed2027

Epitope Tagging and Coimmunoprecipitation to Identify Viral Protein Interactors.

Affinity purification-mass spectrometry (AP-MS) is a powerful proteomic approach for dissecting the interaction network between virus and host. Traditional AP-MS employs overexpression of viral proteins as baits to enrich host interactors. However, overexpressed viral proteins may mislocalize to inappropriate cellular compartments and trigger endoplasmic reticulum stress by overwhelming the protein-folding machinery, which leads to false identification of host factors. To overcome these limitations, we introduce an AP-MS strategy based on direct infection with an epitope-tagged chikungunya virus (CHIKV/myc-E2), which we used to successfully uncover two new antiviral factors in CHIKV cellular reservoirs-macrophages. In this protocol, we will describe this technique step by step: (1) design and construction of myc-tagged virus by advanced multi-fragment assembly, (2) in vitro transcription and preparation of infectious myc-tagged virus stocks, and (3) immunoprecipitation of myc-tagged viral protein and its interactome for mass spectrometry analysis. This strategy enables accurate identification of viral interactors in a physiologically relevant context, providing a framework for future proteomic studies using tagged viruses.

Chikungunya virus↗
PubMed2001

The US3 protein kinase of herpes simplex virus 1 mediates the posttranslational modification of BAD and prevents BAD-induced programmed cell death in the absence of other viral proteins.

Earlier studies have shown that the d120 mutant of herpes simplex virus 1, which lacks both copies of the alpha4 gene, induces apoptosis in all cell lines tested. In some cell lines d120-induced apoptosis, manifested by the release of cytochrome c, activation of caspase 3, and fragmentation of cellular DNA, is blocked by the overexpression of Bcl-2. In these cells viral protein kinase U(S)3 delivered in trans blocks apoptosis induced by the mutant virus at a premitochondrial stage. We report that the U(S)3 protein kinase targets the pro-apoptotic BAD member of the Bcl-2 family. Specifically, the U(S)3 protein kinase mediates a posttranslational modification of BAD and blocks its cleavage, which is reported to activate apoptosis. Thus, U(S)3 protein kinase is the sole viral protein required to block activation of caspase 3, prevent cleavage of poly(ADP-ribose) polymerase, and block fragmentation of cellular DNA induced by BAD.

Animals↗
PubMed1992

Serodiagnostic assay of hepatitis C virus infection using viral proteins expressed in Escherichia coli.

Infection with hepatitis C virus (HCV) was analyzed by an enzyme-linked immunosorbent assay based on recombinant viral proteins encoded by regions of the putative viral core, NS3, NS4 and NS5, which were expressed in E. coli. Results showed that 106 of 124 cases (85.5%) of non-A, non-B chronic hepatitis and 43 of 45 cases (95.5%) of hepatocellular carcinoma, negative for HBV marker, were positive for antibodies against at least one of these viral proteins. One of 87 healthy individuals with normal alanine aminotransferase activity was positive for antibody against only the viral core, but was negative for HCV RNA. The serum of one patient with chronic hepatitis was positive for one of these proteins, but negative for HCV RNA. These findings in combination with results on detection of HCV RNA in the sera of patients with non-A, non-B chronic hepatitis indicated that 105 of 124 cases (84.6%) were positive for HCV infection. Sera that were negative for HCV antibodies against all these proteins were also negative for HCV RNA assayed by reverse transcription followed by the polymerase chain reaction. Screening of HCV infection by detecting viral antibodies in circulating blood using all these viral proteins is useful for reducing the number of ambiguous results in screening for viral infection. Thus, this assay system may be useful diagnostic purposes.

Carcinoma, Hepatocellular↗
PubMed1983

Humoral antibody response to individual viral proteins after murine cytomegalovirus infection.

The purpose of this study was to identify viral proteins that played an important role in the humoral immune response to murine cytomegalovirus (MCMV). Viral proteins were separated from a purified virus preparation on polyacrylamide gels, were blotted onto nitrocellulose strips, and were reacted with antisera collected from mice on various days post infection. No antibody response was detected in serum obtained 5 days post infection, but by 10 days there was a faint response to five different proteins. Thereafter, the number of proteins eliciting an antibody response, as well as the intensity of the response, increased with time so that by 42 days post infection a response to 13 major antigens was detected. This method provides a means of separating out important immunogens from the more than 30 different MCMV proteins originally identified by polyacrylamide gel electrophoresis. Such information may improve our understanding of the pathogenesis of MCMV infection as well as host immune responses to the virus.

Animals↗
PubMed2000

Identification of effective constituents of influenza vaccine by immunization with plasmid DNAs encoding viral proteins.

The use of plasmid DNA encoding influenza viral proteins to vaccinate animals constitutes a promising approach to the development of effective subunit vaccines. This review describes the results obtained by the immunization of mice with such plasmid DNAs. (i) Both hemagglutinin (HA)- and neuraminidase (NA)-expressing DNAs for the surface glycoproteins from A/PR/8/34 (H1N1) or B/Ibaraki/2/85 virus can provide the most effective protection against influenza A-type or B-type virus infection among the various viral protein-expressing DNAs tested in BALB/c mice. (ii) A mixture of plasmid DNAs encoding HA and NA can provide more effective protection against virus challenge than plasmid DNA encoding HA or NA alone in BALB/c mice. (iii) NA-DNA can provide protection against infection not only by homologous virus but also by drift viruses. (iv) HA-DNA from A/PR/8/34 (H1N1) virus provides significant protection only in BALB/c (H-2(b)) mice, whereas HA-DNA from B/Ibaraki/2/85 virus affords significant protection in BALB/c, B10 (H-2(d)), and C3H (H-2(k)) mice. NA-DNA from both A-type and B-type viruses provides significant protection in the three strains of mice. These results suggest that both HA and NA molecules should be used as vaccine components to provide effective protection against influenza A-type and B-type virus infection in genetically heterogeneous humans.

Animals↗
PubMed2004

The changes in the T helper 1 (Th1) and T helper 2 (Th2) cytokine balance during HIV-1 infection are indicative of an allergic response to viral proteins that may be reversed by Th2 cytokine inhibitors and immune response modifiers--a review and hypothesis.

The HIV-1 infection in humans induces an early cellular immune response to react to the viral proteins with a cytotoxic T cell (CTL) response that fails to inhibit virus replication and the spread of the virus. It became evident that the progression of the disease causes chronic changes to the immune system of which a gradual increase in IgE antibodies is one of its features. When the HIV-1 epidemic began, the relation between the gradual increase in IgE content and AIDS was not understood, but later it became a marker for disease prognosis. The advances in the knowledge on T helper 1 (Th1) and T helper 2 (Th2) cells revealed that Th1 cells produce cytokines that stimulate the proliferation of CTLs. Th2 cells produce cytokines that are responsible for the activation of the humoral immune response in healthy people. Studies on both Th1 and Th2 cytokine synthesis revealed an aberration in HIV-1 infected people. Clerici and Shearer presented a hypothesis (1993) whereby Th1 cell activity declines and Th2 activity increases (the Th1 --> Th2 switch hypothesis) in HIV-1 infected people. In fact, experiments concerning this hypothesis ultimately supported the premise that the switch involves a critical change in the cytokine balance, which leads to the contraction of AIDS. However, the research community must still discern why such a Th1 --> Th2 switch takes place in infected people and how it can be reversed. The present review points to the fact that a similar Th1 --> Th2 switch constitutes the response of allergic people to environmental allergens. HIV-1 patients and allergic people that are exposed to allergens respond with an increased synthesis of Th2 cytokines and IgE, together with a decrease in Th1 cytokines. The studies on allergen-induced Th2 cells revealed that the Th2 cytokine IL-4 induces B cells to synthesize IgE, and cytokine IL-5 is the inducer of eosinophilia, just as in HIV-1 infection. The difference between the HIV-1 infection and allergies is the ability of IL-4 to induce the synthesis in T cells of the HIV-1 coreceptor CXCR4 that selects from the replicating virus a syncytium-inducing (SI) virus, a variant virus that replicates rapidly. The present hypothesis implicates the viral proteins in the induction of Th2 cytokine synthesis. This suggests that in viral proteins, allergen-like domains may be responsible for the activation of Th2 cytokine synthesis. Based on the analogy of the responses of humans to allergens and HIV-1, the following hypotheses is suggested: (a) Removal of allergen-like domains from viral genes by genetic engineering may provide viral proteins for vaccine development. (b) Attempts to treat allergic patients with IL-4 receptor inhibitors suggests that the "Th2 --> Th1 Reversion" constitutes a possible approach to inhibiting the Th2 cytokines and inducing a revival of the anti-viral Th1 response.

Adjuvants, Immunologic↗
PubMed1987

The poliovirus-induced shut-off of cellular protein synthesis persists in the presence of 3-methylquercetin, a flavonoid which blocks viral protein and RNA synthesis.

In poliovirus-infected cells, the viral protein and RNA synthesis were severely reduced, provided 3-methylquercetin was present between 1 and 2 h post-infection. Under these conditions, the virally induced host shut-off remained in effect. On the other hand, in uninfected HeLa cells, protein and RNA synthesis was inhibited only slightly by 3-methylquercetin. The inhibition of poliovirus cytopathogenicity in Vero cells by 3-methylquercetin exhibited a similar time dependence.

Animals↗
PubMedDate not supplied

[Isolation of a complex of tick-borne encephalitis viral proteins by affinity chromatography using monoclonal antibodies to proteins NS5 and NS3].

Affinity chromatography of lysates of continuous porcine embryo cells infected with tick-borne encephalitis (TBE) virus on sepharose with immobilized monoclonal antibodies to TBE virus proteins NS5 and NS3 results in isolation of a stable protein complex. This complex contains viral proteins NS5, NS3, p49, and, probably, two more cell proteins. This complex is not detected at the early stage of infection, and 24 h after infection its structure does not depend on the specificity of immobilized monoclonal antibodies used in affinity chromatography and on the time elapsed after the infection. Immunoprecipitates of infected cells phosphorylated TBE virus protein NS5 in vitro, but possessed no RNA-polymerase activity. Elution of the complex with buffers with pH 3.5 or 11.1 or with 2M urea failed to purify the active replicase. The complex of viral and cellular proteins isolated by affinity chromatography on different immunoadsorbents did not possess RNA-polymerase activity because of inactivation or absence of additional subunit(s).

Animals↗
PubMed2006

Porcine reproductive and respiratory syndrome virus-infected alveolar macrophages contain no detectable levels of viral proteins in their plasma membrane and are protected against antibody-dependent, complement-mediated cell lysis.

Porcine reproductive and respiratory syndrome virus (PRRSV) can evade the host immune system, which results in prolonged virus replication for several weeks to several months. To date, the mechanisms of PRRSV immune evasion have not been investigated in detail. One possible immune-evasion strategy is to avoid incorporation of viral proteins into the plasma membrane of infected cells, as this prevents recognition by virus-specific antibodies and consequent cell lysis either by the classical complement pathway or by antibody-dependent, cell-mediated cytotoxicity. In this study, viral proteins were not observed in the plasma membrane of in vitro-infected macrophages by using confocal microscopy or flow cytometry. Subsequently, the sensitivity of PRRSV-infected macrophages towards antibody-dependent, complement-mediated cell lysis (ADCML) was determined by using an ADCML assay. A non-significant percentage of PRRSV-infected cells were killed in the assay, showing that in vitro PRRSV-infected macrophages are protected against ADCML. PRRSV proteins were not detected in the plasma membrane of in vivo-infected alveolar macrophages and ADCML was also not observed. Together, these data indicate that viral proteins are not incorporated into the plasma membrane of PRRSV-infected macrophages, which makes infected cells invisible to PRRSV-specific antibodies. This absence of viral proteins on the cell surface could explain the protection against ADCML observed for in vitro and in vivo PRRSV-infected macrophages, and may play a role in virus persistence.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1977

Viral protein synthesis in Friend erythroleukemia cell lines.

Viral protein synthesis was studied in two Friend virus-induced erythroleukemia cell lines (Ostertag cell lines FSD1-F4 and B8) by the technique of immuno-precipitation with monospecific antisera to the major envelope glycoprotein gp70 and major core protein p30. One of the cell lines (F4) releases active Friend virus complex to the growth medium, where release of virus from the other cell line (B8) is barely or nondetectable. It was found that in the nonproducer cell line B8, a large-molecular-weight protein of about 65,000 containing p30 antigenic determinants is synthesized, yet no p30 is produced upon prolonged incubation and chase, suggesting that this might be the actual lesion that prevents mature virus production by these cells. In both cell lines, the predominant protein species that is immunoprecipitated with monospecific anti-gp70 serum is a protein of 55,000 to 60,000 daltons that is labeled with glucosamine to a much lesser extent that gp70 and appears to become heterogeneous with time. Large amounts of gp70 can be detected in the cell-free medium, but none of the unstable species of 55,00 to 60,000 molecular weight.

Cell Line↗
PubMed1989

The large viral RNA segment of California serogroup bunyaviruses encodes the large viral protein.

Reassortant bunyaviruses derived from two members of the California serogroup (La Crosse/original and Tahyna/181-57) viruses were used to demonstrate that the large Mr viral protein (L) is encoded by the L RNA segment. Radiolabelled viral proteins were analysed by discontinuous SDS-PAGE. The L protein of La Crosse virus was observed to migrate ahead of its Tahyna virus counterpart when electrophoresed through a 5% acrylamide resolving gel. Among the reassortant viruses, the L protein phenotype segregated with the viral L RNA segment. After confirming the genotype of the viruses used in this study, it was concluded that the L RNA species of California serogroup viruses codes for the L protein, the presumed viral polymerase.

Animals↗
PubMed1975

Lifetimes of mRNA molecules directing the synthesis of viral proteins in herpes simplex virus-infected cells.

Possible variations in the functional lifetimes of herpes simplex virus type 1 mRNA molecules in infected HeLa cells were studied. As shown by the rate of decrease of radioactive amino acid incorporation into viral proteins after the addition of actinomycin, the average lifetime of early viral mRNA's are shorter than those for the late messenger species. In addition, when the viral proteins made after the addition of actinomycin were further analyzed by gel electrophoresis, it was found that messengers for individual viral proteins translated within the early or late time period also had some differences in their functional lifetimes. These results indicate that the synthesis of herpes simplex virus type 1 proteins during the replicative cycle is regulated in part by mechanisms controlling the functional lifetimes of viral mRNA's.

Cycloheximide↗
PubMed2006

Herpes simplex virus DNA synthesis is not a decisive regulatory event in the initiation of lytic viral protein expression in neurons in vivo during primary infection or reactivation from latency.

The herpes simplex virus genome can enter a repressed transcriptional state (latency) in sensory neurons of the host nervous system. Although reduced permissiveness of the neuronal environment is widely accepted as a causal factor, the molecular pathway(s) directing and maintaining the viral genome in the latent state remains undefined. Over the past decade, the field has been strongly influenced by the observations of Kosz-Vnenchak et al., which have been interpreted to indicate that, in sensory neurons in vivo, a critical level of viral DNA synthesis within the neuron is required for sufficient viral immediate-early (IE) and early (E) gene expression (M. Kosz-Vnenchak, J. Jacobson, D. M. Coen, and D. M. Knipe, J. Virol. 67:5383-5393, 1993). The levels of IE and E genes are, in turn, thought to regulate the decision to enter the lytic cycle or latency. We have reexamined this issue using new strategies for in situ detection and quantification of viral gene expression in whole tissues. Our results using thymidine kinase-null and rescued mutants as well as wild-type strains in conjunction with viral DNA synthesis blockers demonstrate that (i) despite inhibition of viral DNA replication, many neurons express lytic viral proteins, including IE proteins, during acute infection in the ganglion; (ii) at early times postinoculation, the number of neurons expressing viral proteins in the ganglion is not reduced by inhibition of viral DNA replication; and (iii) following a reactivation stimulus, the numbers of neurons and apparent levels of lytic viral proteins, including IE proteins, are not reduced by inhibition of viral DNA replication. We conclude that viral DNA replication in the neuron per se does not regulate IE gene expression or entry into the lytic cycle.

Animals↗
PubMed2000

Short synthetic peptides derived from viral proteins compete with HIV gp120 for the binding to CD4 receptors.

In the complex mechanism of adhesion, internalization, and infection of cells by human immunodeficiency virus (HIV) viral particles, a determinant role is played by the viral envelope glycoprotein gp120, which binds to CD4 receptors of T cells and monocytes. We tested the ability of a panel of 7- to 12-residue synthetic peptides, selected from the region 414-434 of the HIV-1 gp120, to inhibit the binding of the viral protein to CD4 receptors of cultured human lymphoid cells. The assay was based on the observation that the binding of gp120 to the receptors interferes with the binding of a specific anti-CD4 monoclonal antibody, as a result of the masking of the antibody epitope; thus, we tested whether preincubation of cells with the peptides before gp120 addition might restore the recognition of the CD4 molecule by the antibody. High expression of CD4 receptors was thus assumed as indication that the binding of the viral protein had been inhibited. Maximum activity was displayed by a 9-residue peptide located near the amino terminal end of the 414-434 fragment. In addition, several fragments deduced from other viral proteins, possessing partial amino acid sequence homology with the HIV gp120 fragment, exhibited a similar type of interaction with the CD4 receptor. All active peptides contain the Cys residue (position 423 of gp120). This residue is essential, although not sufficient, for inhibiting gp120 binding, as few other amino acid residues within the fragment play a complementary role in increasing or decreasing the inhibitory ability.

Amino Acid Sequence↗
PubMed2005

The anaphase promoting complex: a critical target for viral proteins and anti-cancer drugs.

The study of animal viruses has provided extraordinary insights into cell cycle dynamics and tumor biology. The significance of the p53 and Rb tumor suppressor proteins, for example, was discovered due to their interactions with viral oncogenes. In the past several years, investigations with four viral proteins, human immunodeficiency virus type 1 (HIV-1) vpr, adenovirus E4orf4, chicken anemia virus (CAV) apoptin and human T lymphotropic virus type I (HTLV-I) Tax, have indicated that there are also critical viral targets involved in G2/M control. In particular, recent studies with E4orf4 and apoptin have shown that they induce G2/M arrest by targeting and inhibiting the anaphase-promoting complex/cyclosome (APC/C). Notably, these two viral proteins induce apoptosis selectively in transformed cells in a p53-independent manner; thus pathways affected by these proteins are of significant therapeutic interest. Further investigation of the underlying mechanism of G2/M arrest and subsequent apoptosis induced by viral APC/C inhibitors may shed light on the mechanisms of current cancer therapies and provide the foundation for developing novel therapeutic targets.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2003

Adenoviral-expressed GP5 of porcine respiratory and reproductive syndrome virus differs in its cellular maturation from the authentic viral protein but maintains known biological functions.

The ORFs 5, 6 and 7, encoding for the three major structural proteins, GP(5), M and N, of the IAF-Klop strain of PRRSV were cloned and expressed in 293 cells using replication-defective human type 5 adenoviral vectors (hAdVs). Although the M protein gene could be cloned into hAdVs and expressed constituvely in 293 cells under the control of the hCMV immediate early promotor/enhancer, hAdVs expressing N and GP(5) proteins, which appeared to be toxic or interfered with adenovirus replication, could only be generated by inclusion of a tetracycline-regulatable promotor in the transfer vector pAdTR5. The recombinant (rec) proteins appeared similar to the authentic viral proteins in regards to their M(r)s and antigenicities. However, the recGP(5) apparently possesses different N-linked oligosaccharides residues. Its sensitivity to endo-beta-galactosidase digestion indicates that poly-N-acetyllactosamine is present on the individually-expressed protein, but not on the authentic GP(5) anchored into the virion envelope. The recGP(5) apparently accumulates within the ER compartment as a glycoprotein that possesses high-mannose N-linked oligosaccharide side chains sensitive to endo-beta-N-acetylglucosaminidase H treatment, by contrast to its viral counterpart for which N-linked oligosaccharide side chains are of both high-mannose and complex types. Coinfection of 293 cells with hAdVs expressing the M and GP(5) did not lead to M-GP(5) heterodimer formation, as demonstrated in PRRSV-infected cells. Moreover, cells infected with inducible hAdV/ORF5 showed that GP(5) of the North American strain is proapoptotic. Indeed, when the expression cassette was turned-on, caspase 3 activity in hAdV/ORF5 infected cells was enhanced and DNA fragmentation could be detected by TUNEL assays. Pigs intradermally injected twice with hAdV/ORF5 developed antibody titers to the authentic viral GP(5) as soon as 10 days following challenge with the homologous virulent PRRSV strain, as revealed by Western blot and virus neutralization tests, suggesting the establishment of a specific immune memory.

Adenoviridae↗