Aidan Dolan

The Complete DNA Sequence of the Long Unique Region in the Genome of Herpes Simplex Virus Type 1

We have determined the DNA sequence of the long unique region (UL) in the genome of herpes simplex virus type 1 (HSV-1) strain 17. The UL sequence contained 107,943 residues and had a base composition of 66.9% G + C. Together with our previous work, this completes the sequence of HSV-1 DNA, giving a total genome length of 152,260 residues of base composition 68.3% G + C. Genes in the UL region were located by the use of published mapping analyses, transcript structures and sequence data, and by examination of DNA sequence characteristics. Fifty-six genes were identified, accounting for most of the sequence. Some 28 of these are at present of unknown function. The gene layout for UL was found to be very similar to that for the corresponding part of the genome of varicella-zoster virus, the only other completely sequenced alphaherpesvirus, and the amino acid sequences of equivalent proteins showed a range of similarities. In the whole genome of HSV-1 we now recognize 72 genes which encode 70 distinct proteins.

Toward a Comprehensive Phylogeny for Mammalian and Avian Herpesviruses

ABSTRACT 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 (theAlpha-, Beta-, andGammaherpesvirinae) 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- andBetaherpesvirinae showed extensive cospeciation with host lineages by criteria of congruence in branching patterns and consistency in extent of divergence. The Gammaherpesvirinaepresented 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.

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.

Evolution and diversity in human herpes simplex virus genomes

ABSTRACT Herpes simplex virus 1 (HSV-1) causes a chronic, lifelong infection in >60% of adults. Multiple recent vaccine trials have failed, with viral diversity likely contributing to these failures. To understand HSV-1 diversity better, we comprehensively compared 20 newly sequenced viral genomes from China, Japan, Kenya, and South Korea with six previously sequenced genomes from the United States, Europe, and Japan. In this diverse collection of passaged strains, we found that one-fifth of the newly sequenced members share a gene deletion and one-third exhibit homopolymeric frameshift mutations (HFMs). Individual strains exhibit genotypic and potential phenotypic variation via HFMs, deletions, short sequence repeats, and single-nucleotide polymorphisms, although the protein sequence identity between strains exceeds 90% on average. In the first genome-scale analysis of positive selection in HSV-1, we found signs of selection in specific proteins and residues, including the fusion protein glycoprotein H. We also confirmed previous results suggesting that recombination has occurred with high frequency throughout the HSV-1 genome. Despite this, the HSV-1 strains analyzed clustered by geographic origin during whole-genome distance analysis. These data shed light on likely routes of HSV-1 adaptation to changing environments and will aid in the selection of vaccine antigens that are invariant worldwide.

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.

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.

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.

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.

Novel cytomegaloviruses in free-ranging and captive great apes: phylogenetic evidence for bidirectional horizontal transmission

Wild great apes often suffer from diseases of unknown aetiology. This is among the causes of population declines. Because human cytomegalovirus (HCMV) is an important pathogen, especially in immunocompromised individuals, a search for cytomegaloviruses (CMVs) in deceased wild and captive chimpanzees, gorillas and orang-utans was performed. By using a degenerate PCR targeting four conserved genes (UL54-UL57), several distinct, previously unrecognized CMVs were found for each species. Sequences of up to 9 kb were determined for ten novel CMVs, located in the UL54-UL57 block. A phylogenetic tree was inferred for the ten novel CMVs, the previously characterized chimpanzee CMV, HCMV strains and Old World and New World monkey CMVs. The primate CMVs fell into four clades, containing New World monkey, Old World monkey, orang-utan and human CMVs, respectively, plus two clades that each contained both chimpanzee and gorilla isolates (termed CG1 and CG2). The tree loci of the first four clades mirrored those for their respective hosts in the primate tree, suggesting that these CMV lineages arose through cospeciation with host lineages. The CG1 and CG2 loci corresponded to those of the gorilla and chimpanzee hosts, respectively. This was interpreted as indicating that CG1 and CG2 represented CMV lineages that had arisen cospeciationally with the gorilla and chimpanzee lineages, respectively, with subsequent transfer within each clade between the host genera. Divergence dates were estimated and found to be consistent with overall cospeciational development of major primate CMV lineages. However, CMV transmission between chimpanzees and gorillas in both directions has also occurred.

Patterns of divergence in the vCXCL and vGPCR gene clusters in primate cytomegalovirus genomes.

Primate cytomegalovirus (CMV) genomes contain tandemly repeated gene clusters putatively encoding divergent CXC chemokine ligand-like proteins (vCXCLs) and G protein-coupled receptor-like proteins (vGPCRs). In human, chimpanzee and rhesus CMVs, respectively, the vCXCL cluster contains two, three and six genes, and the vGPCR cluster contains two, two and five genes. We report that (i) green monkey CMV strains fall into two groups, containing either eight and five genes or seven and six genes in the respective clusters, and (ii) owl monkey CMV has two and zero genes. Phylogenetic analysis suggested that the vCXCL cluster evolved from a CXCL chemokine gene (probably GRO-alpha) that was captured in an incompletely spliced form by an ancestor of Old and New World primate CMVs, and that the vGPCR cluster evolved from a GPCR gene captured by an Old World primate CMV. Both clusters appear to have evolved via complex duplication and deletion events.