Relationships and host range of human, canine, simian and porcine isolates of simian virus 5 (parainfluenza virus 5).

Sequence comparison of the V/P and F genes of 13 human, canine, porcine and simian isolates of simian virus 5 (SV5) revealed a surprising lack of sequence variation at both the nucleotide and amino acid levels (0-3%), even though the viruses were isolated over 30 years and originated from countries around the world. Furthermore, there were no clear distinguishing amino acid or nucleotide differences among the isolates that correlated completely with the species from which they were isolated. In addition, there was no evidence that the ability of the viruses to block interferon signalling by targeting STAT1 for degradation was confined to the species from which they were isolated. All isolates had an extended cytoplasmic tail in the F protein, compared with the original W3A and WR monkey isolates. Sequence analysis of viruses that were derived from human bone-marrow cells isolated in London in the 1980s revealed that, whilst they were related more closely to one another than to the other isolates, they all had identifying differences, suggesting that they were independent isolates. These results therefore support previous data suggesting that SV5 can infect humans persistently, although the relationship of SV5 to any human disease remains highly contentious. Given that SV5 has been isolated on multiple occasions from different species, it is proposed that the term simian virus 5 is inappropriate and suggested that the virus should be renamed parainfluenza virus 5.

Latent gene sequencing reveals familial relationships among Chinese Epstein-Barr virus strains and evidence for positive selection of A11 epitope changes.

Epstein-Barr virus (EBV) strains from the highly HLA-A11-positive Chinese population are predominantly type 1 and show a variety of sequence changes (relative to the contemporary Caucasian prototype strain B95.8) in the nuclear antigen EBNA3B sequences encoding two immunodominant HLA-A11 epitopes, here called IVT and AVF. This has been interpreted by some as evidence of immune selection and by others as random genetic drift. To study epitope variation in a broader genomic context, we sequenced the whole of EBNA3B and parts of the EBNA2, 3A, and 3C genes from each of 31 Chinese EBV isolates. At each locus, type 1 viruses showed <2% nucleotide divergence from the B95.8 prototype while type 2 sequences remained even closer to the contemporary African prototype Ag876. However, type 1 isolates could clearly be divided into families based on linked patterns of sequence divergence from B95.8 across all four EBNA loci. Different patterns of IVT and AVF variation were associated with the different type 1 families, and there was additional epitope diversity within families. When the EBNA3 gene sequences of type 1 Chinese strains were subject to computer-based analysis, particular codons within the A11-epitope-coding region were among the few identified as being under positive or diversifying selection pressure. From these results, and the observation that mutant epitopes are consistently nonimmunogenic in vivo, we conclude that the immune selection hypothesis remains viable and worthy of further investigation.

Molecular evolution of the gamma-Herpesvirinae.

Genomic sequences available for members of the gamma-Herpesvirinae allow analysis of many aspects of the group's evolution. This paper examines four topics: (i) the phylogeny of the group; (ii) the histories of gamma-herpesvirus-specific genes; (iii) genomic variation of human herpesvirus 8 (HHV-8); and (iv) the relationship between Epstein-Barr virus types 1 and 2 (EBV-1 and EBV-2). A phylogenetic tree based on eight conserved genes has been constructed for eight gamma-herpesviruses and extended to 14 species with smaller gene sets. This gave a generally robust assignment of evolutionary relationships, with the exception of murine herpesvirus 4 (MHV-4), which could not be placed unambiguously on the tree and which has evidently experienced an unusually high rate of genomic change. The gamma-herpesviruses possess a variable complement of genes with cellular homologues. In the clearest cases these virus genes were shown to have originated from host genome lineages in the distant past. HHV-8 possesses at its left genomic terminus a highly diverse gene (K1) and at its right terminus a gene (K15) having two diverged alleles. It was proposed that the high diversity of K1 results from a positive selection on K1 and a hitchhiking effect that reduces diversity elsewhere in the genome. EBV-1 and EBV-2 differ in their alleles of the EBNA-2, EBNA-3A, EBNA-3B and EBNA-3C genes. It was suggested that EBV-1 and EBV-2 may recombine in mixed infections so that their sequences outside these genes remain homogeneous. Models for genesis of the types, by recombination between diverged parents or by local divergence from a single lineage, both present difficulties.

Evolution of the dUTPase gene of mammalian and avian herpesviruses.

Sequences of dUTPases encoded by Alpha- and Gammaherpesviruses resemble other dUTPases in their possession of five conserved motifs, but differ in having greater chain lengths (about twice as long) and in the location of Motif 3 at an N terminal location relative to the other motifs. It was proposed that the herpesvirus gene arose by intragenic duplication of a standard dUTPase coding sequence and subsequent loss of one copy of each motif from the double length chain, and that the resulting enzyme was active as a monomer. With knowledge of the trimeric 3D structure of standard dUTPases, it is possible to suggest transformations that occurred in evolutionary development of the herpesvirus dUTPase. The distinct location of Motif 3 can indeed be seen to be consistent with it contributing to a single intramolecular active site with the other motifs. Separately, the occurrence in herpesvirus dUTPases of around 20 to 40 additional residues between Motifs 4 and 5 allows the C-terminal Motif 5 to reach the active site intramolecularly. The driving force behind these evolutionary changes remains obscure. We speculate that they may have allowed acquisition of a novel, presently unknown function by the protein. Consistent with this idea is the observation that in Alpha- and Gammaherpesvirus dUTPases the original locus of Motif 3 is occupied by a distinct conserved sequence (Motif 6); perhaps this element constitutes part of a separate functional capability. Notably, the apparently orthologous protein in Betaherpesviruses lacks the standard motifs while Motif 6 is still present.

Toward a comprehensive phylogeny for mammalian and avian herpesviruses.

With the aim of deriving a definitive phylogenetic tree for as many mammalian and avian herpesvirus species as possible, alignments were made of amino acid sequences from eight conserved and ubiquitously present genes of herpesviruses, with 48 virus species each represented by at least one gene. Phylogenetic trees for both single-gene and concatenated alignments were evaluated thoroughly by maximum-likelihood methods, with each of the three herpesvirus subfamilies (the Alpha-, Beta-, and Gammaherpesvirinae) examined independently. Composite trees were constructed starting with the top-scoring tree based on the broadest set of genes and supplemented by addition of virus species from trees based on narrower gene sets, to give finally a 46-species tree; branching order for three regions within the tree remained unresolved. Sublineages of the Alpha- and Betaherpesvirinae showed extensive cospeciation with host lineages by criteria of congruence in branching patterns and consistency in extent of divergence. The Gammaherpesvirinae presented a more complex picture, with both higher and lower substitution rates in different sublineages. The final tree obtained represents the most detailed view to date of phylogenetic relationships in any family of large-genome viruses.

The descent of human herpesvirus 8.

Analysis of conserved herpesvirus genes shows that human herpesvirus 8 (HHV8) belongs to a clade of primate herpesviruses within the gamma2 sublineage. Overall, this clade has developed by cospeciation with host species. Seventeen genes of HHV8 and its nearest relatives lack simple homologues in other herpesviruses, and eight of these have known mammalian homologues, presumed to be the sources of the HHV8 versions. Comparative analyses yield further insights into the source or time of acquisition for several of these genes. All were acquired by the HHV8 lineage in the very distant past. Two unusual processes are manifest in the contemporary evolution of HHV8. First, the K1 gene is uniquely undergoing a process of extensive and positively selected substitution, of unknown significance. Secondly, the K15 gene has been substituted in some lineages by a distant homologue, presumably by recombination with an unknown herpesvirus. While available data are otherwise compatible with a cospeciational relationship between HHV8 and the human species, they do not exclude relatively recent transfer to humans from another primate host.

The genome sequence of herpes simplex virus type 2.

The genomic DNA sequence of herpes simplex virus type 2 (HSV-2) strain HG52 was determined as 154,746 bp with a G+C content of 70.4%. A total of 74 genes encoding distinct proteins was identified; three of these were each present in two copies, within major repeat elements of the genome. The HSV-2 gene set corresponds closely with that of HSV-1, and the HSV-2 sequence prompted several local revisions to the published HSV-1 sequence (D. J. McGeoch, M. A. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. McNab, L. J. Perry, J. E. Scott, and P. Taylor, J. Gen. Virol. 69:1531-1574, 1988). No compelling evidence for the existence of any additional protein-coding genes in HSV-2 was identified.

The published DNA sequence of human cytomegalovirus strain AD169 lacks 929 base pairs affecting genes UL42 and UL43.

Compared with the published DNA sequence (M. S. Chee, et al. Curr. Top. Microbiol. Immunol. 154:125-170, 1990), most isolates of human cytomegalovirus strain AD169 contain an additional 929 bp after nucleotide 54612. This results in a changed reading frame for the 5'-terminal 50 codons of gene UL42 and expansion of gene UL43 (a US22 family member) from 187 (3'-truncated) to 423 (full-length) codons. The UL42 and UL43 gene products are nonessential for growth in culture.

The active site of ICP47, a herpes simplex virus-encoded inhibitor of the major histocompatibility complex (MHC)-encoded peptide transporter associated with antigen processing (TAP), maps to the NH2-terminal 35 residues.

The herpes simplex virus (HSV) immediate early protein ICP47 inhibits the transporter associated with antigen processing (TAP)-dependent peptide translocation. As a consequence, empty major histocompatibility complex (MHC) class I molecules are retained in the endoplasmic reticulum and recognition of HSV-infected cells by cytotoxic T lymphocytes is abolished. We chemically synthesized full-length ICP47 (sICP47) and show that sICP47 inhibits TAP-dependent peptide translocation in human cells. Its biological activity is indistinguishable from that of recombinant ICP47 (rICP47). By using synthetic peptides, we mapped the core sequence of ICP47 minimally required for TAP inhibition to residues 2-35. This segment is located within the region of the molecule conserved between ICP47 from HSV-1 and HSV-2. Through alanine scanning substitution we identified three segments within this region that are critical for the ability to inhibit TAP function. The interaction of ICP47 with TAP is unlikely to mimic precisely that of the transported peptides, as deduced from differential labeling of the TAP1 and TAP2 subunits using sICP47 fragments with chemical cross-linkers.

Primary characterization of a herpesvirus agent associated with Kaposi's sarcomae.

Detection of novel DNA sequences in Kaposi's sarcoma (KS) and AIDS-related body cavity-based, non-Hodgkin's lymphomas suggests that these neoplasms are caused by a previously unidentified human herpesvirus. We have characterized this agent using a continuously infected B-lymphocyte cell line derived from an AIDS-related lymphoma and a genomic library made from a KS lesion. In this cell line, the agent has a large episomal genome with an electrophoretic mobility similar to that of 270-kb linear DNA markers during clamped homogeneous electric field gel electrophoresis. A 20.7-kb region of the genome has been completely sequenced, and within this region, 17 partial and complete open reading frames are present; all except one have sequence and positional homology to known gammaherpesvirus genes, including the major capsid protein and thymidine kinase genes. Phylogenetic analyses using both single genes and combined gene sets demonstrated that the agent is a gamma-2 herpesvirus (genus Rhadinovirus) and is the first member of this genus known to infect humans. Evidence for transient viral transmission from infected to uninfected cells is presented, but replication-competent virions have not been identified in infected cell lines. Sera from patients with KS have specific antibodies directed against antigens of infected cell lines, and these antibodies are generally absent in sera from patients with AIDS without KS. These studies define the agent as a new human herpesvirus provisionally assigned the descriptive name KS-associated herpesvirus; its formal designation is likely to be human herpesvirus 8.

Molecular phylogeny and evolutionary timescale for the family of mammalian herpesviruses.

A detailed phylogenetic analysis for mammalian members of the family Herpesviridae, based on molecular sequences is reported. Sets of encoded amino acid sequences were collected for eight well conserved genes that are common to mammalian herpesviruses. Phylogenetic trees were inferred from alignments of these sequence sets using both maximum parsimony and distance methods, and evaluated by bootstrap analysis. In all cases the three recognised subfamilies (Alpha-, Beta- and Gammaherpesvirinae), and major sublineages in each subfamily, were clearly distinguished, but within sublineages some finer details of branching were incompletely resolved. Multiple-gene sets were assembled to give a broadly based tree. The root position of the tree was estimated by assuming a constant molecular clock and also by analysis of one herpesviral gene set (that encoding uracil-DNA glycosylase) using cellular homologues as outgroups. Both procedures placed the root between the Alphaherpesvirinae and the other two subfamilies. Substitution rates were calculated for the combined gene sets based on a previous estimate for alphaherpesviral UL27 genes, where the time base had been obtained according to the hypothesis of cospeciation of virus and host lineages. Assuming a constant molecular clock, it was then estimated that the three subfamilies arose approximately 180 to 220 million years ago, that major sublineages within subfamilies were probably generated before the mammalian radiation of 80 to 60 million years ago, and that speciations within sublineages took place in the last 80 million years, probably with a major component of cospeciation with host lineages.

Molecular phylogeny of the alphaherpesvirinae subfamily and a proposed evolutionary timescale.

Phylogenetic trees were derived for the Alphaherpesvirinae subfamily of the Herpesviridae using molecular sequences. Sequences from the families of genes encoding glycoprotein B, thymidine kinase, S region protein kinase, immediate-early transcriptional regulator IE175 and ribonucleotide reductase large subunit were examined by means of both maximum parsimony and distance methods, and for both protein and DNA alignments. Trees obtained were evaluated by bootstrap analysis. A clear consensus tree was obtained, with most detail coming from 14 sequences in the glycoprotein B gene set. The tree showed two avian viruses branching first from the lineage leading to the mammalian alphaherpesviruses. The mammalian viruses were split into two groups, which corresponded to the Simplexvirus and Varicellovirus genera. A timescale for events in alphaherpesvirus evolution was tested, based on the proposition that most of the lineages arose by ancient cospeciation with hosts. The virus phylogenetic tree was unambiguously compatible with cospeciation for ten of the 12 mammalian viruses. The tree was also supported by demonstration of an approximate proportionality between magnitudes of pairwise divergences of viral sequences and times since lineages of corresponding pairs of hosts split. On the basis of this timescale it was estimated that the two mammalian alphaherpesvirus groups diverged around the period of the mammalian radiation, and that alphaherpesviral genome sequences have evolved faster than those of mammals by a factor of one to two orders of magnitude.

HLA class I molecules are not transported to the cell surface in cells infected with herpes simplex virus types 1 and 2.

To assess the effect of herpes simplex virus (HSV) on assembly and transport of class I MHC molecules, we compared class I MHC immunoprecipitated from metabolically labeled infected and uninfected human dermal fibroblasts. The immunoprecipitates were analyzed by isoelectric focusing, allowing identification of individual class I alleles and assessment of transport through the Golgi apparatus by the sialation of carbohydrate residues. In cells infected with wild-type HSV, class I synthesis was reduced or abolished because of the host protein synthesis shutoff function of the UL41 gene product. In cells infected with mutant viruses of both HSV-2 strain G and HSV-1 strain 17 that lack the UL41 gene, class I HLA molecules failed to become sialated, suggesting that they were not transported to the Golgi apparatus. In contrast, transferrin receptor was normally sialated in both infected and uninfected cells. Drug treatments of cells to restrict viral gene expression suggested that an early gene or genes were responsible for the effect. A pulse chase showed that class I molecules were synthesized in normal amounts in infected cells, but that heavy chains were retained in a sialyl transferase negative compartment either stably associated with beta 2m or as free heavy chain in a pattern that is characteristic for each class I allele. HSV is thus the fourth example of a DNA virus that interferes with class I assembly or transport.

A mutant of herpes simplex virus type 1 in which the UL13 protein kinase gene is disrupted.

Gene UL13 of herpes simplex virus type 1 (HSV-1) has previously been proposed to encode a protein kinase. An HSV-1 mutant with UL13 inactivated by insertion of the Escherichia coli lacZ gene was constructed. This UL13-lacZ mutant was found to grow to near wild-type (wt) titres in tissue culture. Comparison of silver-stained SDS-PAGE profiles of wt and UL13-lacZ virions demonstrated that the UL13 protein is a readily detectable component of wt virions, located in the tegument and probably equivalent to the previously described species VP18.8. Studies of in vitro phosphorylation with nuclear extracts of virus-infected cells and with detergent-treated virions showed that the UL13 protein is involved in phosphorylation of the tegument protein VP22. Extracts of cells engineered to express UL13, and infected with UL13-lacZ virus, were also capable of VP22 phosphorylation.

A novel herpes simplex virus gene (UL49A) encodes a putative membrane protein with counterparts in other herpesviruses.

Comparative analysis of DNA sequences located between the coding regions of genes UL49 and UL50 of herpes simplex virus types 1 and 2 (HSV-1 and -2) has revealed a small open reading frame (ORF) of 91 and 87 codons respectively with the characteristics of a genuine protein-coding region. The predicted protein products are clearly related and exhibit features of membrane-inserted proteins, with potential N-proximal signal peptides and C-proximal membrane anchor regions. Counterparts are present in the other sequenced alphaherpesviruses, namely varicella-zoster virus (a previously undescribed gene, 9A) and equine herpesvirus type 1 (gene 10), in the betaherpesvirus human cytomegalovirus (gene UL73) and in the gammaherpesvirus Epstein-Barr virus (gene BLRF1). Therefore, we consider that this ORF represents an additional HSV gene (UL49A) with counterparts in all sequenced alpha-, beta- and gammaherpesviruses.

Status of the ICP34.5 gene in herpes simplex virus type 1 strain 17.

In the published DNA sequence of herpes simplex virus type 1 (HSV-1) strain 17 the coding region for the neurovirulence factor ICP34.5 is disrupted by a 2 bp insertion relative to the corresponding sequence in HSV-1 strain F; this difference would render 60% of the ICP34.5 coding sequence out of frame in strain 17. Re-investigation of the strain 17 sequence showed that the plasmid clone used as the major sequencing substrate for the region was atypical in that other clones did not possess the 2 bp insertion. It was concluded that the coding sequence for ICP34.5 is intact in HSV-1 strain 17 and that the HSV-1 DNA sequence should be revised at this locus.

The myristylated virion proteins of herpes simplex virus type 1: investigation of their role in the virus life cycle.

Herpes simplex virus type 1 (HSV-1) gene UL11 encodes a myristylated virion protein. In this paper we have characterized the UL11 product further and investigated its role in the virus life cycle. Wild-type HSV-1 strain 17syn+ expresses three electrophoretically distinguishable UL11 polypeptide species. Analysis of single plaque isolates demonstrated that two virus populations exist within the 17syn+ stock: a major population encoding only the two higher Mr species, and a minor population encoding the lowest Mr species alone. DNA sequence analysis suggests that the latter polypeptide differs from the former ones at a single amino acid residue only. The UL11 polypeptides are synthesized as delayed early gene products and are phosphorylated in vitro. Following subcellular fractionation of infected cells, they are found predominantly associated with membranes. Within the virus particle, they appear to reside within the tegument. An insertion mutant containing the lacZ gene from Escherichia coli within the UL11 open reading frame is viable in tissue culture, although it gives smaller plaques and is impaired for growth compared to the wild-type parent or revertant viruses; it does not have a temperature-sensitive or host-range phenotype. Thus, although required for efficient replication, the myristylated HSV-1 virion protein, in contrast to those of many other viruses, is not essential for virus growth in tissue culture.

The UL13 virion protein of herpes simplex virus type 1 is phosphorylated by a novel virus-induced protein kinase.

Herpes simplex virus type 1 (HSV-1) induces a protein kinase (PK) activity in infected cell nuclei. In vitro, the enzyme is able to phosphorylate exogenous casein (albeit inefficiently) but not protamine, can use ATP or GTP as a phosphate donor, is stimulated by high salt concentrations and is insensitive to inhibition by heparin. On the basis of these properties, the PK appears to be distinct from previously described cellular enzymes and from the cytoplasmic PK encoded by the viral US3 gene. A major substrate of the enzyme in vitro is a virus-induced protein with an Mr of 57000 (Vmw57). The gene encoding Vmw57 was mapped using recombinants between HSV-1 and HSV-2 to a region of the virus genome containing genes UL9 to UL15. Use of a monospecific rabbit antiserum showed that Vmw57 is a virion structural protein encoded by gene UL13. These results, in conjunction with previous reports that the UL13 protein contains PK sequence motifs, support the notions that the nuclear PK and Vmw57 are identical, and that the observed reactivity is due to autophosphorylation.

Comparative sequence analysis of the long repeat regions and adjoining parts of the long unique regions in the genomes of herpes simplex viruses types 1 and 2.

We report the determination of the DNA sequence of the long repeat (RL) region and adjacent parts of the long unique (UL) region in the genome of herpes simplex virus type 2 (HSV-2) strain HG52. The DNA sequences and genetic content of the extremities of HSV-2 UL were found to be closely similar to those determined previously for HSV-1. The 5658 bp sequenced at the left end of HSV-2 UL contained coding regions for genes UL1 to UL4 plus part of UL5. The 4355 bp sequenced at the right end of UL contained coding regions for part of gene UL53, and the whole of genes UL54 to UL56. Comparison of the HSV-1 and HSV-2 UL56 sequences led to a correction in the published HSV-1 UL56 reading frame. The HSV-2 RL region, including one copy of the a sequence, was determined to be 9263 bp, with a base composition of 75.4% G+C and with many repetitive sequence elements. In HSV-2 RL, sequences were identified corresponding to HSV-1 genes encoding the immediate early IE110 (ICP0) transcriptional regulator and the ICP34.5 neurovirulence factor; the former HSV-2 gene was proposed to contain two introns, and the latter one intron. Downstream of the HSV-2 immediate early gene, the RL sequence encoding the latency-associated transcripts (LATs) was found to be dissimilar to that in HSV-1; the probable LAT promoter regions, however, showed similarities to HSV-1. Properties of the LAT sequences in both HSV-1 and HSV-2 were consistent with LATs being generated as an intron excised from a longer transcript.