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1.
The herpes simplex virus type 1 (HSV-1) tegument protein VP22 encoded by the UL49 gene is essential for HSV-1 infection. However, its precise functions in the virus life cycle are unknown. A relatively important tool for disclosing these functions is an antiserum specifically detecting VP22 in the infected cell. To this end, a recombinant truncated VP22 protein consisting of C-terminal 45 aa fused to EYFP (enhanced yellow fluorescent protein) and His-tag was expressed in Escherichia coli, purified by the Ni2+-NTA affinity chromatography, and used for the preparation of antiserum in rabbits. Western blot and immunofluorescence assay showed that this antiserum specifically detected purified truncated VP22 as well as full-length VP22 in the HSV-1 infected cells. These results indicate that the prepared antiserum could serve as a valuable tool for further studies of VP22 functions. 相似文献
2.
We constructed a recombinant thymidine kinase-negative herpes simplex virus type 1 (HSV-1) that expressed the rotavirus major outer capsid glycoprotein, VP7. In the recombinant HSV-1, a promoter from the 5' noncoding region of the HSV-1 glycoprotein B locus regulated the expression of VP7 as a HSV-1 gamma 1 gene product. HSV-1-expressed VP7 resembled rotavirus-expressed VP7 in its SDS-PAGE mobility, high mannose-type glycosylation, disulfide bonding, perinuclear to cytoplasmic localization, intracellular retention, and reactivity with polyclonal antisera and nonneutralizing antibodies. Unlike rotavirus-expressed VP7, HSV-1-expressed VP7 lacked several neutralizing epitopes by immuno-histochemical staining and by ELISA. One neutralizing epitope identified on HSV-1-expressed VP7 by ELISA was masked by paraformaldehyde fixation of recombinant HSV-1- but not rotavirus-infected cells. Neutralizing epitopes were restored to HSV-1-expressed VP7 by coinfection of cells with the HSV-1 recombinant and a heterologous rotavirus that lack the neutralizing epitopes. The recovered neutralizing epitopes were detected on double-shelled rotavirus particles produced in the coinfected cells. This study indicates that the formation of several neutralizing epitopes on rotavirus VP7 requires interaction of VP7 with other rotavirus proteins. In addition, HSV-1 was a useful vector for studying the localization, processing, and antigenicity of an RNA virus glycoprotein. 相似文献
3.
Complement-mediated cytolysis of HSV-1 and HSV-2 infected cells: plasma membrane antigens reactive with type-specific and cross-reactive antibody. 总被引:11,自引:0,他引:11
Surface antigens of BHK-21 cells infected with HSV-1 or HSV-2 were radioiodinated (125I) with lactoperoxidase, immune precipitated and analysed by polyacrylamide gel electrophoresis (PAGE). Experiments using antiserum to HSV-1 or HSV-2, absorbed with appropriate hemotypic or heterotypic antigens, revealed that both type-specific (homotypic) and cross-reactive antibody combined with surface glycoproteins to form a single large radioactive peak. This peak, which constituted the major glycoprotein region (region a) observed in electropherograms, represented a range in mol. wt. from 115000 to 130000. Sensitization of cells to complement lysis, neutralization of infectious virus and immune precipitation of surface glycoproteins (region a) were found to be generally correlated properties of all the antibody preparations analysed, including antibody prepared specifically against region a antigens. These findings suggest a major immunological role for the surface glycoproteins migrating in PAGE region a. 相似文献
4.
P Raj D O Matson B S Coulson R F Bishop K Taniguchi S Urasawa H B Greenberg M K Estes 《Journal of clinical microbiology》1992,30(3):704-711
Three sets of neutralizing monoclonal antibodies (MAbs) used to type the outer capsid protein VP7 of four group A rotavirus serotypes (1 through 4) were compared in competition immunoassays. Reciprocal competition was observed for each of the VP7 type 2-, 3-, and 4-specific MAbs. The VP7 type 1 MAbs exhibited variable competition patterns with other VP7 type 1 MAbs. MAb RV4:3, which has been used to recognize antigenic variants within VP7 type 1 strains, showed reciprocal competition with the four VP7 type 3 MAbs (RV3:1, YO-1E2, 4F8, and 159) using a VP7 type 3 virus (SA11) as antigen. MAb 2C9, also prepared against VP7 type 1, reacted with VP7 type 3 strains and competed with a VP7 type 3 MAb, 159, using RRV as antigen. Use of the different sets of VP7 type-specific MAbs in the enzyme-linked immunosorbent assay permitted the recognition of six antigenic variants within VP7 types 1, 2, and 3 among specimens whose VP7 type could not be determined previously with only one set of typing MAbs. These results demonstrate differences of typing ability among these VP7-specific MAbs and emphasize the need to improve the sensitivity of typing systems by incorporating panels of MAbs reacting with several neutralizing epitopes. 相似文献
5.
Kerstin Borchers M. Özel G. Pauli H. R. Gelderblom H. Ludwig 《Archives of virology》1990,111(1-2):1-14
Summary Some major structural components of simian herpesvirus SA 8 were analyzed and the relationship of SA 8 with HSV-1 and especially with BHV-2 was further characterized using a panel of SA 8- and BHV-2-specific monoclonal antibodies directed against gB, gD, gE, and ICP 8.It could be shown that SA 8 and BHV-2 expressed gB-1 equivalents, which differ in electrophoretic mobility, but share common epitopes with HSV-1. The antigenic determinants were detectable in the cytoplasm, on the surface of infected cells and on the virus envelope. Monoclonal antibodies reactive with epitopes of gB-SA 8 and gB-BHV-2 neutralized the homologous virus and cross-neutralized only HSV-1 and HSV-2, suggesting differences in accessability of the corresponding epitope on SA 8 and BHV-2, respectively. A second protein with conserved epitopes on SA 8, BHV-2, and HSV-1 was identified as ICP 8. This nucleus associated protein was additionally detected on the envelope of SA 8 and HSV-1. The results imply that ICP 8 might have a function not only in virus replication, but also in virus assembly.We could furthermore define type-specific epitopes on two SA 8 envelope proteins which are analogous to gD-1 and gE-1, respectively. The gD-specific epitope induced a type-specific neutralizing antibody, making it interesting for differentiation of closely related herpesviruses. 相似文献
6.
A monospecific rabbit antiserum has been prepared to the denatured 145K major capsid protein of strain Colburn cytomegalovirus (CMV). Using this antiserum to analyze proteins electrophoretically transferred to nitrocellulose, it was found that infected cell preparations from all primate CMVs examined contain a single cross-reactive protein band that ranged in molecular weight from 144K to 153K, depending upon the isolate. No reactivity was observed when similar preparations of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), and herpes zoster virus (HZV) were tested, suggesting that this antiserum may be CMV group-specific. A second antiserum prepared to the denatured Colburn 66K matrix protein was found to be more selective in its reactivity toward other simian CMVs, and without reactivity to human CMV preparations. 相似文献
7.
Cell-mediated immunity to herpes simplex virus: recognition of type-specific and type-common surface antigens by cytotoxic T cell populations. 总被引:10,自引:2,他引:10
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In this communication, we examine the specificity of anti-herpes simplex virus (HSV) cytotoxic T lymphocytes (CTL). Serological studies of the two related HSV serotypes (HSV-1 and HSV-2) have revealed both type-specific and cross-reactive antigenic determinants in the viral envelope and on the surface of infected cells. By analysis of cytotoxicity of CTL, generated in vitro by restimulation of splenocytes from mice primed with one or the other HSV serotype, the recognition of both type-specific and cross-reactive determinants on infected target cells by anti-HSV CTL was detectable. Thus, effector cells generated by priming and restimulating with the same virus recognized both type-specific and cross-reactive determinants on target cells infected with the homologous virus, but only cross-reactive determinants on target cells infected with the heterologous HSV serotype. CTL generated by restimulation with the heterologous virus were capable of recognizing only the cross-reactive determinants on either HSV-1- or HSV-2-infected target cells. These results indicate that two subpopulations of CTL exist in a population of anti-HSV immune spleen cells--those which recognize type-specific determinants and those specific for cross-reactive antigenic determinants present on the surface of HSV infected cells. The type-specific subset of anti-HSV CTL was shown to recognize the gC glycoprotein of HSV-1 infected target cells. In addition to the gC glycoprotein, at least one other type-specific surface antigen was also recognized by anti-HSV CTL in addition to the cross-reactive determinants recognized by anti-HSV CTL. 相似文献
8.
Isolation and characterization of a large molecular-weight polypeptide of herpes simplex virus type 1 总被引:47,自引:0,他引:47
Infection of human embryonic lung cells at the nonpermissive temperature (39°) with tsB2, a DNA-negative mutant of herpes simplex virus type 1 (HSV-1) resulted in the accumulation of a large molecular-weight (175,000) virus-induced polypeptide detectable by SDS-polyacrylamide gel electrophoresis. This polypeptide (VP175) was detectable only in small amounts and was found mainly in the nuclear fraction of cells infected with tsB2 at the permissive temperature (34°) or with wild-type HSV-1 at both 34° and 39°. However at 39° VP175 accumulated to become the major component in both the cytoplasmic and nuclear fractions of tsB2-infected cells. Identical results were obtained with a second mutant (tsB21) in the same complementation group. Temperature-shift studies suggest that the events responsible for the accumulation of VP175 at 39° occur early in the replicative cycle. With the use of a combination of SDS-preparative and analytical disc gel electrophoresis, VP175 was isolated and rabbit antisera to this polypeptide were prepared. By immunofluorescence assay, anti-VP175 sera reacted only with the nucleus of wild-type HSV-1-infected cells, whereas tsB2-infected cells cultured at 39° showed both cytoplasmic and nuclear immunofluorescence. In addition, the anti-VP175 serum reacted preferentially with HSV-1 as compared with HSV-2-infected cells, suggesting a possible type specificity for the reagent. 相似文献
9.
Synthesis of proteins in cells infected with herpesvirus. X. Proteins excreted by cells infected with herpes simplex virus, types 1 and 2. 总被引:5,自引:0,他引:5
Uninfected primary rabbit kidney cells, as well as cells infected with herpes simplex virus (HSV), types 1 and 2, excrete glycoproteins. The PAGE profiles of the glycoproteins excreted by the infected and by the uninfected cells are different from those of the glycoproteins that remain cell-associated. Furthermore, the glycoproteins excreted by cells infected with HSV-1 have different polyacrylamide-gel electrophoretic (PAGE) profiles from those of cells infected with HSV-2. Both profiles differ from the PAGE profile of glycoproteins excreted by uninfected cells.Indirect radioimmune precipitation tests revealed that antisera prepared against glycoproteins excreted by cells infected with HSV-1 or HSV-2 are highly type-specific. Sera prepared against the glycoproteins excreted by cells infected with HSV-1 or HSV-2 react significantly only with the homologous glycoproteins. Sera prepared against mature HSV-1 virions react to a considerable extent with HSV-1 and HSV-2 virions, as well as with the cell-associated glycoproteins of cells infected with both virus types; these sera, however, react to a significant extent only with the glycoproteins excreted by cells infected with HSV-1 but not those excreted by cells infected with HSV-2. Thus, cells infected with HSV excrete considerable amounts of virus-specific glycoproteins that are mainly type-specific. The potential value of these excreted glycoproteins as diagnostic tools is discussed. 相似文献
10.
Herpes simplex virus 2 VP22 phosphorylation induced by cellular and viral kinases does not influence intracellular localization 总被引:2,自引:0,他引:2
Phosphorylation of the herpes simplex virus (HSV) VP22 protein is regulated by cellular kinases and the UL13 viral kinase, but the sites at which these enzymes induce phosphorylation of HSV-2 VP22 are not known. Using serine-to-alanine mutants to map phosphorylation sites on HSV-2 VP22 in cells, we made three major observations. First, phosphorylation by a cellular kinase mapped to serines 70, 71, and/or 72 within CKII consensus sites analogous to previously identified phosphorylation sites in HSV-1 VP22. Second, we mapped UL13-mediated phosphorylation of HSV-2 VP22 to serines 28 and 34, describing for the first time UL13-dependent phosphorylation sites on VP22. Third, previously identified VP22-associated cellular kinase sites in HSV-1 VP22 (serines 292 and 294) were not phosphorylated in HSV-2 VP22 (serines 291 and 293). VP22 expressed alone accumulated in the cytoplasm and to a lesser extent in the nucleus. Phosphorylation by endogenous cellular kinase(s) did not alter the localization of VP22. Co-expression of HSV-2 VP22 with active UL13, but not with enzymatically inactive UL13, resulted in nuclear accumulation of VP22 and altered nuclear morphology. Surprisingly, redistribution of VP22 to the nucleus occurred independently of UL13-induced phosphorylation of VP22. The altered nuclear morphology of UL13-expressing cells was not due to apoptosis. These results demonstrate that phosphorylation of HSV-2 VP22 at multiple serine residues is induced by UL13 and cellular kinase(s), and that the nuclear/cytoplasmic distribution of VP22 is independent of its phosphorylation status but is controlled indirectly by UL13 kinase activity. 相似文献
11.
Sylvain Meylan Daniel Robert Christine Estrade Valérie Grimbuehler Olivier Péter Pascal R Meylan Roland Sahli 《Journal of clinical virology》2008,41(2):87-91
BACKGROUND: HSV-1 and HSV-2 cause CNS infections of dissimilar clinico-pathological characteristics with prognostic and therapeutic implications. OBJECTIVES: To validate a type-specific real-time PCR that uses MGB/LNA Taqman probes and to review the virologico-clinical data of 25 eligible patients with non-neonatal CNS infections. RESULTS: This real-time PCR was evaluated against conventional PCR (26 CSF and 20 quality controls), and LightCycler assay (51 mucocutaneous, 8 CSF and 32 quality controls) and culture/immunofluorescence (75 mucocutaneous) to assess typing with independent methods. Taqman real-time PCR detected 240 HSV genomes per ml CSF, a level appropriate for the management of patients, and provided unambiguous typing for the 104 positive (62 HSV-1 and 42 HSV-2) out the 160 independent clinical samples tested. HSV type diagnosed by Taqman real-time PCR predicted final diagnosis (meningitis versus encephalitis/meningoencephalitis, p<0.001) in 24/25 patients at time of presentation, in contrast to clinical evaluation. CONCLUSIONS: Our real-time PCR, as a sensitive and specific means for type-specific HSV diagnosis, provided rapid prognostic information for patient management. 相似文献
12.
Herpes simplex virus type-selective enzyme-linked immunosorbent assay with Helix pomatia lectin-purified antigens 总被引:5,自引:1,他引:5
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B Svennerholm S Olofsson S Jeansson A Vahlne E Lycke 《Journal of clinical microbiology》1984,19(2):235-239
Helix pomatia lectin-purified antigens with specific reactivity to herpes simplex virus type 1 (HSV-1) and HSV-2 antibodies in human sera were used in an enzyme-linked immunosorbent assay. The type specificity of the antigens was assessed by double immunodiffusion precipitation in gel against rabbit HSV-1 and HSV-2 hyperimmune sera, and by enzyme-linked immunosorbent assay with human reference sera containing antibodies to either type of HSV. Fifty-two sera from patients with documented infection with either HSV-1 or HSV-2 were assayed for HSV type-specific immunoglobulin G antibodies. The reactivity of the sera against lectin-purified antigens correlated completely with the results of virus typing. We conclude that HSV type-specific immunoglobulin G antibodies can easily be measured by enzyme-linked immunosorbent assay with the use of Helix pomatia lectin-purified HSV-1 and HSV-2 antigens. 相似文献
13.
Detection of type-specific antibody to herpes simplex virus type 1 by radioimmunoassay with herpes simplex virus type 1 glycoprotein C purified with monoclonal antibody. 总被引:10,自引:0,他引:10
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Herpes simplex virus type 1 (HSV-1) and HSV-2 specify at least four glycoproteins designated gA/gB, gC, gD, and gE. Previous studies have shown that gC produced by HSV-1 is antigenically distinct from the corresponding HSV-2 glycoprotein. With the exception of gC, the glycoproteins of both serotypes share antigenic sites. Standard serological assays fail to differentiate the antibody to the shared antigenic determinants from the type-specific antibody. In this paper, we describe a procedure for purifying gC from HSV-1-infected cell extracts with an immunoadsorbent prepared with an HCL monoclonal antibody. When used in a solid-phase radioimmunoassay, gC proved to be a type-specific antigen for quantitation of antibody to HSV-1. Among individuals who had no antibody to HSV at the onset of infection, all of those with primary HSV-1 infection developed antibody to gC. Subjects with primary HSV-2 infection failed to develop antibody reactive with gC of HSV-1 (P less than 0.01). Both immunoglobulin G and M antibodies against gC were detected in sera from subjects with either primary or recurrent HSV-1 infection. Higher antibody titers to gC were found in sera from individuals with recurrent infection than in sera from those with primary HSV-1 infection. 相似文献
14.
Analysis of herpes simplex virus type 1 antigens exposed on the surface of infected tissue culture cells. 总被引:11,自引:0,他引:11
Crossed immunoelectrophoretic analysis of herpes simplex virus type 1 (HSV-1) antigens solubilized from infected tissue culture cells has previously identified five viral specific precipitates: the type-common Ag-11, Ag-8, and Ag-3, the type-specific Ag-6, and Ag-7, which may be type specific. In the present study, the antigens exposed at the surface of infected cells were analyzed by peroxidase-catalyzed 125I labeling of the cell surface at different times postinfection. The antigens were detectable in the following sequence: Ag11 (5 hr postinfection), Ag-8 (8 hr postinfection) and Ag-6 and Ag-7 (between 8 and 18 hr postinfection). Ag-3 was not exposed at the surface of infected cells and was not present in the HSV-1 virion. Polypeptide analysis of the individual HSV-1 antigens after iodination of the surface of infected cells showed that mainly the glycopolypeptides of the protein complexes were present at the surface. No immunochemical cross-reactivity between the viral and the host cell antigens was demonstrated even though sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis indicated that polypeptides of equal molecular weight were present in Ag-8 and the host cell membrane. 相似文献
15.
Nozawa N Daikoku T Yamauchi Y Takakuwa H Goshima F Yoshikawa T Nishiyama Y 《Virus genes》2002,24(3):257-266
We have raised a rabbit polyclonal antiserum against a recombinant 6× His-tagged herpes simplex virus type 2 (HSV-2) UL7 fusion protein expressed in Escherichia coli. The antiserum specifically reacted with a 33 kDa protein in HSV-1 and HSV-2-infected cell lysates, and was used to characterize the UL7 gene product of HSV-2. The UL7 protein was produced in the late phase of infection, and its synthesis was highly inhibited, but not abolished by the addition of acyclovir (ACV). The UL7 protein associated with extracellular virions and also with all types of capsids, including A, B, and C capsids, though the association seemed to be weak. Indirect immunofluorescence studies revealed that at 9 h postinfection, UL7 specific fluorescence was detected in part or all of the nucleus, and the specific fluorescence colocalized with the scaffold protein ICP35. However, at later times postinfection, the UL7 protein was mainly detected as a mass in a juxtanuclear cytoplasmic region. In addition, transmission immunoelectron microscopy (TIEM) confirmed the association of the UL7 protein with intracellular capsids and virions in HSV-2-infected cells. The HSV-2 UL7 protein contained a domain highly conserved in all herpesviruses, part of which exhibited a homology with domains in the fission yeast Schizosaccharomyces pombe DNA topoisomerase III. We discuss the possibility that the UL7 protein may play a supplementary role in the viral DNA cleavage/packaging process. 相似文献
16.
The authors prepared 156 mouse hybridomas producing monoclonal (MCA) antibodies to type- and group-specific antigenic determinants of HSV-1 and HSV-2. Seven of them were studied at length by western blot and radioimmunoprecipitation methods. The cell lines 1H97 and 2H141 were shown to produce immunoglobulins of G2 beta and M class, respectively, and were directed against group-specific antigenic determinants of the major nucleocapsid protein p150. The cell lines 1H38 and 1H110 produced immunoglobulins of M and G2 beta, respectively, and were directed against type-specific antigens of HSV-1 glycoprotein gB. At the same time, the presence of group-specific antigenic determinants on glycoprotein gB molecule was indicated by MCA 1H188 belonging to immunoglobulins of G2 alpha class. Two cell lines, 2H208 and 1H225, produced immunoglobulins G2 alpha directed against type-specific antigenic determinants of HSV-2 glycoprotein gD and group-specific antigenic determinants of HSV-1 gD, respectively. The results of immunoelectron microscopy indicated that MCA 1H110 and 2H208 were directed against virus envelope proteins. 相似文献
17.
18.
The location of nucleotide sequences within the bovid herpesvirus 1 (BHV-2) genome homologous to herpes simplex virus 1 (HSV-1) DNA were investigated. BHV-2 DNA was digested with restriction endonucleases and blotted to nitrocellulose paper. The blots were then probed with plasmids containing HSV-1 genes for thymidine kinase (TK), the major DNA binding protein (ICP8), the major capsid protein (VP5) and genes for HSV-1 glycoproteins gB, gD, and gC. Except for HSV-1 gC, each HSV-1 gene tested hybridized to BHV-2 nucleotide sequences that were located either on both sides of a restriction endonuclease cleavage site, within a small restriction endonuclease fragment, or to an area common to two overlapping restriction fragments. Thus, we were able to localize BHV-2 nucleotide sequences homologous to the HSV-1 ICP8 gene between 0.38 and 0.41 map units (m.u.), and BHV-2 nucleotide sequences homologous to the HSV-1 VP5 gene between 0.24 and 0.27 m.u. In addition, BHV-2 nucleotide sequences homologous to HSV-1 genes for TK, gB and gD were found to lie on both sides of restriction endonuclease cleavage sites at 0.30 0.35, and 0.94 m.u., respectively. 相似文献
19.
ICP1/2 (also designated VP1/2) is a 270-kDa structural protein of herpes simplex virus type 1 (HSV-1) which is located in the tegument region of the virion. In this report we describe the production of a polyclonal antiserum specific for ICP1/2 and the use of this antiserum to examine the synthesis, processing, and intracellular localization of the viral polypeptide. Pulse-labeling studies indicated that ICP1/2 is synthesized late during infection, being initially detectable between 8 and 9 hr postinfection with the rate of synthesis continuing to increase until 11 to 12 hr postinfection. Further studies on the expression of ICP1/2 in the presence or absence of viral DNA replication indicated that the synthesis of the polypeptide is absolutely dependent on viral DNA replication. These results suggest that ICP1/2 represents a gamma 2 (true late) gene product. Additionally, we have performed experiments to determine if ICP1/2 is post-translationally modified in HSV-infected cells. These studies indicated that ICP1/2 is phosphorylated on serine residues; however, we found no evidence to suggest that the protein is glycosylated. Using subcellular fractionation and indirect immunofluorescence techniques, we have determined that ICP1/2 is diffusely distributed throughout the nucleus and cytoplasm of HSV-infected cells with no specific compartmentalization of the polypeptide. 相似文献
20.
Summary. We developed a rabbit polyclonal antiserum reactive against a recombinant 6x His-UL46 fusion protein expressed in*Escherichia coli, and using this antiserum identified the UL46 gene product of herpes simplex virus type 2 (HSV-2) to be phosphoproteins with
apparent molecular masses of 82-, 84-, and 86-kDa in infected Vero cells. The UL46 protein was produced in the late phase
of infection in a manner highly dependent on viral DNA synthesis, and was mainly distributed at the edge of the nucleus in
the cytoplasm. Although its kinetics of production and its progress of distribution were different from those of the major
tegument protein VP16 (the UL48 gene product or α-trans-inducing factor (αTIF)), most of the UL46 protein colocalized with
VP16 in the late phase of infection, and copurified with it in column chromatography. Moreover, our data showed that the HSV-2
UL46 protein, when coexpressed with VP16, enhanced α4 promotor-regulated gene expression in a transient luciferase reporter
assay, while the expression of the UL46 protein alone suppressed it.
Received December 16, 1999 Accepted February 23, 2000 相似文献