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1.
HSV-1 virions contain a proteinaceous layer termed the tegument that lies between the nucleocapsid and viral envelope. The molecular mechanisms that facilitate incorporation of tegument proteins are poorly characterized. The tegument protein VP22 interacts with VP16 and the cytoplasmic tail of glycoprotein E (gE). Virion incorporation of VP22 occurs independently of interaction with VP16; however, the contribution of gE binding remains undefined. Site-directed mutagenesis was used to identify VP22 mutants which abrogate interaction with gE but retain VP16 binding. Virion incorporation assays demonstrated that failure to bind gE did not abrogate VP22 packaging. A region of VP22 which binds to both VP16 and gE failed to be packaged efficiently, with wild-type levels of incorporation only attained when residues 43-86 of VP22 were present. Mutational analysis of an acidic cluster of amino acids within this region indicates that this motif facilitates trans-Golgi network (TGN) localization and optimal virion incorporation of VP22. 相似文献
2.
Herpes simplex virus type 1 (HSV-1) virions contain a proteinaceous layer termed the tegument that lies between the nucleocapsid and viral envelope. The mechanisms underlying tegumentation remain largely undefined for all herpesviruses. Using glutathione S-transferase (GST) pulldowns and coimmunoprecipitation studies, we have identified a domain of the tegument protein VP22 that facilitates interaction with VP16. This region of VP22 (residues 165-225) overlaps the glycoprotein E (gE) binding domain of VP22 (residues 165-270), which is sufficient to mediate VP22 packaging into assembling virus particles. To ascertain the contribution of the VP16 and gE binding activities of VP22 to its virion incorporation, a transfection/infection based virion incorporation assay, using point mutants that discern between the two binding activities, was utilized. Our results suggest that interaction with VP16 is not required for incorporation of VP22 into virus particles and that binding to the cytoplasmic tail of gE is sufficient to facilitate packaging. 相似文献
3.
The herpes simplex virus (HSV) major tegument structural protein VP22 resides in multiple subcellular regions during productive infection. During an analysis of the molecular determinants of these localizations, we observed that a transfected fusion of the C-terminal portion of VP22, containing its pat4 nuclear localization signal, with GFP lacked nucleolar sparing compared to GFP alone. Thus, the initial goal was to determine whether VP22 associates with nucleoli. Using an optimized indirect immunofluorescence system to visualize nucleolin and viral proteins, we observed that VP22 present in VP22-expressing Vero (V49) cells "surrounded" nucleolin. These two initial findings implied that VP22 might associate directly with nucleoli. We next analyzed HSV-infected cells and observed that at late times, anti-nucleolin immune reactivity was dispersed throughout the nuclei while it retained uniform, circular staining in mock-infected cells. Time course infection experiments indicated that nucleolin initiated its transition from uniform to dispersed structures between 2 and 4 hpi. Comparison of Hoechst stained nuclei showed bright anti-nucleolin staining localized to regions of marginalized chromatin. These effects required de novo infected cell protein synthesis. A portion of VP22 detected in nuclei at 4 and 6 hpi localized to these areas of altered nucleolin and marginalized chromatin. VP22 was excluded from viral replication compartments containing the viral regulatory protein ICP22. Finally, altered nucleolin and marginalized chromatin were detected with a VP22-null virus, indicating that VP22 was not responsible for these nuclear architecture alterations. Thus, we conclude that nuclear VP22 targets unique subnuclear structures early (<6hpi) during herpes simplex virus 1 (HSV-1) infection. 相似文献
4.
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
5.
Evidence of DNA: protein interactions that mediate HSV-1 immediate early gene activation by VP16 总被引:50,自引:0,他引:50
The viral genes first expressed upon lytic infection by herpes simplex virus type 1 (HSV-1) encode the five immediate early (IE) proteins. IE gene expression is potently and specifically induced by a virion protein termed VP16. Previous studies have shown that the activating properties of VP16 are IE gene specific and mediated by upstream regulatory elements common to each IE gene. Paradoxically, however, VP16 does not appear to be a sequence-specific DNA-binding protein. To understand the specificity of VP16 activation, we identified the cis-regulatory sequences of an IE gene that mediate VP16 response. Two distinct DNA sequence motifs enable the ICP4 gene to respond to VP16. Biochemical fractionation of nuclear proteins from uninfected cells revealed the existence of cellular proteins that bind directly to each of these VP16 cis-response elements. These observations, in concert with the identification of functional domains of the VP16 protein, lead to the hypothesis that VP16 achieves activation specificity via protein: protein, rather than protein: DNA, interactions. 相似文献
6.
7.
The molecular mechanisms responsible for the addition of tegument proteins into nascent herpesvirus particles are poorly understood. To better understand the tegumentation process of herpes simplex virus type 1 (HSV-1) virions, we initiated studies that showed the tegument protein pUL46 (VP11/12) has a similar cellular localization to the membrane-associated tegument protein VP22. Using membrane flotation analysis we found that pUL46 associates with membranes in both the presence and absence of other HSV-1 proteins. However, when purified virions were stripped of their envelope, the majority of pUL46 was found to associate with the capsid fraction. This strong affinity of pUL46 for capsids was confirmed by an in vitro capsid pull-down assay in which purified pUL46-GST was able to interact specifically with capsids purified from the nuclear fraction of HSV-1 infected cells. These results suggest that pUL46 displays a dynamic interaction between cellular membranes and capsids. 相似文献
8.
Herpes simplex virus type 1 (HSV-1) virions, contain a proteinaceous layer termed the tegument that lies between the nucleocapsid and viral envelope. Current evidence suggests that viral glycoprotein tails play a role in the recruitment of tegument-coated capsids to the site of final envelopment; vesicles derived from the trans-Golgi network. We have identified an interaction between VP22, an abundant tegument protein and the cytoplasmic tail of glycoprotein E (gE). This interaction was identified by coimmunoprecipitation studies and confirmed by a glutathione-S-transferase (GST) pulldown from infected cell lysates. Truncation mutagenesis suggests that residues 165-270 of VP22 facilitate the interaction with the cytoplasmic tail of gE. In fact, this region of VP22 is sufficient to bind to gE in the absence of additional viral proteins. Using a transfection/infection-based virion incorporation assay, residues 165-270 of VP22 fused to GFP competed efficiently with wild-type VP22 for packaging into assembling virus particles. 相似文献
9.
Summary. A full-length cDNA clone of olive latent virus 1 (OLV-1), a member of the genus Necrovirus, family Tombusviridae, was subjected to site-directed mutagenesis, and coat protein gene mutants were constructed. A mutant clone, denoted Δ3297,
was obtained by deleting the nucleotide in position 3297, thus inducing a frameshift and replacing the last 49 amino acids
of the viral coat protein (CP) by a shorter sequence of 39 amino acids. This mutant was viable, stable, able to synthesize
a smaller CP, and able to give rise to the formation of apparently intact virus particles. Cell-to-cell movement of Δ3297
in Nicotiana benthamiana leaves was not affected, but, contrary to wild type OLV-1, it failed to spread systemically. These results indicate that
virion formation is necessary but not sufficient for long-distance movement for OLV-1 and highlights the role of the CP carboxy-terminal
domain in systemic infection. 相似文献
10.
背景:VP22是单纯疱疹病毒1型(Herpes simplex virus type 1,HSV-1)UL49基因编码的碱性蛋白质,具有蛋白转导结构域(protein transduction domain, PTD),能够把与之融合的蛋白或与之结合的DNA 等大分子跨膜送递到邻近细胞,在基因靶向预防中表现出优势。
目的:构建表达单纯疱疹病毒1型VP22与柯萨奇病毒B3主要中和抗原VP1融合蛋白的重组腺病毒载体疫苗,观察外源基因在HEK293细胞中的良好表达。
方法:PCR法扩增目的基因HSV-1 VP22和CVB3 VP1,经Linker连接,将VP22-L-VP1插入腺病毒穿梭载体pAdTrack-CMV,构建重组穿梭质粒AdTrack-CMV/VP22-L-VP1。再将此载体与腺病毒骨架载体pAdEasy-1在大肠杆菌BJ5183中进行同源重组,生成重组腺病毒质粒pAd/VP22-L-VP1,脂质体介导pAd/VP22-L-VP1转染HEK293细胞包装重组腺病毒rAd/VP22-L-VP1。HEK293细胞上进行病毒扩增和滴定并检测外源基因的表达。
结果与结论:构建的重组腺病毒载体pAd/VP22-L-VP1经过第4轮扩增,其滴度达到6.77×107 pfu/mL,体外感染293细胞可见VP22和VP1融合蛋白的表达。说明实验成功构建并包装重组腺病毒rAd/VP22-L-VP1。 相似文献
11.
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
12.
HSV-1 B capsids are composed of seven major proteins, designated VP5, VP19C, 21, 22a, VP23, VP24, and VP26. VP indicates that the capsid protein is also a component of the infectious virion. Capsid proteins 21, 22a, and VP24 are specified by a single open reading frame (UL26) that encodes 635 amino acids. An objective of the work in our laboratory is to identify and map interactions among and between capsid proteins. In the present studies we employed the yeast GAL4 two-hybrid system developed by Fields and his colleagues (Nature240, 245–246 (1989)) for this purpose. DNA corresponding to the capsid open reading frames was derived as a PCR product and fused to sequences of the GAL4 activation and DNA binding domains. Using this system each of the capsid proteins has been tested for interactions with all of the other capsid proteins. Three interactions have been identified: a relatively strong self-interaction between 22a molecules (residues 307–635 of UL26), bimolecular interactions between 22a and VP5, and another between VP19C and VP23. The interactions were detected by the expression of β-galactosidase enzyme activity, and yielded 289, 86, and 63 units of enzyme activity, respectively. For the 22a self-interaction, elimination of residues 611–635 resulted in an approximately twofold decrease in enzyme activity. The C-terminal 25 amino acids of 22a were also essential for the bimolecular interaction between 22a and VP5. 相似文献
13.
Analysis of two VP60 capsid protein genes of rabbit hemorrhagic disease virus from viruses obtained from the same farm 总被引:1,自引:0,他引:1
Two distinct clones of the VP60 capsid protein gene of rabbit hemorrhagic disease virus were amplified from mixed liver tissue
of rabbits collected from the same farm in the Xinjiang Uygur Autonomous Region of China in 2002. The results of DNA sequence
analysis showed that the length of the VP60 gene in the first clone was 1,740 bp, similar to other VP60 genes. The length
of the VP60 gene in the second clone was only 1,536 bp. The two clones were predicted to encode 579 and 511 amino acids, respectively. 相似文献
14.
We previously reported that at least eight HSV-1 and five HSV-2 proteins were tyrosine phosphorylated in infected human and mouse cells and the first phosphotyrosine-modified gene product identified was the ICP22 regulatory protein (Blaho, J. A., Zong, C. S., and Mortimer, K. A., 1997, J. Virol. 71, 9828-9832). All electrophoretic forms of ICP22 are tyrosine phosphorylated with the exception of the fastest migrating (unmodified) isoform. We now report the following. (i) ICP22 that reacted with a specific anti-phosphotyrosine antibody contained a significant amount of phosphotyrosine based on phospho-amino acid analysis. These results validate the discovery of ICP22 tyrosine phosphorylation. (ii) Wild-type ICP22 extracted from infected HEp-2 cells migrated as at least seven isoforms, termed ICP22a-g, in denaturing gels. (iii) The primary structure of ICP22 possesses a sequence that is homologous to protein tyrosine kinase recognition sites. A virus, termed RF141, was generated in which ICP22 tyrosine(193) in the kinase target site was mutated to an alanine. (iv) Biochemical analyses of infected HEp-2 and primary HFF cells indicated that the distributions of ICP22 isoforms differed between RF141 and control HSV-1(F). (v) The accumulations of representative viral polypeptides in RF141-infected HEp-2 cells appeared similar to wild-type virus. (vi) RF141 had reduced efficiencies of plating in HFF cells compared to control Vero cells. These differences increased as the multiplicity of infection decreased. Based on these results, we conclude (vii) that ICP22 tyrosine(193) is required for optimal posttranslational modification of the protein in HSV-1 infected human epithelial HEp-2 and primary human fibroblast cells. 相似文献
15.
The intracellular trafficking of different VP22-enhanced yellow fluorescent protein (EYFP) fusion proteins expressed by bovine herpesvirus-1 (BHV-1) recombinants was examined by live-cell imaging. Our results demonstrate that (i) the fusion of EYFP to the C terminus of VP22 does not alter the trafficking of the protein in infected cells, (ii) VP22 expressed during BHV-1 infection translocates to the nucleus through three different pathways, namely early mitosis-dependent nuclear translocation, late massive nuclear translocation that follows a prolonged cytoplasmic stage of the protein in non-mitotic cells, and accumulation of a small subset of VP22 in discrete dot-like nuclear domains during its early cytoplasmic stage, (iii) the addition of the SV40 large-T-antigen nuclear localization signal (NLS) to VP22-EYFP abrogates its early cytoplasmic stage, and (iv) the VP22 131PRPR134 NLS is not required for the late massive nuclear translocation of the protein, but this motif is essential for the targeting of VP22 to discrete dot-like nuclear domains during the early cytoplasmic stage. These results show that the amount of VP22 in the nucleus is precisely regulated at different stages of BHV-1 infection and suggest that the early pathways of VP22 nuclear accumulation may be more relevant to the infection process as the late massive nuclear influx starts when most of the viral progeny has already emerged from the cell. 相似文献
16.
RNA interference (RNAi) is the process by which double-stranded RNA directs sequence-specific degradation of homologous mRNA.
Short interfering RNAs (siRNAs) are the mediators of RNAi and represent powerful tools to silence gene expression in mammalian
cells including genes of viral origin. In this study, we applied siRNAs targeting the VP7 gene of African horse sickness virus
(AHSV) that encodes a structural protein required for stable capsid assembly. Using a VP7 expression reporter plasmid and
an in vitro model of infection, we show that synthetic siRNA molecules corresponding to the AHSV VP7 gene silenced effectively
VP7 protein and mRNA expression, and decreased production of infectious virus particles as evidenced by a reduction in the
progeny virion titres when compared to control cells. This work establishes RNAi as a genetic tool for the study of AHSV and
offers new possibilities for the analysis of viral genes important for AHSV physiology. 相似文献
17.
Costello F Steenfos N Jensen KT Christensen J Gottschalck E Holm A Aasted B 《Scandinavian journal of immunology》1999,49(4):347-354
Six overlapping fragments of the Aleutian Mink Disease parvoVirus (AMDV) virion protein VP1 and 2 (VP1/2) gene were inserted into the expression vector pMAL-c2. Four of the clones carried large overlapping fragments covering the entire VP1/2 gene. The remaining two clones covered specifically chosen regions within the VP1/2 gene. Using a Western blotting detection system, sera from AMDV-infected mink were tested against the recombinant polypeptides. These studies showed reactions primarily directed against the two AMDV polypeptides ranging from amino acids 297 to 518. Weaker reactions against other regions of the VP1/2 were also observed. The small fusion protein designed to cover the presumed AMDV VP1/2 loop 4 was purified by affinity chromatography and used to develop solid-phase immunoassays. Twelve small synthetic peptides were constructed and used as inhibitors. A peptide covering amino acids S428 to T448 was shown to block the reactivity of a pool of positive mink sera, indicating the presence of one dominant linear epitope. 相似文献
18.
Herpes simplex virus type 1 (HSV-1) UL37 is a 1123 amino acid tegument protein that self-associates and binds to the tegument protein UL36 (VP1/2). Studies were undertaken to identify regions of UL37 involved in these protein-protein interactions. Coimmunoprecipitation assays showed that residues within the carboxy-terminal half of UL37, amino acids 568-1123, are important for interaction with UL36. Coimmunoprecipitation assays also revealed that amino acids 1-300 and 568-1123 of UL37 are capable of self-association. UL37 appears to self-associate only under conditions when UL36 is not present or is present in low amounts, suggesting UL36 and UL37 may compete for binding. Transfection-infection experiments were performed to identify domains of UL37 that complement the UL37 deletion virus, K?UL37. The carboxy-terminal region of UL37 (residues 568-1123) partially rescues the K?UL37 infection. These results suggest the C-terminus of UL37 may contribute to its essential functional role within the virus-infected cell. 相似文献
19.
VP22, encoded by the U(L)49 gene, is one of the most abundant proteins of the herpes simplex virus type 1 (HSV-1) tegument and has been shown to be important for virus replication and spread. However, the exact role(s) played by VP22 in the HSV-1 replication cycle have yet to be delineated. The lack of a procedure to purify full-length VP22 has limited molecular studies on VP22 function. A procedure was developed for the purification of soluble, full-length VP22 from cells infected with HSV-1. A recombinant virus encoding His-tagged VP22 was generated and found to express VP22 at levels comparable to the wild type virus upon infection of Vero cells. By experimenting with a wide variety of cell lysis buffer conditions, several buffers that promote the solubility of full-length VP22 were identified. Buffers that gave the highest levels of solubility were then used in immobilized metal ion affinity chromatography experiments to identify conditions that provided the greatest level of VP22 binding and recovery from cobalt and nickel affinity resins. Using this strategy soluble, full-length VP22 was purified from cells infected with HSV-1. 相似文献
20.
R J Courtney 《Virology》1976,73(1):286-294
The proteins and glycoproteins induced by herpes simplex virus type 1 (HSV-1) were labeled with [14C]amino acids or [14C]glucosamine in the presence or absence of 2-deoxy-d-glucose (deoxyglucose) and analyzed by slab gel electrophoresis. In the presence of 0.1% deoxyglucose (6.1 mM), the major envelope glycoprotein (VP123, MW 123,000) labeled with [14C]glucosamine was shifted to a component of an apparent lower molecular weight (VP123′). In the presence of increasing concentrations of deoxyglucose, there was a progressive decrease in the amount of [14C]amino acids incorporated into polypeptides which normally band in the VP123 region. Concomitant with this decrease was an increase in [14C]amino acids incorporated into a polypeptide(s) of greater electrophoretic mobility and of an apparently lower molecular weight. The polypeptide(s) was designated DG92 (MW 92,000) and was found to be predominantly associated with the nuclear fraction of HSV-1-infected cells cultured in the presence of deoxyglucose. The effects of deoxyglucose on HSV-1 polypeptide synthesis could be prevented by the addition of mannose. 相似文献