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1.
Mouse L cell fibroblasts were infected with vaccinia virus and labeled 2 to 3 h postinfection with [35S]methionine. Labeled proteins were fractionated on native and denatured DNA-cellulose columns and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Twenty-four 90,000 to 12,500, were detected. VDP-12A (molecular weight, 29,750) had affinity for denatured but not native DNA, and its synthesis was dependent on viral DNA replication. VDP-20 (molecular weight, 41,000) bound very tightly to native and denatured DNA and was displaced only after boiling the protein-DNA-cellulose matrix in 1% sodium dodecyl sulfate. VDP-8,-11,-12,-13, -and-14 behaved electrophoretically like the polypeptide species previously shown to be present in DNA-protein complexes prepared from infected cells. The molecular weights of VDP-10 (50,000), VDP-11 (36,000), and VDP-8 (67,000) were similar to the polypeptide subunits of polyadenylate polymerase and phosphohydrolase I, enzymes purified from virions which have also been shown to have affinity for DNA.  相似文献   

2.
A recombinant vaccinia virus containing cloned DNA sequences coding for the three structural proteins and nonstructural proteins NS1 and NS2a of dengue type 4 virus was constructed. Infection of CV-1 cells with this recombinant virus produced dengue virus structural proteins as well as the nonstructural protein NS1. These proteins were precipitated by specific antisera and exhibited the same molecular size and glycosylation patterns as authentic dengue virus proteins. Infection of cotton rats with the recombinant virus induced NS1 antibodies in 1 of 11 animals. However, an immune response to the PreM and E glycoproteins was not detected. A reduced level of gene expression was probably the reason for the limited serologic response to these dengue virus antigens.  相似文献   

3.
E Paez  S Dallo    M Esteban 《Journal of virology》1987,61(8):2642-2647
To investigate the genetic stability of vaccinia virus DNA, we have tested whether alterations occurred in the polypeptide composition of this complex virus during persistent infections. We found that variants isolated at various passages in Friend erythroleukemia cells persistently infected with vaccinia virus contained, in addition to an 8-megadalton (MDa) deletion on the left terminus of the viral genome, major alterations in the sizes of three structural proteins with molecular masses of about 39, 21, and 14 kDa. Alterations in isoelectric points were also observed in proteins of 48, 27, and 14 kDa. The 14-kDa protein is part of the virus envelope, and the variants increased the size of this protein from 0.5 to 3 kDa with increasing passage number. Alteration in size of the 14-kDa protein is a dominant trait since it appeared in the whole virus population by passage 48. With more passages, some variants were found to increase or decrease the size of a 39-kDa core protein by about 2 kDa and to decrease the size of an envelope protein of 21 kDa by about 2 kDa. These three proteins were immunogenic in mice and elicited a strong host immune response. Major alterations in the sizes of these proteins were prevented by continuous treatment of the persistently infected cultures with interferon. However, after interferon was removed, protein modifications appeared with increasing passage number. Generation of the 8-MDa deletion and alterations in the size of the 14-kDa protein correlated with a marked decrease in virulence of these variants. Our findings suggest that during virus persistence, specific mutations are introduced in the vaccinia virus genome that lead to protein alterations and to highly attenuated viruses.  相似文献   

4.
P Lee  D E Hruby 《Journal of virology》1993,67(7):4252-4263
The three major vaccinia virus (VV) virion proteins (4a, 4b, and 25K) are proteolytically matured from larger precursors (P4a, P4b, and P25K) during virus assembly. Within the precursors, Ala-Gly-X motifs have been noted at the putative processing sites, with cleavage apparently taking place between the Gly and X residues. To identify the sequence and/or structural parameters which are required to define an efficient cleavage site, a trans-processing assay system has been developed by tagging the carboxy terminus of the P25K polypeptide (precursor of 25K) with an octapeptide FLAG epitope, which can be specifically recognized by a monoclonal antibody. By using transient expression assays with cells coinfected with VV, the proteolytic processing of the chimeric gene product (P25K:FLAG) was monitored by immunoblotting procedures. The relationship between the P25K:FLAG precursor and the 25K:FLAG cleavage product was established by pulse-chase experiments. The in vivo cleavage of P25K:FLAG was inhibited by the drug rifampin, implying that the reaction was utilizing the same pathway as authentic VV core proteins. Moreover, the 25K:FLAG protein was found in association with mature virions in accord with the notion that cleavage occurs concomitantly with virion assembly. Site-directed mutagenesis of the Ala-Gly-Ala motif at residues 31 to 33 of the P25K:FLAG precursor to Ile-Asp-Ile blocked production of the 25K:FLAG product. The efficiency of 25K:FLAG production (33.71%) is, however, approximately only half of the production of 25K (63.98%) within VV-infected cells transfected with pL4R:FLAG. One explanation for the lower efficiency of 25K:FLAG production was suggested by the observation in the immunofluorescent-staining experiment that 25K:FLAG-related proteins were not specifically localized to the virus assembly factories (virosomes) within VV-infected cells, although virosome localization was prominent for P25K-related polypeptides. Since VV core protein proteolytic processing is believed to take place during virion maturation, only the P25K:FLAG which was assembled into immature virions could undergo proteolytic maturation. Furthermore during these experiments, a potential cleavage intermediate (25K') of P25K was identified. Amino acid residues 17 to 19 (Ala-Gly-Ser) of the P25K precursor were implicated as the intermediate cleavage site, since no 25K':FLAG product was produced from a mutant precursor in which the sequence was altered to Ile-Asp-Ile. Taken together, these results provide biochemical and genetic evidence to support the hypothesis that the Ala-Gly-X cleavage motif plays a critical role in VV virion protein proteolytic maturation.  相似文献   

5.
Fatty acid acylation of vaccinia virus proteins.   总被引:1,自引:6,他引:1       下载免费PDF全文
Labeling of vaccinia virus-infected cells with [3H]myristic acid resulted in the incorporation of label into two viral proteins with apparent molecular weights of 35,000 and 25,000 (designated M35 and M25, respectively). M35 and M25 were expressed in infected cells after the onset of viral DNA replication, and both proteins were present in purified intracellular virus particles. Virion localization experiments determined M25 to be a constituent of the virion envelope, while M35 appeared to be peripherally associated with the virion core. M35 and M25 labeled by [3H]myristic acid were stable to treatment with neutral hydroxylamine, suggesting an amide-linked acylation of the proteins. Chromatographic identification of the protein-bound fatty acid moieties liberated after acid methanolysis of M25, isolated from infected cells labeled during a 4-h pulse, resulted in the recovery of 25% of the protein-bound fatty acid as myristate-associated label and 75% as palmitate, indicating that interconversion of myristate to palmitate had occurred during the labeling period. Similar analyses of M25 and M35, isolated from infected cells labeled during a 0.5-h pulse, determined that 46 and 43%, respectively, of the protein-bound label had been elongated to palmitate even during this brief labeling period. In contrast, M25 and M35 isolated from purified intracellular virions labeled continuously during 24 h of growth contained 75 and 70%, respectively, myristate-associated label, suggesting greater stability of these proteins or a favored interaction of the proteins containing myristate with the maturing or intracellular virion.  相似文献   

6.
A unique abundant protein, designated P by analogy to the putative polymerase proteins of other paramyxoviruses, was identified in purified Newcastle disease virus. Under nonreducing conditions the P proteins could be separated from other viral proteins on sodium dodecyl sulfate-polyacrylamide gels. The P proteins were isolated from detergent-solubilized virions as 53,000- to 55,000-dalton monomers and disulfide-linked trimers. Distinct forms of P having four different isoelectric points and two different electrophoretic mobilities were resolved by two-dimensional electrophoresis. Two forms of P were phosphorylated, as were the nucleocapsid protein and non-glycosylated membrane protein. In addition to disulfide-linked forms of P, dimers of the hemagglutinin-neuraminidase glycoprotein and two disulfide-linked versions of the fusion glycoprotein were identified. Several electrophoretic variants of the nucleocapsid protein that were probably created by intrachain disulfide bonding were also isolated from virions under nonreducing conditions. The locations of the newly identified proteins were determined by detergent-salt fractionation of virions and by surface-selective radioiodination of the viral envelope. The P proteins were associated with nucleocapsids and were not detected at the surface of virions. Both forms of the fusion glycoproteins were on the exterior of the viral envelope. Herein the properties of the P proteins are compared with similar proteins of rhabdoviruses and other paramyxoviruses, and a role for multiple forms of proteins in the genetic economy of newcastle disease virus is discussed.  相似文献   

7.
Polyacrylamide gel electrophoresis of purified Junin virus revealed six distinct structural polypeptides, two major and four minor ones. Four of these polypeptides appeared to be covalently linked with carbohydrate. The molecular weights of the six proteins, estimated by coelectrophoresis with marker proteins, ranged from 25,000 to 91,000. One of the two major components (number 3) was identified as a nucleoprotein and had a molecular weight of 64,000. It was the most prominent protein and was nonglycosylated. The other major protein (number 5), with a molecular weight of 38,000, was a glucoprotein and a component of the viral envelope. The location on the virion of three additional glycopeptides with molecular weights of 91,000, 72,000, and 52,000, together with a protein with a molecular weight of 25,000, was not well defined.  相似文献   

8.
9.
Previous studies have shown that at least three vaccinia virus (VV) late proteins (with apparent molecular asses of 37, 35, and 25 kDa) label with myristic acid. Time course labeling of VV-infected cells with [3H]myristic acid reveals at least three additional putative myristylproteins, with apparent molecular masses of 92, 17, and 14 kDa. The 25-kDa protein has previously been identified as that encoded by the L1R open reading frame, leaving the identities of the remaining proteins to be determined. Sequence analysis led to the preliminary identification of the 37-, 35-, and 17-kDa proteins as G9R, A16L, and E7R, respectively. Using synthetic oligonucleotides and PCR techniques, each of these open reading frames was amplified by using VV DNA as a template and then cloned individually into expression vectors behind T7 promoters. These plasmid constructs were then transcribed in vitro, and the resulting mRNAs were translated in wheat germ extracts and radiolabeled with either [35S]methionine or [3H]myristic acid. Each wild-type polypeptide was labeled with [35S]methionine or [3H]myristic acid in the translation reactions, while mutants containing an alanine in place of glycine at the N terminus were labeled only with [35S]methionine, not with myristic acid. This result provided strong evidence that the open reading frames had been correctly identified and that each protein is myristylated on a glycine residue adjacent to the initiating methionine. Subcellular fractionations of VV-infected cells suggested that A16L and E7R are soluble, in contrast to L1R, which is a membrane-associated protein.  相似文献   

10.
Three avian nuclear proteins which bind to the Rous sarcoma virus long terminal repeat have been detected. Two of the proteins bind to sequences within the enhancer, and the third protein binds to a sequence spanning the enhancer and an upstream promoter region.  相似文献   

11.
12.
In a coimmunoprecipitation assay with monospecific antisera, the two major envelope proteins GL and M of equine arteritis virus were found to occur in heteromeric complexes in virions and infected cells. While the GL protein associated with M rapidly and efficiently, newly synthesized M protein was incorporated into complexes at a slower rate, which implies that it interacts with GL molecules synthesized earlier. Analysis under nonreducing conditions revealed that the GL/M complexes consist of disulfide-linked heterodimeric structures. Pulse-chase experiments showed that virtually all GL monomers ended up in heterodimers, whereas a fraction of the M protein persisted as monomers. The M protein also formed covalently linked homodimers, but only the heterodimers were incorporated into virus particles.  相似文献   

13.
The monospecific antiserums to the major proteins p12, p19, p20, p42, p61 of the vaccinia virus coat were obtained and analyzed. The dynamics of the proteins accumulation in the infected cells has been studied. Products of the cell-free translation of the total viral mRNA precipitated by the obtained antiserums were identified. The p20 protein has been found to be a result of p12 protein reversible oligomerization under the conditions of electrophoresis. The antigenic relation of p12 and p20 proteins to a p42 protein, also a p12 oligomer, has been demonstrated. The possibility is discussed to use the obtained antiserums for mapping of the genes corresponding to structural proteins in the genome of the vaccinia virus.  相似文献   

14.
We investigated whether the VP1 protein of simian virus 40 binds to DNA. In vitro DNA-binding experiments clearly indicate that VP1 bound strongly to double-stranded and single-stranded DNA, with a higher affinity for the latter; additional experiments show that VP1 did not bind to a specific sequence of simian virus 40 DNA.  相似文献   

15.
Genes encoding virus-specific late proteins with molecular mass 36 kDa and 12 kDa were mapped in HindIII-P DNA fragment of vaccinia virus strain L-IVP by hybrid selection of RNA to cloned DNA fragments followed by in vitro translation. RNA origin site of the 36K protein was detected in HindIII-J fragment. Nucleotide sequences of these genes were determined. Amino acid sequences of the 36K and 12K polypeptides were compared with the protein bank PIR.  相似文献   

16.
Robust methods to detect DNA-binding proteins from structures of unknown function are important for structural biology. This paper describes a method for identifying such proteins that (i) have a solvent accessible structural motif necessary for DNA-binding and (ii) a positive electrostatic potential in the region of the binding region. We focus on three structural motifs: helix–turn-helix (HTH), helix–hairpin–helix (HhH) and helix–loop–helix (HLH). We find that the combination of these variables detect 78% of proteins with an HTH motif, which is a substantial improvement over previous work based purely on structural templates and is comparable to more complex methods of identifying DNA-binding proteins. Similar true positive fractions are achieved for the HhH and HLH motifs. We see evidence of wide evolutionary diversity for DNA-binding proteins with an HTH motif, and much smaller diversity for those with an HhH or HLH motif.  相似文献   

17.
18.
I Guizani  M P Kieny  R Lathe  P Clertant 《Gene》1988,73(1):163-173
We previously reported that live recombinant vaccinia viruses (VV) encoding either the large T (LT) or middle T (MT) antigens of polyoma virus (PyV) were able to induce rejection of tumors caused by PyV-transformed cells [Lathe et al., Nature 326 (1987) 878-880]. Here we present evidence that PyV early proteins expressed by the recombinants retain the biochemical characteristics of their authentic counterparts despite the cytopathic effect of VV infection. VV-encoded LT is a nuclear phosphoprotein, with specific DNA binding, ATPase and nucleotide-binding activities. VV-expressed MT associates with cellular kinases, particularly with pp60c-src, by which it is phosphorylated in vitro. Expression levels of LT and MT reached 10(6) molecules per infected cell. The use of VV as a vector is encouraged by the high expression level obtained and because VV infection does not seem to prevent appropriate post-translational processing of proteins encoded by VV recombinants.  相似文献   

19.
Densonucleosis virus structural proteins   总被引:1,自引:0,他引:1       下载免费PDF全文
The protein coats of two densonucleosis viruses (types 1 and 2) were examined by a variety of biophysical, biochemical, and serological techniques. The viruses were 24 nm in diameter, contained at least four polypeptides, were remarkably stable to extremes of pH and denaturing agents, and were serologically closely related. The two viruses could, however, be distinguished serologically and by differences in migration of their structural polypeptides. For each virus the “top component” (i.e., the protein coat minus DNA, found occurring naturally in infections) appeared to have a composition identical to that of the coat of the virus and was a more stable structure. Electrometric titration curves of the virus particles and top components demonstrated that the DNA phosphate in densonucleosis virus particles was neutralized by cations other than basic amino acid side chains of the protein coat. Circular dichroism studies showed that there was a conformational difference between the protein coats of top components and virus particles.  相似文献   

20.
One approach for a safer smallpox vaccine is to utilize recombinant subunits rather than live vaccinia virus (VACV). The products of the VACV envelope genes A27L, L1R, B5R, and A33R induce protective antibodies in animal models. We propose that proteins that elicit T-cell responses, as well as neutralizing antibodies, will be important to include in a molecular vaccine. To evaluate VACV-specific memory T-cell responses, peripheral blood mononuclear cells (PBMC) from four VACV vaccinees were tested against whole VACV and the individual envelope proteins A27, B5, L1, and A33, using gamma interferon enzyme-linked immunospot and cytokine flow cytometry assays. PBMC were stimulated with autologous dendritic cells infected with VACV or electroporated with individual VACV protein mRNAs. T-cell lines from all donors, vaccinated from 1 month to over 20 years ago, recognized all four VACV envelope proteins. Both CD4(+) and CD8(+) T-cell responses to each protein were detected. Further analysis focused on representative proteins B5 and A27. PBMC from a recent vaccinee exhibited high frequencies of CD4(+) and CD8(+) T-cell precursors to both B5 (19.8 and 20%, respectively) and A27 (6.8 and 3.7%). In comparison, B5- and A27-specific T-cell frequencies ranged from 0.4 to 1.3% in a donor vaccinated 3 years ago. Multiple CD4(+) and CD8(+) T-cell epitopes were identified from both A27 and B5, using overlapping 15-mer peptides. These data suggest that all four VACV envelope proteins may contribute to protective immunity, not only by inducing antibody responses, but also by eliciting T-cell responses.  相似文献   

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