We have mapped the location in herpes simplex virus (HSV) DNA of (i) three mutations at different loci ( syn loci) which alter the social behavior of infected cells from clumping of rounded cells to polykaryocytosis, (ii) a mutation which determines the accumulation of one major glycoprotein [VP8.0(C 2 )], and (iii) the sequences encoding four major virus glycoproteins [VP8.0(C 2 ), VP7(B 2 ), VP8.5(A), and VP19E(D 2 )]. The experimental design and results were as follows. (i) Analysis of HSV-1 × HSV-2 recombinants showed that the sequences encoding the VP19E(D 2 ) glycoprotein map in the S component, whereas the sequences encoding the other three major glycoproteins are in two locations in the L component of HSV DNA. The templates specifying the HSV-1 and HSV-2 glycoprotein VP8.0(C 2 ) appear not to be colinear; we isolated recombinants specifying glycoproteins comigrating in sodium dodecyl sulfate-polyacrylamide gels with VP8.0(C 2 ) of both HSV-1 and HSV-2. (ii) Marker rescue of a ts mutant defective in accumulation of glycoprotein VP7(B 2 ) showed that the mutation maps within a region containing the sequences encoding that glycoprotein. (iii) Marker transfer experiments involving transfection of rabbit skin cells with donor HSV-1(F) DNA and fragments from several donor strains causing fusion of Vero or both Vero and HEp-2 cells revealed the existence of three syn loci specifying the social behavior of cells and one locus ( Cr ) determining the accumulation of glycoprotein VP8.0(C 2 ). The Cr locus maps to the right of the template specifying VP8.0(C 2 ) glycoprotein. Loci syn 1 and syn 2 map at or near the Cr locus but can be segregated from it. Locus syn 3 maps at or near the template specifying glycoproteins VP7(B 2 ) and VP8.5(A). The expression of mutations in the syn 1 and syn 3 loci appear to be cell type dependent, in that recombinants with these mutations fuse Vero cells but not HEp-2 cells. Recipients of the syn 2 locus or of both syn 2 and syn 1 loci fuse both Vero and HEp-2 cells.

Originally identified as an essential component of the herpes simplex virus immediate early (IE) gene enhancer complex, the transcriptional coactivator host cell factor-1 (HCF-1) has been implicated in a broad range of cellular regulatory circuits. The protein mediates activation through multiple interactions with transcriptional activators, coactivators, and chromatin remodeling complexes. However, the mechanisms involved in HCF-1-dependent transcriptional stimulation were undefined. By using a minimal HCF-1-dependent promoter and a model activator, the varicella zoster IE62 protein, it was determined that HCF-1 was not required for the assembly of the RNAPII basal complex, which depended solely on IE62 in conjunction with the cellular factor Sp1. In contrast, HCF-1 was required for recruitment of the histone methyltransferases Set1 and MLL1 (mixed-lineage leukemia 1), leading to histone H3K4 trimethylation and transcriptional activation. Similarly, in a varicella zoster virus lytic infection, HCF-1, Set1, and MLL1 were recruited to the viral genomic IE promoter, suggesting an essential role for HCF-1 in chromatin modification and remodeling during initiation of lytic infection. The results indicate that one biological rationale for the incorporation of the viral IE activators in the viral particle is to recruit HCF-1/histone methyltransferase complexes and promote assembly of the viral IE gene promoters into transcriptionally active chromatin. These studies also contribute to the model whereby the induced nuclear transport of HCF-1 in sensory neurons may be critical to the reactivation of latent herpesviruses by promoting the activation of chromatin modifications.chromatin ͉ histone methyltransferase ͉ chromatin modifications ͉ Sp1 ͉ transcription T he cellular transcriptional coactivator host cell factor-1 (HCF-1) was originally isolated as a component of the herpes simplex virus (HSV) immediate early (IE) gene enhanceosome complex containing the cellular POU domain protein Oct-1 and the viral transactivator VP16 (1-6). The protein has been most thoroughly studied in this context where it mediates the VP16 transcriptional activation of the viral IE genes (7,8). In an analogous manner, HCF-1 also mediates the induction of the related varicella zoster virus (VZV) IE genes by the viral transactivators ORF10 and IE62 (8). The transcription of these ␣-herpesvirus genes is regulated by multiple mechanisms and factors via complex combinatorial enhancer-promoter domains. Strikingly, HCF-1 has been shown to be essential for IE gene expression, suggesting that it mediates a common rate-limiting step. Most intriguingly, both HSV and VZV establish latency in the neurons of sensory ganglia. In these cells, HCF-1 is uniquely sequestered in the cytoplasm of sensory neurons and is rapidly transported to the nucleus upon stimulation that results in viral reactivation (9). Therefore, whereas the protein is essential for viral lytic replication, it may also be a key component in the ␣-herpesvirus latency reactivation cycle.Sinc...

The DNAs of here sim lex virus (HSV) 1 and 2 consist of two components, L and S, each composed of unique sequences bracketed by inverted repeats. In this study we have probed the structure of the reiterated regionsof the S component in marker rescue experiments involving transfection of cells with mixtures of intact HSV-1 mutant viral DNA and individual DNA fragments generated by restriction endonuclease digestion of wild-type HSV-1 or HSV-2 DNAs. The results were as foflows: (i) HSV is diploid for the wild-type sequences that rescue two temperature-sensitive (ts) mutants. DNA fragments from both reiterated regions of theS component of HSV-1(F) DNA can rescue tsLB2 and tsD mutants. (ii) Identity of the entire reiterated sequence at both ends of S is not obligatory because only one end of the S component of wild 1)notype virus HSV-1(1061) rescues tsD even though both ends rescue tsLB2.(ill) Genes in both reiterated sequences can be expressed. We produced, by marker rescue experiments, recombinants with heterotypic ends of the S component, and these specified correspon in polypeptides characteristic of both HSV-1 and HSV2. (iv) The reiterated sequences of the S component may contain a region of obligatory identity. Thus, several recombinant clones produced by rescue with HSV-2 DNA contained identical HSV-2 DNA insertions within both reiterated regions of the HSV-1 S component. Consistent with this conclusion, the termini of the S component in the heterodiploids described in iii were identical by restriction enzyme analysis. (v) The observation that HSV DNA can be expanded by at least 5 X 100 by means of insertion in the S component suggests that it can be a vehicle for exogenous DNA. Herpes simplex virus 1 (HSV-1) specifies approximately 50 polypeptides which form at least three groups, designated a, fl, and y, whose synthesis is coordinately regulated and sequentially ordered (1). Its DNA (97 X 106 molecular weight) consists of two covalently linked components designated L and S. comprising 82 and 18% of total DNA, respectively (Fig. 1A). Both L and S consist of unique sequences (UL and Us) bracketed by inverted repeats (2). The reiterated sequences of L, designated ab and b'a', each consists of 6% of total DNA, whereas the reiterated sequences of S, designated a'c' and ca, each consists of 4.3% of total DNA (3). HSV-1 DNA extracted from virions consists of four equimolar populations differing in the orientation of the L and S components relative to each other (4, 5). The four populations have been designated P (prototype), IL (inversion of L), Is (inversion of S), and ISL (inversion of both S and L) (Fig. 1A). One consequence of this DNA arrangement is that restriction endonucleases that do not cleave within the reiterated regions generate three classes of fragments. The first class, corresponding in concentration to the molarity of intact DNA, comprises fragments mapping between the first and last cleavage within the unique L and unique S regions. The second class comprises the four terminal fragmen...

Varicella-zoster virus (VZV) open reading frame 63 (ORF63), located between nucleotides 110581 and 111417 in the internal repeat region, encodes a nuclear phosphoprotein which is homologous to herpes simplex virus type 1 (HSV-1) ICP22 and is duplicated in the terminal repeat region as ORF70 (nucleotides 118480 to 119316). We evaluated the role of ORFs 63 and 70 in VZV replication, using recombinant VZV cosmids and PCR-based mutagenesis to make single and dual deletions of these ORFs. VZV was recovered within 8 to 10 days when cosmids with single deletions were transfected into melanoma cells along with the three intact VZV cosmids. In contrast, VZV was not detected in transfections carried out with a dual deletion cosmid. Infectious virus was recovered when ORF63 was cloned into a nonnative AvrII site in this cosmid, confirming that failure to generate virus was due to the dual ORF63/70 deletion and that replication required at least one gene copy. This requirement may be related to our observation that ORF63 interacts directly with ORF62, the major immediate-early transactivating protein of VZV. ORF64 is located within the inverted repeat region between nucleotides 111565 and 112107; it has some homology to the HSV-1 Us10 gene and is duplicated as ORF69 (nucleotides 117790 to 118332). ORF64 and ORF69 were deleted individually or simultaneously using the VZV cosmid system. Single deletions of ORF64 or ORF69 yielded viral plaques with the same kinetics and morphology as viruses generated with the parental cosmids. The dual deletion of ORF64 and ORF69 was associated with an abnormal plaque phenotype characterized by very large, multinucleated syncytia. Finally, all of the deletion mutants that yielded recombinants retained infectivity for human T cells in vitro and replicated efficiently in human skin in the SCIDhu mouse model of VZV pathogenesis.Varicella-zoster virus (VZV) is a ubiquitous human herpesvirus that causes varicella during primary infection of susceptible individuals (2). VZV is a lymphotropic virus, with the capacity to infect CD4 and CD8 T cells, permitting its spread to mucocutaneous sites and producing the vesicular rash commonly referred to as chicken pox. VZV is a member of the alphaherpesvirus group and also exhibits the neurotropism characteristic of these viruses; it establishes latency in sensory nerve ganglia, and its reactivation results in herpes zoster, a localized dermatomal exanthem.The VZV genome is a double-stranded DNA molecule with open reading frames (ORFs) that are known or predicted to encode at least 69 distinct gene products. The genome consists of two main coding regions, the unique long (U L ) and unique short (U S ) segments, each of which is flanked by internal repeat (IR) and terminal repeat (TR) sequences. Functions have been assigned to only about half of the VZV gene products, and many of these are presumed because of their partial sequence homologies with herpes simplex virus type 1 (HSV-1), which is the prototype of the alphaherpesviruses. Whereas generating mutant ...

The varicella zoster virus (VZV) IE62 protein is involved in the activation of expression of all three kinetic classes of VZV proteins. Analysis of the viral promoter for VZV glycoprotein I has shown that the cellular factor Sp1 is involved in or required for the observed IE62 mediated activation. Co-immunoprecipitation experiments show that the two proteins are present in a complex in VZV-infected cells. Protein affinity pull-down assays using recombinant proteins showed that IE62 and Sp1 interact in the absence of any other viral and cellular proteins. Mapping studies using GST-fusion proteins containing truncations of IE62 and Sp1 have delimited the interacting regions to amino acids 612-778 in Sp1 and amino acids 226 -299 in IE62. The region identified in Sp1 is involved in DNA-binding, synergistic Sp1 activation, and Sp1 interaction with cellular transcription factors. The interacting region identified in IE62 overlaps with or borders on sites involved in interactions with the VZV IE4 protein and the cellular factors TBP and TFIIB. Assays using wild-type and mutant promoter elements indicate that Sp1 is involved in recruitment of IE62 to the gI promoter and IE62 enhances Sp1 and TBP binding. Varicella zoster virus (VZV)1 is a member of the alphaherpesvirinae and the causative agent of chicken pox (varicella) and shingles (zoster). The VZV genome is a linear doublestranded DNA molecule, which encodes approximately seventy proteins (1). The entire complement of VZV genes is believed to be expressed during lytic infection in three broad kinetic classes, immediate early (IE), early (E), and late (L). Transcription of VZV genes is performed by the host cell RNA polymerase II, as is the case with all other herpes viruses. Efficient expression of the VZV genome is driven by a small group of VZV gene products including those encoded by open reading frames (ORFs) 62, 4, 61, 63, and 10 (2-11). The major viral transactivator is the product of ORF 62 and its complement, ORF 71, which lie within the inverted repeats bracketing the Us region of VZV DNA. This protein is commonly designated IE62 since it is synthesized in the immediate early phase of lytic VZV gene expression. IE62 contains a potent N-terminal acidic transactivation domain and is capable of activating the expression of all three kinetic classes of VZV genes (12)(13)(14).While IE62 is involved in transactivation of VZV promoters, careful analysis of a limited number of individual viral promoters has shown that cellular transcription factors acting at sites upstream of the coding regions of the viral genes are also involved in the mechanism of IE62 activation. These proteins include the ubiquitous, sequence specific cellular factor Sp1. Sp1 is the protoype of a family of closely related factors which bind to GC-rich elements including the GC-box (GGGCGG or GGGCGGG) and the related GT/CACCC-box. Sp1 contains five distinct domains, four of which (A, B, C, and D) are involved in various aspects of transcriptional activation as well as a DNA binding region conta...

Varicella-zoster virus (VZV) codes for a protein serine kinase called ORF47; the herpes simplex virus (HSV) homolog is UL13. No recombinant alphaherpesvirus serine kinase has been biologically active in vitro. We discovered that preservation of the intrinsic kinase activity of recombinant VZV ORF47 required unusually stringent in vitro conditions, including physiological concentrations of polyamines. In this assay, ORF47 phosphorylated two VZV regulatory proteins: the ORF62 protein (homolog of HSV ICP4) and the ORF63 protein (homolog of HSV ICP22). Of interest, ORF47 kinase also coprecipitated ORF63 protein from the kinase assay supernatant.Varicella-zoster virus (VZV) contains two genes, the ORF47 gene and the ORF66 gene, with classical serine/threonine kinase motifs (26). Two VZV immediate-early proteins, ORF62 and ORF63, are functional homologs of herpes simplex virus (HSV) regulatory proteins ICP4 and ICP22, respectively.

The varicella zoster virus (VZV) IE63 protein is required for growth of the virus in cell culture and is expressed during both lytic and latent phases of VZV infection. We have investigated the physical and functional interaction of this protein with the major VZV transactivating protein IE62. The region of the IE63 protein required for interaction with the IE62 protein has been identified and encompasses the N-terminal 142 amino acids. We have found that the interaction is stable at physiological ionic strength. We have also shown that a portion of the IE63 and IE62 proteins colocalize in VZV-infected cells at both 15 and 48 h postinfection. IE63 was found to have no transcriptional activating or repressing activity within the context of a minimal VZV glycoprotein promoter. The presence of the IE63, however, upmodulated the IE62 transactivation of this promoter. Finally, we show that the IE63 protein can be coimmunoprecipitated with the cellular RNA polymerase II from infected cell extracts, indicating that it is present in a complex with that enzyme.

Varicella-zoster virus is considered to have one of the most stable genomes of all human herpesviruses. In 1998, we reported the unanticipated discovery of a wild-type virus that had lost an immunodominant B-cell epitope on the gE ectodomain (VZV-MSP); the gE escape mutant virus exhibited an unusual pattern of egress. Further studies have now documented a markedly enhanced cell-to-cell spread by the mutant virus in cell culture. This property was investigated by laser scanning confocal microscopy combined with a software program that allows the measurement of pixel intensity of the fluorescent signal. For this new application of imaging technology, the VZV immediate early protein 62 (IE 62) was selected as the fluoresceinated marker. By 48 h postinfection, the number of IE 62-positive pixels in the VZV-MSP-infected culture was nearly fourfold greater than the number of pixels in a culture infected with a low-passage laboratory strain. Titrations by infectious center assays supported the above image analysis data. Confirmatory studies in the SCID-hu mouse documented that VZV-MSP spread more rapidly than other VZV strains in human fetal skin implants. Generally, the cytopathology and vesicle formation produced by other strains at 21 days postinfection were demonstrable with VZV-MSP at 14 days. To assess whether additional genes were contributing to the unusual VZV-MSP phenotype, approximately 20 kb of the VZV-MSP genome was sequenced, including ORFs 31 (gB), 37 (gH), 47, 60 (gL), 61, 62 (IE 62), 66, 67 (gI), and 68 (gE). Except for a few polymorphisms, as well as the previously discovered mutation within gE, the nucleotide sequences within most open reading frames were identical to the prototype VZV-Dumas strain. In short, VZV-MSP represents a novel variant virus with a distinguishable phenotype demonstrable in both infected cell cultures and SCID-hu mice.