[Show abstract][Hide abstract] ABSTRACT: The glycoprotein complex gp82-gp105 is a major virion envelope glycoprotein complex of human herpesvirus 6 variant A (HHV-6A) and consists of a number of related polypeptides. Monoclonal antibodies (MAbs) 2D4, 2D6, and 13D6 against this glycoprotein complex neutralized HHV-6A infectivity. We have previously reported the isolation, mapping, and characterization of a portion of the viral genomic DNA fragment encoding the gp82-gp105 complex and the identification of the neutralizing epitope (B. Pfeiffer, Z. N. Berneman, F. Neipel, C. K. Chang, S. Tirwatnapong, and B. Chandran, J. Virol. 67:4611-4620, 1993). This gene was further characterized by the identification of a 2.3-kb genomic fragment and by the identification of a 2.5-kb cDNA clone. The genomic sequence contains a short open reading frame (ORF) encoding the epitope recognized by the MAbs. The identified cDNA showed specificity for HHV-6 in Southern blot analysis with viral DNA. In Northern (RNA) blot analysis with total RNA from HHV-6A(GS)-infected cells, the cDNA insert specifically hybridized with several RNA species. Restriction mapping analysis localized this cDNA to the HHV-6A(U1102) genomic BamHI G fragment, at the right end of the unique long segment of the genome and to the SalI L and SalI O fragments within the left and right terminal direct repeat regions, respectively. In vitro transcription and translation of the cDNA revealed a polypeptide of about 88.5 kDa which was glycosylated in the presence of microsomal membranes to a polypeptide of approximately 104.2 kDa. Both polypeptides were immunoprecipiated by MAb 2D6, verifying the identity of the cDNA as encoding the gp105 in the gp82-gp105 complex. Sequence analysis of the cDNA revealed a large ORF potentially encoding a 650-amino-acid protein with 11 potential N-linked glycosylation sites and 18 cysteine residues. A potential membrane-spanning domain is located only near the amino terminus of the putative protein, indicating that gp105 may be a class 2 glycoprotein. Comparison of the cDNA nucleotide sequence with sequences from HHV-6A(U1102) genomic BamHI G and SalI L fragments revealed that the gene encoding gp105 contains 12 exons, spanning over 20 kb of the viral genome, with intron 1 spanning about 8 kb of genomic DNA. The first exon of the cDNA mapped to the right and left terminal direct repeats, while the other exons mapped within the unique long segment of the genome.(ABSTRACT TRUNCATED AT 400 WORDS)
Journal of Virology 07/1995; 69(6):3490-500. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The complete DNA sequence was determined for strain U1102 of human herpesvirus-6, a CD4+ T-lymphotropic virus with disease associations in immunodeficient settings and a possible complicating factor in AIDS. The genome is 159,321 bp in size, has a base composition of 43% G + C, and contains 119 open reading frames. The overall structure is 143 kb bounded by 8 kb of direct repeats, DRL (left) and DRR (right), containing 0.35 kb of terminal and junctional arrays of human telomere-like simple repeats. Since eight open reading frames are duplicated in the repeats, six span repetitive elements and three are spliced, the genome is considered to contain 102 separate genes likely to encode protein. The genes are arranged colinearly with those in the genome of the previously sequenced betaherpesvirus, human cytomegalovirus, and has a distinct arrangement of conserved genes relative to the sequenced gammaherpesviruses, herpesvirus saimiri and Epstein-Barr virus, and the alphaherpesviruses, equine herpesvirus-1, varicella-zoster virus, and herpes simplex virus. Comparisons of predicted amino acid sequences allowed the functions of many human herpesvirus-6 encoded proteins to be assigned and showed the closest relationship in overall number and similarity to human cytomegalovirus products, with approximately 67% homologous proteins as compared to the 21% identified in all herpesviruses. The features of the conserved genes and their relative order suggested a general scheme for divergence among these herpesvirus lineages. In addition to the "core" conserved genes, the genome contains four distinct gene families which may be involved in immune evasion and persistence in immune cells: two have similarity to the "chemokine" chemotactic/proinflammatory family of cytokines, one to their peptide G-protein-coupled receptors, and a fourth to the immunoglobulin superfamily.
[Show abstract][Hide abstract] ABSTRACT: The unit-length genome of human herpesvirus 6 (HHV-6) consists of a single unique component (U) bounded by direct repeats DRL and DRR and forms head-to-tail concatemers during productive infection. cis-elements which mediate cleavage and packaging of progeny virions (a sequences) are found at the termini of all herpesvirus genomes. In HHV-6, DRL and DRR are identical and a sequences may therefore also occur at the U-DR junctions to give the arrangement aDRLa-U-aDRRa. We have sequenced the genomic termini, the U-DRR junction, and the DRR.DRL junction of HHV-6 strain variants U1102 and Z29. A (GGGTTA)n motif identical to the human telomeric repeat sequence (TRS) was found adjacent to, but did not form, the termini of both strain variants. The DRL terminus and U-DRR junction contained sequences closely related to that of the well-conserved herpesvirus packaging signal Cn-Gn-Nn-Gn (pac-1), followed by tandem arrays of TRSs separated by single copies of a hexanucleotide repeat. HHV-6 strain U1102 contained repeat sequences not found in HHV-6 Z29. In contrast, the DRR terminus of both variants contained a simple tandem array of TRSs and a close homolog of a herpesvirus pac-2 signal (GCn-Tn-GCn). The DRR.DRL junction was formed by simple head-to-tail linkage of the termini, yielding an intact cleavage signal, pac-2.x.pac-1, where x is the putative cleavage site. The left end of DR was the site of intrastrain size heterogeneity which mapped to the putative a sequences. These findings suggest that TRSs form part of the a sequence of HHV-6 and that the arrangement of a sequences in the genome can be represented as aDRLa-U-a-DRRa.
Journal of Virology 06/1994; 68(5):3007-14. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The nucleotide sequence of a 12 kbp HindIII fragment (HindIII C) from the right end of the unique component of the genome of human herpesvirus 6 (HHV-6) (strain U1102) was determined. The sequence has a mean G + C content of 42% and contains approximately 28 copies of a tandemly repeated 104 to 107 bp element, which, with a single exception, contain a cleavage site for KpnI (the KpnI repeats). Each of these elements contains potential binding sites for transcription factors NF-kappa B and AP2. The KpnI repeats lie immediately upstream of a region previously identified as a candidate immediate early (IE) gene locus and therefore may constitute an IE gene enhancer element. One incomplete and six complete open reading frames (ORFs) were identified in the unique sequence of the HindIII C fragment. The predicted products of these ORFs do not include homologues of proteins encoded by members of the alpha- or gamma-herpesvirus sub-family. However, the HindIII C fragment does contain a homologue of the US22 gene family, previously found only in the beta-herpesvirus human cytomegalovirus (HCMV). These findings provide evidence that the close phylogenetic relationship between HHV-6 and HCMV is not confined to the beta-herpesvirus-specific arrangement of conserved replicative and structural genes which has been demonstrated previously.
Journal of General Virology 08/1992; 73 ( Pt 7):1649-60. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sequencing studies have indicated that the unique component of the human herpesvirus 6 (HHV-6) genome and the unique long segment of the human cytomegalovirus genome are genetically colinear. Of particular interest is the identification of a region of local CpG dinucleotide suppression in the genome of HHV-6, a feature conserved in the genomes of human cytomegalovirus, murine cytomegalovirus, and simian cytomegalovirus, and a characteristic of the major immediate-early loci of these viruses. Adjacent to this region in HHV-6 are approximately 30 copies of a 103- to 108-bp sequence element, which contains consensus binding sites for the transcription factors AP2 and NF kappa B, in addition to a single KpnI recognition site. Together, these KpnI repeat units may compose an immediate-early enhancer, analogous to those found in the cytomegaloviruses. We present the sequence of this region of HHV-6 and demonstrate that a transactivating function is encoded by this region. We have used polymerase chain reaction to synthesize fragments containing open reading frames and 5' sequences with or without the upstream KpnI repeat units. Effector plasmids containing these HHV-6 coding and 5' sequences were able to effect activation of heterologous promoter-chloramphenicol acetyltransferase (CAT) constructs, including adenovirus E3-CAT and E4-CAT, human T-cell lymphotropic virus type I long terminal repeat (LTR)-CAT, and human immunodeficiency virus LTR-CAT, in cotransfection experiments in Vero cells and peripheral blood lymphocytes. Furthermore, we have identified the major open reading frame (RF4; 2.3 kb) as being essential for activation, and we have shown that the NF kappa B, SP1, and TATA box motifs in the human immunodeficiency virus LTR are all required for full induction of the promoter by the HHV-6-encoded transactivator.
Journal of Virology 11/1991; 65(10):5381-90. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human herpesvirus type-6 (HHV-6) is a recently isolated herpesvirus which is highly prevalent in adult populations around the world. HHV-6 was first isolated from the peripheral blood of six individuals with lymphoproliferative disorders, two of whom were also infected with human immunodeficiency virus. HHV-6, in common with other herpesviruses, transactivates the HIV long terminal repeat linked to reporter genes and has in addition been shown to accelerate HIV gene expression and CD4 cell death in cultures co-infected with both viruses. The virus is tropic for CD4+ lymphocytes and persists in the peripheral blood of most seropositive individuals. We have now identified a gene in HHV-6 encoding a 490-amino-acid polypeptide homologous to the human adeno-associated virus type-2 (AAV-2) rep gene. This gene has an essential role in AAV-2 DNA replication, can trans-regulate homologous and heterologous gene expression, and inhibits cellular transformation. The acquisition of rep by HHV-6 could be due to natural transfer of genetic information between DNA viruses of eukaryotes and is likely to have important consequences for the life-cycle of HHV-6 and for the host CD4 cell.
[Show abstract][Hide abstract] ABSTRACT: More than 50 fragments resulting from complete digestion of the DNA of human herpesvirus 6 (HHV-6, strain U1102) with BamHI, EcoRI, HindIII, KpnI, NruI, SalI or SmaI have been isolated as clones in M13, plasmid, cosmid and lambda vectors. Using these clones, maps have been constructed for the fragments produced by nine restriction enzymes from unit-length virus genomes and from their concatemeric precursors. The unit-length genome is a linear, double-stranded molecule of 161.5 kbp composed of a central segment of a largely unique sequence of 141 kbp (U) with a sequence of 10 kbp duplicated in the same orientation at both 'left' and 'right' genomic termini (i.e. 'left' and 'right' copies of the direct repeat; DRL and DRR). Adopting as standard an orientation in which the major capsid protein gene is 'left' of the gene for alkaline exonuclease, then the 'right' genome termini and DRL. U junctions occur close to or within repetitive (GGGTTA)n sequences. Repetitions of short sequence motifs are present in at least two other regions of the genome. One of these regions consists of a simple repeat (TC/G) of approximately 1.5 kbp in length and is unstable as clones in bacterial vectors. The second region is stably maintained in such vectors and consists of a tandem array of at least 25 copies of a 110 bp sequence containing a single KpnI site. Comparisons of fragments arising from unit-length DNA with those from virus DNA from the nuclei of infected cells have shown that the concatemeric junctions in intracellular DNA contain head-to-tail dimers of the terminal duplications (i.e. ...U1.DRR1.DRL2.U2...). The gross structure established here for the genome from the U1102 isolate of HHV-6 resembles closely that suggested by Pellett and his colleagues for the Z29 isolate and differs from that of the five previously characterized human herpesviruses. This structure of HHV-6 DNA bears a superficial resemblance to that proposed for DNA from channel catfish virus and equine cytomegalovirus.
Journal of General Virology 02/1991; 72 ( Pt 1):157-68. · 3.53 Impact Factor