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1.
Irradiation of chicken muscle cells with ultraviolet light (254 nm) to cross-link RNA and protein moieties was used to examine the polypeptide complements of cytoplasmic mRNA-protein complexes (mRNP). The polypeptides of translationally active mRNP complexes released from polysomes were compared to the repressed nonpolysomal cytoplasmic (free) mRNP complexes. In general, all of the polypeptides present in free mRNPs were also found in the polysomal mRNPs. In contrast to polysomal mRNPS, polypeptides of Mr 28 000, 32 000, 46 000, 65 000 and 150 000 were either absent or present in relatively smaller quantities in free mRNP complexes. On the other hand, the relative proportion of polypeptides of Mr 130 000 and 43 000 was higher in free mRNPs than in polysomal mRNP complexes. To examine the role of cytoplasmic mRNP complexes in protein synthesis or mRNA metabolism, the changes in these complexes were studied following (a) inhibition of mRNA synthesis and (b) heat-shock treatment to alter the pattern of protein synthesis. Actinomycin D was used to inhibit mRNA synthesis in chick myotubes. The possibility of newly synthesized polypeptides of cytoplasmic mRNP complexes being assembled into these complexes in the absence of mRNA synthesis was examined. These studies showed that the polypeptides of both free and polysomal mRNP complexes can bind to pre-existing mRNAs, therefore suggesting that polypeptides of mRNP complexes can be exchanged with a pool of RNA-binding proteins. In free mRNP complexes, this exchange of polypeptides is significantly slower than in the polysomal mRNP complexes. Heat-shock treatment of chicken myotubes induces the synthesis of three polypeptides of Mr = 81 000, 65 000 and 25 000 (heat-shock polypeptides). Whether this altered pattern of protein synthesis following heat-shock treatment could affect the polypeptide composition of translationally active polysomal mRNPs was examined. The results of these studies show that, compared to normal cells, more newly synthesized polypeptides were assembled into polysomal mRNPs following heat-shock treatment. A [35S]methionine-labeled polypeptide of Mr = 80 000 was detected in mRNPs of heat-shocked cells, but not of normal cells. This polypeptide was, however, detected by AgNO3 staining of the unlabeled polypeptide of mRNP complexes of normal cells. These results, therefore, suggest that the assembly of newly synthesized 80 000-Mr polypeptide to polysomal mRNPs was enhanced following induction of new heat-shock mRNAs. The results of these studies reported here have been discussed in relation to the concept that free mRNP complexes are inefficiently translated in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

2.
Proteins associated with mRNA in cells infected with herpes simplex virus   总被引:2,自引:0,他引:2  
The structure of messenger ribonucleoprotein (mRNP) complexes in herpes simplex virus type 1 (HSV-1) infected cells was analyzed by examining the proteins that could be crosslinked to polyadenylated mRNAs by irradiation of intact cells with ultraviolet light. The profiles of crosslinked proteins were qualitatively similar for mRNPs from mock infected and infected cells. However, infection with wild type HSV-1 caused a decrease in the abundance of a major 52 kda protein and an increase in a 49 kda protein. These changes were observed at early times after infection. They occurred following infection with wild type HSV-1 under conditions that blocked viral gene expression, but not following infection with the virion host shutoff mutant vhs 1.  相似文献   

3.
4.
Chromatin isolated from herpes simplex virus type 1-infected baby hamster kidney cells contains a number of tightly associated virus-induced polypeptides. A subset of these proteins bind to immobilized DNA in vitro (Vmw 175, 155, 130, 63, 43, 38/39). Virus-induced polypeptides extractable with acid from infected cell chromatin include Vmw 155, the major capsid protein of herpes simplex virus type 1 virions, and Vmw 63 and 38/39 which are heterogeneous with respect to charge and are phosphorylated. These chromatin preparations, in the presence of deoxynucleoside triphosphates and MgCl2 were capable of synthesizing viral and cell DNA in a reaction which was stimulated by the addition of ATP, riboNTPs and potassium acetate. In vitro synthesized viral DNA co-sedimented with prelabelled parental DNA but the single-stranded product was smaller than parental DNA. Density labelling indicated that extensive synthesis was taking place and all BamHI fragments of viral DNA were represented by the DNA synthesized in vitro.  相似文献   

5.
6.
At present, the effect of herpes simplex virus infection on the entire proteomes of infected cells is very poorly documented. Following several studies performed over the past few years, the modifications of a sub-cellular fraction induced by herpes simplex virus type 1 can be documented. These studies were performed in order to characterize the virally-induced modifications of a major component of the translational apparatus, the ribosomes. The very basic nature of most of the ribosomal proteins renders them very difficult to separate using isoelectric focusing (IEF). Therefore these studies were achieved using several different but related two-dimensional electrophoretic systems which allowed several two-dimensional ribosomal protein maps to be built. Comparison of the ribosomal protein maps built from non-infected cells with those built from infected cells demonstrated that infection by herpes simplex virus type 1 (HSV-1) induces important modifications of ribosomes: (i) non-reversible phosphorylation of ribosomal protein S6; (ii) unusual phosphorylation of several proteins of the small and the large subunits; and (iii) association of viral and cellular proteins to the ribosomal fraction. An overview of these published studies is presented in this review.  相似文献   

7.
The major glycosphingolipids (GSLs) of a line of African green monkey kidney cells (BGM) were characterized as glucosylceramide, lactosylceramide, galactosyl-galactosyl-glucosylceramide, and N-acetylgalactosaminyl-galactosyl-galactosyl-glucosylceramide. Neutral GSLs accounted for approximately 80% of the total GSLs isolated. The predominant gangliosides were N-acetylneuraminyl-galactosyl-glucosylceramide, N-acetylgalactosaminyl-N-acetylneuraminyl-galactosyl- glucosylceramide, and galactosyl-N-acetylgalactosaminyl-N-acetylneuraminyl -galactosyl-glucosylceramide. The incorporation of labeled galactose into GSLs was compared in mock-infected and herpes simplex virus type 1-infected BGM cells. Herpes simplex virus type 1 infection resulted in a three- to four-fold increase in galactose incorporation into glucosylceramide and a decrease in galactose incorporation into galactosyl-galactosyl-glucosylceramide and N-acetyl-galactosaminyl-galactosyl-galactosyl-glucosylceramide. The virus-induced alteration in the GSL labeling pattern occurred early in infection, before the release of infectious virus, and was not prevented by the presence of cytosine arabinoside. Treatment of uninfected BGM cells with cycloheximide resulted in alterations in the GSL pattern which were similar to those observed in herpes simplex virus type 1-infected cells. These observations suggest that an early virus function such as inhibition of host cell protein synthesis is responsible for the observed alterations of GSL metabolism. Experiments with a syncytium-producing strain of herpes simplex virus type 1, herpes simplex virus type 2, and pseudorabies virus indicated that other herpes viruses altered GSL metabolism in a manner similar to herpes simplex virus type 1.  相似文献   

8.
Fluorescence photobleaching recovery measurements showed that herpes simplex virus type 1 attachment to target cells rapidly induced an anchorage modulation of cell surface protein mobility, an activity mediated by the cytoskeleton and associated with the multivalent attachment of other ligands (e.g., cells, lectins, or anti-immunoglobulin) to cell surfaces. The restriction in cell surface protein mobility was released concurrently with virus penetration. The effects of attachment and penetration on cell surface protein mobility and cytoskeletal function are some of the earliest cellular changes induced by herpes simplex virus infection.  相似文献   

9.
We expressed the bovine herpesvirus 1 (BHV-1) glycoprotein IV (gIV) in bovine cells. The protein expressed was identical in molecular mass and antigenic reactivity to the native gIV protein but was localized in the cytoplasm. Expressing cells were partially resistant to BHV-1, herpes simplex virus, and pseudorabies virus, as shown by a 10- to 1,000-fold-lower number of plaques forming on these cells than on control cells. The level of resistance depended on the level of gIV expression and the type and amount of challenge virus. These data are consistent with previous reports by others that cellular expression of the BHV-1 gIV homologs, herpes simplex virus glycoprotein D, and pseudorabies virus glycoprotein gp50 provide partial resistance against infection with these viruses. We have extended these findings by showing that once BHV-1 enters gIV-expressing cells, it replicates and spreads normally, as shown by the normal size of BHV-1 plaques and the delayed but vigorous synthesis of viral proteins. Our data are consistent with the binding of BHV-1 gIV to a cellular receptor required for initial penetration by all three herpesviruses and interference with the function of that receptor molecule.  相似文献   

10.
Summary Modifications of ribosomes have been investigated in human epidermoid carcinoma-2 cells at different stages of herpes simplex virus type 1 infection. Very early in infection, there is an increase in ribosomal protein S6 phosphorylation even in the absence of serum. The same result is obtained in the presence of actinomycin D. At early infection time, ribosomal proteins S2, S3a and Sa are newly phosphorylated. At early and early-late times, three phosphorylated non-ribosomal proteins (v1, v2 and v3) are differently associated temporally to ribosomes. Analyses of proteins extracted from 40S subunits, 80S ribosomes and polysomes show that v1 and v2 are distributed differently among the different ribosomal populations. S6 phosphopeptides were found to be identical after serum stimulation and after viral infection. In every case phosphoserine and phosphothreonine were identified in S6. Only phosphoserine was found in other phosphorylated proteins. Our results indicate that herpes simplex virus type 1 is able to modify pre-existing ribosomes: (i) by stimulating a pre-existing kinase for S6 phosphorylation even in the absence of serum and of viral genome expression; (ii) by inducing new specific kinase activity(ies); and (iii) by association of new, phosphorylated proteins to ribosomes. These ribosomal modifications are correlated with changes in protein synthesis, as shown by two-dimensional electrophoretic analyses of newly synthesized 35S-labelled proteins.  相似文献   

11.
Recombination-dependent replication is an integral part of the process by which double-strand DNA breaks are repaired to maintain genome integrity. It also serves as a means to replicate genomic termini. We reported previously on the reconstitution of a recombination-dependent replication system using purified herpes simplex virus type 1 proteins (Nimonkar A. V., and Boehmer, P. E. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10201-10206). In this system, homologous pairing by the viral single-strand DNA-binding protein (ICP8) is coupled to DNA synthesis by the viral DNA polymerase and helicase-primase in the presence of a DNA-relaxing enzyme. Here we show that DNA synthesis in this system is dependent on the viral polymerase processivity factor (UL42). Moreover, although DNA synthesis is strictly dependent on topoisomerase I, it is only stimulated by the viral helicase in a manner that requires the helicase-loading protein (UL8). Furthermore, we have examined the dependence of DNA synthesis in the viral system on species-specific protein-protein interactions. Optimal DNA synthesis was observed with the herpes simplex virus type 1 replication proteins, ICP8, DNA polymerase (UL30/UL42), and helicase-primase (UL5/UL52/UL8). Interestingly, substitution of each component with functional homologues from other systems for the most part did not drastically impede DNA synthesis. In contrast, recombination-dependent replication promoted by the bacteriophage T7 replisome was disrupted by substitution with the replication proteins from herpes simplex virus type 1. These results show that although DNA synthesis performed by the T7 replisome is dependent on cognate protein-protein interactions, such interactions are less important in the herpes simplex virus replisome.  相似文献   

12.
Cloned BglII fragment N (map units 0.58 to 0.625) of herpes simplex virus type 2 DNA has been shown to transform rodent cells to an oncogenic phenotype (Galloway and McDougall, J. Virol. 38: 749-760, 1981). RNA homologous to this fragment directs the synthesis of five polypeptides in a cell-free translation system. The approximate molecular weights of these proteins are 140,000, 61,000, 56,000, 35,000, and 23,500. The 35,000-dalton protein is the major species late in infection and is the only species detected before the onset of viral DNA replication. The arrangement of the sequences encoding these proteins along the herpes simplex virus type 2 genome was determined by hybridization of the RNA to cloned PstI fragment of BglII-N and to single-stranded DNA segments cloned into M13mp7. Both the hybridization experiments and immunoprecipitation with monoclonal antibodies suggested that the 140,000- and 35,000-dalton proteins are at least partially colinear and share antigenic determinants.  相似文献   

13.
The function of proteins that interact with mRNA   总被引:4,自引:0,他引:4  
Specific proteins are associated with mRNA in the cytoplasm of eukaryotic cells. The complement of associated proteins depends upon whether the mRNA is an integral component of the polysomal complex being translated, or, alternatively, whether it is part of the non-translated free mRNP fraction. By subjecting cells to ultraviolet irradiation in vivo to cross-link proteins to mRNA, mRNP proteins have been shown to be associated with specific regions of the mRNA molecule. Examination of mRNP complexes containing a unique mRNA has suggested that not all mRNA contain the same family of associated RNA binding proteins. The function of mRNA associated proteins may include a role in providing stability for mRNA, and/or in modulating translation. With the recent demonstrations that both free and polysomal mRNPs are associated with the cytoskeletal framework, specific mRNP proteins may play a role in determining the subcellular localization of specific mRNPs.  相似文献   

14.
Herpes simplex virus genes form several groups whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Most of the products of the first group, the alpha genes, appear to have regulatory functions. We report that the alpha proteins, infected cell proteins 4, 0, 22, and 27 of herpes simplex virus 1 and 4, 0, and 27 of herpes simplex virus 2, were labeled in the isolated nuclei of infected HeLa cells with [alpha-32P]GTP or [alpha-32P]ATP late in infection and that these proteins represent the largest group of virus-specific proteins labeled in this fashion. Studies with [2-3H]ATP, in which the label is in the purine ring, showed that a portion of the label in alpha proteins and in at least one other infected cell protein is due to nucleotidylylation. Analyses of the labeling reactions in nuclei of (i) cells infected with temperature-sensitive mutants at nonpermissive temperatures, (ii) cells infected with wild-type virus and harvested at different times postinfection, and (iii) cells treated with inhibitors of protein synthesis or of synthesis of viral DNA led to the conclusion that viral gene functions expressed after the synthesis of alpha proteins are required for the labeling of the alpha proteins with [alpha-32P]GTP. We conclude that several of the alpha proteins are extensively posttranslationally modified and that these modifications include nucleotidylylation.  相似文献   

15.
The specificity of herpes simplex virus type 1-specific cytotoxic T cells was examined with target cells expressing either input viral structural antigens or antigens resulting from permissive infection or cells from an interrupted infection in which they expressed predominantly nonstructural immediate-early proteins. These studies indicated that only an insignificant minority of cytotoxic T cells recognized the input viral antigens, whereas a significant proportion (20 to 35%) recognized target cells that expressed the immediate-early proteins despite the absence of serologically detectable viral antigens upon the infected cell surface. The finding that a significant proportion of cytotoxic T-cell populations obtained from the draining lymph nodes of mice acutely infected with herpes simplex virus type 1 also recognized immediately-early gene-expressing target cells indicates the importance of nonstructural herpes simplex virus proteins to antiviral immunity in vivo.  相似文献   

16.
We have assigned eight temperature-sensitive mutants of herpes simplex virus type 1 to complementation group 1-1. Members of this group fail to complement mutants in herpes simplex virus type 2 complementation group 2-2. The mutation of one member of group 1-1, tsHA1 of strain mP, has been shown to map in or near the sequence which encodes the major herpes simplex virus type 1 DNA-binding protein (Conley et al., J. Virol. 37:191-206, 1981). The mutations of five other members of group 1-1 map in or near the sequence in which the tsHA1 mutation maps, a sequence which lies near the center of UL between the genes for the viral DNA polymerase and viral glycoprotein gAgB. These mutants can be divided into two groups; the mutations of one group map between coordinates 0.385 and 0.398, and the mutations of the other group map between coordinates 0.398 and 0.413. At the nonpermissive temperature mutants in group 1-1 are viral DNA negative, and mutant-infected cells fail to react with monoclonal antibody to the 130,000-dalton DNA-binding protein. Taken together, these data indicate that mutants in complementation groups 1-1 and 2-2 define the gene for the major herpes simplex virus DNA-binding protein, an early gene product required for viral DNA synthesis.  相似文献   

17.
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.  相似文献   

18.
P L Ward  W O Ogle    B Roizman 《Journal of virology》1996,70(7):4623-4631
In cells infected with herpes simplex virus 1 (HSV-1), the viral proteins ICP5 (infected-cell protein 5) and VP19c (the product of UL38) are associated with mature capsids, whereas the same proteins, along with ICP35, are components of immature capsids. Here we report that ICP35, ICP5, and UL38 (VP19c) coalesce at late times postinfection and form antigenically dense structures located at the periphery of nuclei, close to but not abutting nuclear membranes. These structures were formed in cells infected with a virus carrying a temperature-sensitive mutation in the UL15 gene at nonpermissive temperatures. Since at these temperatures viral DNA is made but not packaged, these structures must contain the proteins for immature-capsid assembly and were therefore designated assemblons. These assemblons are located at the periphery of a diffuse structure composed of proteins involved in DNA synthesis. This structure overlaps only minimally with the assemblons. In contrast, tegument proteins were located in asymmetrically distributed structures also partially overlapping with assemblons but frequently located nearer to nuclear membranes. Of particular interest is the finding that the UL15 protein colocalized with the proteins associated with viral DNA synthesis rather than with assemblons, suggesting that the association with DNA may take place during its synthesis and precedes the involvement of this protein in packaging of the viral DNA into capsids. The formation of three different compartments consisting of proteins involved in viral DNA synthesis, the capsid proteins, and tegument proteins suggests that there exists a viral machinery which enables aggregation and coalescence of specific viral protein groups on the basis of their function.  相似文献   

19.
Nucleolar modifications induced by herpes simplex virus type 1 (HSV1) infection were studied at the ultrastructural level with special attention to the fate of a family of proteins serologically related to the nucleolar 100 kDa protein. Immunocytochemical techniques revealed that antigenic sites related to these proteins were associated with nucleoli in a pattern similar to that observed with non-infected cells. In addition, the "dense bodies" induced by HSV infection were heavily decorated by antibodies to the 100 kDa protein. Neither DNA nor RNA was detectable in the latter by cytochemical techniques. Therefore, it appears that "dense bodies" are exclusively proteinaceous and contain at least one host protein implicated in ribosomal RNA synthesis. An accumulation of 100 kDa protein in extra-nucleolar structures might account for previously reported defects in ribosomal RNA expression during HSV infection.  相似文献   

20.
The herpes simplex virus type 1 (HSV-1) ICP27 protein is an immediate-early or alpha protein which is essential for the optimal expression of late genes as well as the synthesis of viral DNA in cultures of Vero cells. Our specific goal was to characterize the replication of a virus incapable of synthesizing ICP27 in cultured human cells. We found that infection with an HSV-1 ICP27 deletion virus of at least three separate strains of human cells did not produce immediate-early or late proteins at the levels observed following wild-type virus infections. Cell morphology, chromatin condensation, and genomic DNA fragmentation measurements demonstrated that the human cells died by apoptosis after infection with the ICP27 deletion virus. These features of the apoptosis were identical to those which occur during wild-type infections of human cells when total protein synthesis has been inhibited. Vero cells infected with the ICP27 deletion virus did not exhibit any of the features of apoptosis. Based on these results, we conclude that while HSV-1 infection likely induced apoptosis in all cells, viral evasion of the response differed among the cells tested in this study.  相似文献   

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