共查询到20条相似文献,搜索用时 8 毫秒
1.
2.
3.
An immunoassay for the study of DNA-binding activities of herpes simplex virus protein ICP8. 总被引:16,自引:11,他引:5
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
An immunoassay was used to examine the interaction between a herpes simplex virus protein, ICP8, and various types of DNA. The advantage of this assay is that the protein is not subjected to harsh purification procedures. We characterized the binding of ICP8 to both single-stranded (ss) and double-stranded (ds) DNA. ICP8 bound ss DNA fivefold more efficiently than ds DNA, and both binding activities were most efficient in 150 mM NaCl. Two lines of evidence indicate that the binding activities were not identical: (i) ds DNA failed to complete with ss DNA binding even with a large excess of ds DNA; (ii) Scatchard plots of DNA binding with various amounts of DNA were fundamentally different for ss DNA and ds DNA. However, the two activities were related in that ss DNA efficiently competed with the binding of ds DNA. We conclude that the ds DNA-binding activity of ICP8 is probably distinct from the ss DNA-binding activity. No evidence for sequence-specific ds DNA binding was obtained for either the entire herpes simplex virus genome or cloned viral sequences. 相似文献
4.
Expression of herpes simplex virus type 1 major DNA-binding protein, ICP8, in transformed cell lines: complementation of deletion mutants and inhibition of wild-type virus 总被引:2,自引:16,他引:2
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
To minimize the contribution of residual activity associated with the temperature-sensitive (ts) form of ICP8 specified by available ts mutants, deletion mutations in this gene were constructed. Cells permissive for the generation and propagation of ICP8 deletion mutants were first obtained. Vero cells were cotransfected with pKEF-P4, which contains the gene for ICP8, and pSV2neo or a hybrid plasmid containing the G418 resistance gene linked to pKEF-P4. Of the 48 G418-resistant cell lines, 21 complemented ICP8 ts mutants in plaque assays at the nonpermissive temperature. Four of these were examined by Southern blot analysis and shown to contain 1 to 3 copies of the ICP8 gene per haploid genome equivalent. Cell line U-47 was used as the permissive host for construction of ICP8 deletion mutants. In addition to cell lines which complemented ts mutants, two lines, U-27 and U-35, significantly inhibited plaque formation by wild-type virus, contained 30 and 100 copies of the ICP8 gene per haploid genome equivalent, respectively, and expressed large amounts of ICP8 after infection with wild-type virus. At low but not high multiplicities of infection, this inhibition was accompanied by underproduction of viral polypeptides of the early, delayed-early, and late kinetic classes. For construction of deletion mutants, a 780-base-pair XhoI fragment was deleted from pSG18-SalIA, a plasmid which contains the gene for ICP8, to yield pDX. U-47 cells were then cotransfected with pDX and infectious wild-type DNA. Mutant d61, isolated from the progeny of cotransfection, was found to contain both the engineered deletion in the ICP8 gene and an oriL-associated deletion of approximately 55 base pairs. Because d61 contained two mutations, a second mutant, d21, which carried the engineered ICP8 deletion but an intact oriL, was constructed by cotransfection of U-47 cells with wild-type DNA and an SalI-KpnI fragment purified from pDX. Phenotypic analysis of d21 and d61 revealed that they were similar in all properties examined: both exhibited efficient growth in U-47 cells but not in Vero cells; both induced the synthesis of an ICP8 polypeptide which was smaller than the wild-type form of the protein and which, unlike the wild-type protein, was found in the cytoplasm and not the nucleus of infected Vero cells; and nonpermissive Vero cells infected with either mutant failed to express late viral polypeptides. 相似文献
5.
Genetic evidence for multiple nuclear functions of the herpes simplex virus ICP8 DNA-binding protein 总被引:1,自引:23,他引:1
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
We have isolated several mutant herpes simplex viruses, specifically mutated in the infected cell protein 8 (ICP8) gene, to define the functional domains of ICP8, the major viral DNA-binding protein. To facilitate the isolation of these mutants, we first isolated a mutant virus, HD-2, with the lacZ gene fused to the ICP8 gene so that an ICP8-beta-galactosidase fusion protein was expressed. This virus formed blue plaques on ICP8-expressing cell lines in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Mutated ICP8 gene plasmids cotransfected with HD-2 DNA yielded recombinant viruses with the mutant ICP8 gene incorporated into the viral genome. These recombinants were identified by formation of white plaques. Four classes of mutants were defined: (i) some expressed ICP8 that could bind to DNA but could not localize to the cell nucleus; (ii) some expressed ICP8 that did not bind to DNA but localized to the nucleus; (iii) some expressed ICP8 that neither bound to DNA nor localized to the nucleus; and (iv) one expressed ICP8 that localized to the cell nucleus and bound to DNA in vitro, but the mutant virus did not replicate its DNA. These classes of mutants provide genetic evidence that DNA binding and nuclear localization are distinct functions of ICP8 and that ICP8 has nuclear functions other than binding to DNA. Furthermore, the portion of ICP8 needed for a nuclear function(s) distinct from DNA binding is the part of ICP8 showing sequence similarity to that of the cellular protein cyclin or proliferating cell nuclear antigen. 相似文献
6.
Correct intranuclear localization of herpes simplex virus DNA polymerase requires the viral ICP8 DNA-binding protein. 总被引:17,自引:15,他引:2
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
M Bush D R Yager M Gao K Weisshart A I Marcy D M Coen D M Knipe 《Journal of virology》1991,65(3):1082-1089
We used indirect immunofluorescence to examine the factors determining the intranuclear location of herpes simplex virus (HSV) DNA polymerase (Pol) in infected cells. In the absence of viral DNA replication, HSV Pol colocalized with the HSV DNA-binding protein ICP8 in nuclear framework-associated structures called prereplicative sites. In the presence of viral DNA replication, HSV Pol colocalized with ICP8 in globular intranuclear structures called replication compartments. In cells infected with mutant viruses encoding defective ICP8 molecules, Pol localized within the cell nucleus but showed a general diffuse intranuclear distribution. In uninfected cells transfected with a plasmid expressing Pol, Pol similarly showed a diffuse intranuclear distribution. Therefore, Pol can localize to the cell nucleus without other viral proteins, but functional ICP8 is required for Pol to localize to prereplicative sites. In cells infected with mutant viruses encoding defective Pol molecules, ICP8 localized to prereplicative sites. Thus, Pol or the portions of Pol not expressed by the mutant viruses are not essential for the formation of prereplicative sites or the localization of ICP8 to these structures. These results demonstrate that a specific nuclear protein can influence the intranuclear location of another nuclear protein. 相似文献
7.
8.
Characterization of a major DNA-binding domain in the herpes simplex virus type 1 DNA-binding protein (ICP8). 总被引:15,自引:10,他引:5
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
We have studied the major DNA-binding protein (ICP8) from herpes simplex virus type 1 to identify its DNA-binding site. Since we obtained our protein from a cell line carrying multiple chromosomally located copies of the ICP8 gene, we first analyzed this protein to assess its similarity to the corresponding viral protein. Our protein resembled the viral protein by molecular weight, response to antibody, preference for binding single-stranded DNA, and ability to lower the melting temperature of poly(dA-dT). To define the DNA-binding domain, we subjected the protein to limited trypsin digestion and separated the peptide products on a sodium dodecyl sulfate-polyacrylamide gel. These fragments were then transferred to a nitrocellulose membrane, renatured in situ, and tested for their ability to bind DNA. From this assay, we identified four fragments which both bound DNA and exhibited the expected binding preference for single-stranded DNA. The sequence of the smallest of these fragments was determined and corresponds to a polypeptide spanning residues 300 to 849 in the intact protein. This peptide contains several regions which may be important for DNA binding based on sequence similarities in single-stranded DNA-binding proteins from other herpesviruses and, in one case, on a conserved sequence found in more distant procaryotic and eucaryotic proteins. 相似文献
9.
M Spatz S A Ali M Auer C Graf M M Eibl A Steinkasserer 《Protein expression and purification》1999,16(1):40-46
We have developed a baculovirus expression system for the rapid and efficient production of large quantities (>5 mg/10(8) cells) of ICP8. The recombinant ICP8 is fully functional and binds to single-stranded DNA. Secondary structure calculations from circular dichroism measurements indicate a content of 34.5% alpha-helix and 15.4% beta-sheet. This is the first structural report for ICP8 using CD analysis, which will be very useful for high-throughput assay development and mechanistic studies. 相似文献
10.
ICP8, the herpes simplex virus type-1 single-strand DNA-binding protein, was recently shown to promote strand exchange in conjunction with the viral replicative helicase (Nimonkar, A. V., and Boehmer, P. E. (2002) J. Biol. Chem. 277, 15182-15189). Here we show that ICP8 also catalyzes strand invasion in an ATP-independent manner. Thus, ICP8 promotes the assimilation of a single-stranded donor molecule into a homologous plasmid, resulting in the formation of a displacement loop. Invasion of a homologous duplex by single-stranded DNA requires homology at either 3' or 5' end of the invading strand. The reaction is dependent on the free energy of supercoiling and alters the topology of the acceptor plasmid. Hence, strand invasion products formed by ICP8 are resistant to the action of restriction endonucleases that cleave outside of the area of pairing. The ability to catalyze strand invasion is a novel activity of ICP8 and the first demonstration of a eukaryotic viral single-strand DNA-binding protein to promote this reaction. In this regard ICP8 is functionally similar to the prototypical prokaryotic recombinase RecA and its eukaryotic homologs. This strand invasion activity of ICP8 coupled with DNA synthesis may explain the high prevalence of branched DNA structures during viral replication. 相似文献
11.
Gourves AS Tanguy Le Gac N Villani G Boehmer PE Johnson NP 《The Journal of biological chemistry》2000,275(15):10864-10869
We have carried out solution equilibrium binding studies of ICP8, the major single-stranded DNA (ssDNA)-binding protein of herpes simplex virus type I, in order to determine the thermodynamic parameters for its interaction with ssDNA. Fluorescence anisotropy measurements of a 5'-fluorescein-labeled 32-mer oligonucleotide revealed that ICP8 formed a nucleoprotein filament on ssDNA with a binding site size of 10 nucleotides/ICP8 monomer, an association constant at 25 degrees C, K = 0.55 +/- 0.05 x 10(6) M(-1), and a cooperativity parameter, omega = 15 +/- 3. The equilibrium constant was largely independent of salt, deltalog(Komega)/deltalog([NaCl]) = -2.4 +/- 0.4. Comparison of these parameters with other ssDNA-binding proteins showed that ICP8 reacted with an unusual mechanism characterized by low cooperativity and weak binding. In addition, the reaction product was more stable at high salt concentrations, and fluorescence enhancement of etheno-ssDNA by ICP8 was higher than for other ssDNA-binding proteins. These last two characteristics are also found for protein-DNA complexes formed by recombinases in their active conformation. Given the proposed role of ICP8 in promoting strand transfer reactions, they suggest that ICP8 and recombinase proteins may catalyze homologous recombination by a similar mechanism. 相似文献
12.
The herpes simplex virus type-1 single-strand DNA-binding protein ICP8 is a 128-kDa zinc metalloprotein. In this communication we have shown that unsubstituted and bromodeoxyuridine-substituted oligonucleotides can be specifically crosslinked to ICP8 by UV irradiation. We have used this approach to show that the single-strand DNA-binding site of ICP8 resides within a 53.5-kDa tryptic polypeptide. This polypeptide initiates at alanine 368 and was estimated to extend through arginine 902. A polypeptide encompassing residues 368-902 synthesized in vitro exhibited single-strand DNA-binding activity. We conclude that the region encompassing residues 368-902 contains the single-strand DNA-binding site of ICP8. Moreover, photoaffinity labeling of ICP8 with oligonucleotides provides a means of specifically modifying its single-strand DNA-binding site, thereby facilitating future studies on the importance of its single-strand DNA-binding activity in its interaction with other DNA replication enzymes. 相似文献
13.
Conformational changes in the herpes simplex virus ICP8 DNA-binding protein coincident with assembly in viral replication structures
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The herpes simplex virus (HSV) single-stranded DNA-binding protein, ICP8, is required for viral DNA synthesis. Before viral DNA replication, ICP8 colocalizes with other replication proteins at small punctate foci called prereplicative sites. With the onset of viral genome amplification, these proteins become redistributed into large globular replication compartments. Here we present the results of immunocytochemical and biochemical analysis of ICP8 showing that various antibodies recognize distinct forms of ICP8. Using these ICP8-specific antibodies as probes for ICP8 structure, we detected a time-dependent appearance and disappearance of ICP8 epitopes in immunoprecipitation assays. Immunofluorescence staining of ICP8 in cells infected with different HSV mutant viruses as well as cells transfected with a limited number of viral genes demonstrated that these and other antigenic changes occur coincident with ICP8 assembly at intranuclear replication structures. Genetic analysis has revealed a correlation between the ability of various ICP8 mutant proteins to form the 39S epitope and their ability to bind to DNA. These results support the hypothesis that ICP8 undergoes a conformational change upon binding to other HSV proteins and/or to DNA coincident with assembly into viral DNA replication structures. 相似文献
14.
Herpes simplex virus type 1 single-stranded DNA-binding protein (ICP8) has been crystallized on a positively charged lipid monolayer. The crystals belong to the planar group p2 with a=39 nm, b=23.2 nm and gamma=87.2 degrees. The projected map of ICP8 crystals calculated at a resolution of 3.9 nm shows four ICP8 monomers per unit cell with the crystals formed by a parallel arrangement of 16.2 nm helical ICP8 filaments. This novel filamentous form has not been reported before. The ICP8 monomers show different appearances in projection, suggesting that they may adopt different orientations, probably reflecting the strong intermolecular and lipid-filament interactions in the crystal. When the 23 nm diameter filaments formed by ICP8 in solution at low temperature in the presence of magnesium were generated and then layered on the phospholipid monolayer, highly ordered arrays of an 8.5 nm filament with a shallow 31.2 nm pitch were observed and reconstruction revealed a double-helical structure. 相似文献
15.
Identification of dominant-negative mutants of the herpes simplex virus type 1 immediate-early protein ICP0.
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
ICP0 is a 110,000-molecular-weight immediate-early protein of herpes simplex virus type 1 (HSV-1) which is encoded by three exons. It has been shown to function as a promiscuous transactivator of a variety of different HSV-1 and non-HSV-1 promoters in transient expression assays. Analysis of mutations which truncated the carboxy-terminal end of this 775-amino-acid (aa) protein demonstrated that a polypeptide which contained only aa 1 to 553 still possessed significant transactivation potential. Additional carboxy-terminal truncations which sequentially removed aa 245 to 553 and thus the remainder of the third exon resulted in the eventual loss of transactivation capability in these mutants. However, further analysis of these truncated derivatives demonstrated that they behaved as dominant-negative mutants to the wild-type polypeptide. Moreover, one of the mutants was found to act as a promiscuous repressor, in that it could dramatically inhibit a variety of HSV-1 promoters, non-HSV-1 promoters, and heterologous transactivator proteins in transient expression assays, despite having lost almost the entire third exon. These results indicate that a domain encoded by the first two exons probably interacts with, and can effectively titrate, the unknown cellular factor(s) through which ICP0 mediates transactivation. 相似文献
16.
RNA binding and R-loop formation by the herpes simplex virus type-1 single-stranded DNA-binding protein (ICP8) 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Boehmer PE 《Nucleic acids research》2004,32(15):4576-4584
17.
DNA-binding protein associated with herpes simplex virus DNA polymerase. 总被引:34,自引:30,他引:4
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Purified preparations of herpes simplex virus type 2 DNA polymerase made by many different laboratories always contain at least two polypeptides. The major one, of about 150,000 molecular weight, has been associated with the polymerase activity. The second protein, of about 54,000 molecular weight, which we previously designated ICSP 34, 35, has now been purified. The purified protein has been used to prepare antisera (both polyclonal rabbit serum and monoclonal antibodies). These reagents have been used to characterize the protein, to demonstrate its quite distinct map location from that of the DNA polymerase on the herpes simplex virus genome, and to demonstrate the close association between the two polypeptides. 相似文献
18.
On the mechanism of strand assimilation by the herpes simplex virus type-1 single-strand DNA-binding protein (ICP8) 总被引:2,自引:0,他引:2
ICP8, the herpes simplex virus type-1 encoded single-strand DNA (ssDNA)-binding protein, promotes the assimilation of a single-stranded DNA molecule into a homologous duplex plasmid resulting in the formation of a displacement loop. Here we examine the mechanism of this process. In contrast to the RecA-type recombinases that catalyze strand invasion via an active search for homology, ICP8 acts by a salt-dependent strand annealing mechanism. The active species in this reaction is a ssDNA:ICP8 nucleoprotein filament. There appears to be no requirement for ICP8 to interact with the acceptor DNA. At higher concentrations, ICP8 promotes the reverse reaction, presumably owing to its helix destabilizing activity. ICP8-mediated strand assimilation imparts single-stranded character onto the acceptor DNA, consistent with the formation of a displacement loop. These data suggest that the recombination activity of ICP8 is similar to the mechanism of eukaryotic Rad52. 相似文献
19.
Arana ME Haq B Tanguy Le Gac N Boehmer PE 《The Journal of biological chemistry》2001,276(9):6840-6845
The mechanism of stimulation of a DNA helicase by its cognate single-strand DNA-binding protein was examined using herpes simplex virus type-1 UL9 DNA helicase and ICP8. UL9 and ICP8 are two essential components of the viral replisome that associate into a complex to unwind the origins of replication. The helicase and DNA-stimulated ATPase activities of UL9 are greatly elevated as a consequence of this association. Given that ICP8 acts as a single-strand DNA-binding protein, the simplest model that can account for its stimulatory effect predicts that it tethers UL9 to the DNA template, thereby increasing its processivity. In contrast to the prediction, data presented here show that the stimulatory activity of ICP8 does not depend on its single-strand DNA binding activity. Our data support an alternative hypothesis in which ICP8 modulates the activity of UL9. Accordingly, the data show that the ICP8-binding site of UL9 constitutes an inhibitory region that maintains the helicase in an inefficient ground state. ICP8 acts as a positive regulator by neutralizing this region. ICP8 does not affect substrate binding, ATP hydrolysis, or the efficiency of translocation/DNA unwinding. Rather, we propose that ICP8 increases the efficiency with which substrate binding and ATP hydrolysis are coupled to translocation/DNA unwinding. 相似文献
20.
ICP0, a herpes simplex virus immediate-early gene product, is a highly phosphorylated nuclear protein that is a potent activator of virus and host genes. Using biochemical and genetic assays employing plasmids encoding mutant forms of ICP0 and a recombinant adenovirus that expresses ICP0, we mutant forms of ICP0 and a recombinant adenovirus that expresses ICP0, we provide evidence that the protein multimerizes. Some mutant forms of ICP0 were transdominant and interfered with activation of a target reporter gene or with complementation of an ICP0-minus virus. 相似文献