Herpes simplex virus latency-associated transcript encodes a protein which greatly enhances virus growth, can compensate for deficiencies in immediate-early gene expression, and is likely to function during reactivation from virus latency.
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Herpes simplex virus types 1 and 2 (HSV1 and HSV2) enter and reactivate from latency in sensory neurons, although the events governing these processes are little understood. During latency, only the latency-associated transcripts (LATs) are produced. However, although the LAT RNAs were described approximately 10 years ago, their function remains ambiguous. Mutations affecting the LATs have minimal effects other than a small reduction in establishment of and reactivation from latency in some cases. Mutations in putative LAT-contained open reading frames (ORFs) have so far shown no effect. The LATs consist of a large species from which smaller (approximately 2 kb), nuclear, nonlinear LATs which are abundant during latency are spliced. Thus, translation of ORFs in these smaller LATs would not usually be expected to be possible, and if expressed at all, their expression might be tightly regulated. Here we show that deregulated expression of the largest HSV1 2-kb LAT-contained ORF in various cells of neuronal and nonneuronal origin greatly enhances virus growth in a manner specific to HSV1-the HSV1 LAT ORF has no effect on the growth of HSV2. Similar results of enhanced growth were found when the HSV1 LAT ORF was constitutively expressed from within the HSV1 genome. The mechanism of LAT ORF action was strongly suggested to be by substituting for deficiencies in immediate-early (IE) gene expression (particularly ICP0), because deregulated LAT ORF expression, as well as enhancing wild-type virus growth, was also found to allow efficient growth of viruses with mutations in ICP0 or VMW65. Such viruses otherwise exhibit considerable growth defects. IE gene expression deficiencies are often the block to productive infection in nonpermissive cells and are also evident during latency. These results, which we show to be protein- rather than RNA-mediated effects, strongly suggest a function of the tightly regulated expression of a LAT ORF-encoded protein in the reactivation from HSV latency. (+info)
Modified VP22 localizes to the cell nucleus during synchronized herpes simplex virus type 1 infection.
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The UL49 gene product (VP22) of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) is a virion phosphoprotein which accumulates inside infected cells at late stages of infection. We previously (J. A. Blaho, C. Mitchell, and B. Roizman, J. Biol. Chem. 269:17401-17410, 1994) discovered that the form of VP22 packaged into infectious virions differed from VP22 extracted from infected-cell nuclei in that the virion-associated form had a higher electrophoretic mobility in denaturing gels. Based on these results, we proposed that VP22 in virions was "undermodified" in some way. The goal of this study is to document the biological and biochemical properties of VP22 throughout the entire course of a productive HSV-1 infection. We now report the following. (i) VP22 found in infected cells is distributed in at least three distinct subcellular localizations, which we define as cytoplasmic, diffuse, and nuclear, as measured by indirect immunofluorescence. (ii) Using a synchronized infection system, we determined that VP22 exists predominantly in the cytoplasm early in infection and accumulates in the nucleus late in infection. (iii) While cytoplasmic VP22 colocalizes with the HSV-1 glycoprotein D early in infection, the nuclear form of VP22 is not restricted to replication compartments which accumulate ICP4. (iv) VP22 migrates as at least three unique electrophoretic species in denaturing sodium dodecyl sulfate-DATD-polyacrylamide gels. VP22a, VP22b, and VP22c have high, intermediate, and low mobility, respectively. (v) The relative distribution of the various forms of VP22 derived from infected whole-cell extracts varies during the course of infection such that low-mobility species predominate at early times and high-mobility forms accumulate later. (vi) The highest-mobility forms of VP22 partition with the cytoplasmic fraction of infected cells, while the lowest-mobility forms are associated with the nuclear fraction. (vii) Finally, full-length VP22 which partitions in the nucleus incorporates radiolabel from [32P]orthophosphate whereas cytoplasmic VP22 does not. Based on these results, we conclude that modification of VP22 coincides with its appearance in the nucleus during the course of productive HSV-1 infection. (+info)
Roles of triplex and scaffolding proteins in herpes simplex virus type 1 capsid formation suggested by structures of recombinant particles.
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Typical herpes simplex virus (HSV) capsids contain seven proteins that form a T=16 icosahedron of 1,250-A diameter. Infection of cells with recombinant baculoviruses expressing two of these proteins, VP5 (which forms the pentons and hexons in typical HSV capsids) and VP19C (a component of the triplexes that connect adjacent capsomeres), results in the formation of spherical particles of 880-A diameter. Electron cryomicroscopy and computer reconstruction revealed that these particles possess a T=7 icosahedral symmetry, having 12 pentons and 60 hexons. Among the characteristic structural features of the particle are the skewed appearance of the hexons and the presence of intercapsomeric mass densities connecting the middle domain of one hexon subunit to the lower domain of a subunit in the adjacent hexon. We interpret these connecting masses as being formed by VP19C. Comparison of the connecting masses with the triplexes, which occupy equivalent positions in the T=16 capsid, reveals the probable locations of the single VP19C and two VP23 molecules that make up the triplex. Their arrangement suggests that the two triplex proteins have different roles in controlling intercapsomeric interactions and capsid stability. The nature of these particles and of other aberrant forms made in the absence of scaffold demonstrates the conformational adaptability of the capsid proteins and illustrates how VP23 and the scaffolding protein modulate the nature of the VP5-VP19C network to ensure assembly of the functional T=16 capsid. (+info)
Typing of clinical herpes simplex virus type 1 and type 2 isolates with monoclonal antibodies.
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The purpose of this study was to evaluate the performance of a herpes simplex virus (HSV) type 1-specific anti-glycoprotein C-1 monoclonal antibody (MAb) and a type 2-specific anti-glycoprotein G-2 MAb for typing of 2,400 clinical HSV-1 isolates and 2,400 clinical HSV-2 isolates, respectively, using an enzyme immunoassay. The anti-HSV-1 MAb showed sensitivity and specificity of 100%, and the anti-HSV-2 MAb showed a sensitivity of 99.46% and 100% specificity, indicating that these MAbs are suitable for typing of clinical HSV isolates. (+info)
Activation of the thymidine kinase promoter by herpes simplex virus type 1 immediate early proteins.
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The herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene promoter contains binding sites for the cellular transcription factors such as Spl, CTF, and TFIID, each of which affects basal level expression of the TK gene. The transcription of the TK gene was induced by viral immediate early proteins, ICP0 and ICP4 in an additive manner, but was repressed by ICP22 and ICP27. To gain further insights into the role of ICP0 and ICP4 for expression of the TK gene during virus infection, several mutants with deletions or point mutations in each of the transcriptional regulatory elements were generated starting at -109 and progressing toward +1. According to the CAT assay involving these mutants, the cellular transcription factor (CTF) binding site was necessary for efficient expression in the presence of transfected ICP0 and ICP4 or during virus infection, whereas the Sp1 binding site had a minor effect on ICP0-mediated TK expression. These results indicate that the immediate early proteins of HSV-1 regulate expression of the TK gene during virus infection by modulating activities of cellular transcription factors such as CTF. (+info)
Identification of type-specific domains within glycoprotein G of herpes simplex virus type 2 (HSV-2) recognized by the majority of patients infected with HSV-2, but not by those infected with HSV-1.
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A combination of phage peptide display library mapping and pepscanning, with both murine monoclonal antibodies and a panel of well-characterized human sera, have been used in order to define type-specific epitopes of glycoprotein G of herpes simplex virus type 2 (HSV-2) (gG2). Both techniques revealed an immunodominant region of gG2, centred around amino acids 525-587 of the uncleaved gG2 molecule. A soluble peptide, equivalent to amino acids 551-570, when used as antigen in an ELISA format was recognized by three out of five murine MAbs and by 20/26 (77%) Western blot anti-HSV-2-positive human sera, but by only 1/63 Western blot anti-HSV-2-negative sera (specificity, 98%). The sensitivity of detection of human anti-HSV-2 antibodies was increased to 90% using a peptide derived from this region, presented on a nitrocellulose membrane. This highly antigenic and type-specific domain of gG2 is located at the junction between the 'unique' region of gG2 and its C-terminal end, which has approximately 50% identity with gG1. A second antigenic region of gG2, amino acids 351-427, which lies within the 'unique' part of gG2, was also identified by both techniques employed in this study and is recognized by a proportion of anti-HSV-2-positive sera. These findings demonstrate the feasibility of developing a peptide-based type-specific assay for the detection of anti-HSV-2 antibody in human sera based on type-specific epitopes of gG2 and have implications for the understanding of the three-dimensional topography of gG2. (+info)
Identification and DNA sequence analysis of the Marek's disease virus serotype 2 genes homologous to the herpes simplex virus type 1 UL20 and UL21.
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We determined 3,135 bp of the nucleotide sequence located in an 8.5 kb EcoRI-E fragment in the unique long (UL) genome region of Marek's disease virus serotype 2 (MDV2), and identified UL20 and UL21 homologous genes of herpes simplex virus type 1 (HSV-1). The UL20 and UL21 homologous genes of MDV2 are arranged colinearly with the prototype sequence of HSV-1. In addition, an open reading frame (MDV2 ORF 273), which has been identified within the UL21 homologous gene of MDV2, has no apparent relation to any other known herpesvirus genes. Northern blot analysis and reverse transcriptase polymerase chain reaction confirmed the existance of RNA transcripts related to the UL20 and ORF 273 genes in MDV2-infected cells, except no transcript related to the UL21 gene being detected. The putative protein product of the MDV2 UL20 gene had a relatively low homology but that of the MDV2 UL21 gene had a moderate homology among herpesviruses. Further, the possible functions and features of the predicted proteins encoded within the sequenced region are discussed. (+info)
Identification and sequence analysis of the Marek's disease virus serotype 2 gene homologous to the herpes simplex virus type 1 UL52 protein.
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The gene of Marek's disease virus serotype 2 (MDV2) homologous to the UL52 gene of herpes simplex virus type 1 (HSV-1) was identified and characterized. The MDV2 UL52 homologous gene encodes 1,071 amino acids with a molecular weight of 118.7 kDa, which includes putative metal-binding site and overlapping region with the UL53 homologous gene. Although a putative polyadenylation signal sequence was found in the downstream of the MDV2 UL52 gene, a MDV2 UL52 DNA probe reacted only with the polycistronic 6.3 kb transcript, representing the UL52 and the downstream genes of UL53 and UL54. Transcriptional pattern of this region of MDV2 was somewhat different from corresponding regions of HSV-1 and infectious laryngotracheitis virus. (+info)