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.

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.

Sequence determination and genetic content of the short unique region in the genome of herpes simplex virus type 1.

We have determined the complete DNA sequence of the short unique region in the genome of herpes simplex virus type 1, strain 17, and have interpreted it in terms of messenger RNAs and encoded proteins. The sequence contains variable regions whose length differs between DNA clones. The clones used for most of the analysis gave a short unique length of 12,979 base-pairs. We consider that this region contains 12 genes, which are expressed by mRNAs which have separate promoters, but may share 3'-termination sites, so that all but two mRNAs belong to one of four 3'-coterminal "families": 79% of the sequence is considered to be polypeptide coding. One pair of genes has an extensive out-of-frame overlap of coding sequences. The proteins encoded in the short unique region include two immediate-early species, two virion surface glycoproteins, and a DNA-binding species. Six of the genes have little or no previous characterization. From the nature of the amino acid sequences predicted for their encoded proteins, we deduce that several of these proteins may be membrane-associated.

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.

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.

On the predictive recognition of signal peptide sequences.

DNA sequence analysis of the short unique regions in the genomes of herpes simplex virus (HSV), types 1 and 2, has previously shown that within this region there are four genes, designated US2, US4, US5 and US7, whose functions are unknown but whose predicted amino sequences exhibit hydrophobic N-termini (D.J. McGeoch et al., 1985, J. Mol. Biol. 181, 1-13; D.J. McGeoch, H.W.M. Rixon and D. McNab, unpublished data). In this paper, the possibility was investigated that these hydrophobic sequences might be signal sequences associated with membrane-bound translation of the proteins, and subsequent secretion or insertion into membranes. By using reference sets of protein sequences known to be translated either on membrane-bound or on free ribosomes, criteria were developed to distinguish between these two classes. These criteria comprised: length and net charge of the immediately N-terminal region which often precedes the hydrophobic stretch in membrane-translated proteins; length of the uncharged (hydrophobic) region; and degree of hydrophobicity of the 8-residue maximal hydrophobic region. The latter two parameters were found to be particularly effective when combined as a two dimensional plot, which clearly distinguished 96% of membrane-translated proteins from other classes. When the uncharacterized, predicted HSV protein sequences were judged by these tests, it was found that the products of genes US4, US5 and US7 were convincingly classified as membrane-translated, while the US2 product gave a less definitive result. In conclusion, the US4, US5 and US7 gene products were considered probably to be previously unrecognized, virion membrane-inserted glycoproteins.

Sequence analysis of the splice junction in the transcript of herpes simplex virus type 1 gene UL15.

Gene UL15 of herpes simplex virus type 1 has been proposed to consist of two coding exons separated by an intron of 3587 base pairs. We have generated a DNA fragment copied from the transcript across the proposed splice junction by successive use of reverse transcription and the polymerase chain reaction, and have sequenced this fragment to determine the precise structure of the splice junction.

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.

Protein sequence comparisons show that the 'pseudoproteases' encoded by poxviruses and certain retroviruses belong to the deoxyuridine triphosphatase family.

Amino acid sequence comparisons show extensive similarities among the deoxyuridine triphosphatases (dUTPases) of Escherichia coli and of herpesviruses, and the 'protease-like' or 'pseudoprotease' sequences encoded by certain retroviruses in the oncovirus and lentivirus families and by poxviruses. These relationships suggest strongly that the 'pseudoproteases' actually are dUTPases, and have not arisen by duplication of an oncovirus protease gene as had been suggested. The herpesvirus dUTPase sequences differ from the others in that they are longer (about 370 residues, against around 140) and one conserved element ('Motif 3') is displaced relative to its position in the other sequences; a model involving internal duplication of the herpesvirus gene can account effectively for these observations. Sequences closely similar to Motif 3 are also found in phosphofructokinases, where they form part of the active site and fructose phosphate binding structure; thus these sequences may represent a class of structural element generally involved in phosphate transfer to and from glycosides.

Evolutionary relationships of virion glycoprotein genes in the S regions of alphaherpesvirus genomes.

The short region in the genome of herpes simplex virus type 1 contains a contiguous array of five genes (US4, US5, US6, US7 and US8) which encode known or proposed virion-surface glycoprotein species. Counterparts for certain of these have been described in the genomes of other alphaherpesviruses, namely herpes simplex virus type 2, pseudorabies virus and varicella-zoster virus. Within each of the US4-, US6- and US7-related sets, the amino acid sequences are most conserved in a region containing several cysteine residues. Comparisons in this region among the three sets were carried out by first aligning three cysteine residues which were very similarly placed in each set, and a number of other similarities were then visible. It was concluded that the US4, US6 and US7 sets of genes are related, and thus have evolved by duplication and divergence. The US8-related sequences are distinct from the US4, US6 and US7 sequences, although possible signs of a distant relationship were detected. The US8 set contains two clusters of cysteine residues, and the sequences around these show some similarity, which was interpreted as evidence for occurrence of an intramolecular duplication event.

The genome of herpes simplex virus: structure, replication and evolution.

The objectives of this paper are to discuss the structure and genetic content of the genome of herpes simplex virus type 1 (HSV-1), the nature of virus DNA replicative processes, and aspects of the evolution of the virus DNA, in particular those bearing on DNA replication. We are in the late stages of determining the complete sequence of the DNA of HSV-1, which contains about 155,000 base pairs, and thus the treatment is primarily from a viewpoint of DNA sequence and organization. The genome possesses around 75 genes, generally densely arranged and without long range ordering. Introns are present in only a few genes. Protein coding sequences have been predicted, and the functions of the proteins are being pursued by various means, including use of existing genetic and biochemical data, computer based analyses, expression of isolated genes and use of oligopeptide antisera. Many proteins are known to be virion structural components, or to have regulatory roles, or to function in synthesis of virus DNA. Many, however, still lack an assigned function. Two classes of genetic entities necessary for virus DNA replication have been characterized: cis-acting sequences, which include origins of replication and packaging signals, and genes encoding proteins involved in replication. Aside from enzymes of nucleotide metabolism, the latter include DNA polymerase, DNA binding proteins, and five species detected by genetic assays, but of presently unknown functions. Complete genome sequences are now known for the related alphaherpesvirus varicella-zoster virus and for the very distinct gammaherpesvirus Epstein-Barr virus. Comparisons between the three sequences show various homologies, and also several types of divergence and rearrangement, and so allow models to be proposed for possible events in the evolution of present day herpesvirus genomes. Another aspect of genome evolution is seen in the wide range of overall base compositions found in present day herpesvirus DNAs. Finally, certain herpesvirus genes are homologous to non-herpesvirus genes, giving a glimpse of more remote relationships.

The DNA sequences of the long repeat region and adjoining parts of the long unique region in the genome of herpes simplex virus type 1.

We have determined the DNA sequence of the long repeat region (RL) in the genome of herpes simplex virus type 1 (HSV-1) strain 17, as 9215 bp of composition 71.6% G + C. In addition, the sequences of parts of the long unique region (UL) adjacent to the terminal (TRL) and internal (IRL) copies of RL were determined (2611 and 3836 bp, respectively). Gene organization in these regions of UL was deduced from the sequences and other available data. It was proposed that the region of UL sequenced, adjacent to TRL, contains three complete genes, none with significant previous characterization, and that the region of UL adjacent to IRL also contains three genes, one encoding the immediate early protein IE63. The RL sequence contains one well characterized gene, for the protein IE110, whose organization we have described previously. Between the downstream end of the IE110 gene and UL there is a 3500 bp segment of RL in which we did not find convincing protein-coding sequences, and which thus remains of obscure functionality. Upstream of the IE110 gene is a region previously proposed by others to contain a gene. However, our sequence data are not compatible with their interpretation. We do consider it possible that the region is protein-coding, but regard gene organization here as still unresolved.

Analysis of the 3'-terminal nucleotide sequence of vesicular stomatitis virus N protein mRNA.

The sequence of 205 nucleotides adjacent to the poly(A) tract at the 3'-terminus of the mRNA encoding the N polypeptide of vesicular stomatitis virus has been determined by copying with reverse transcriptase and using 2', 3'-dideoxynucleoside triphosphates as specific chain terminators. The method appears highly suitable for sequence determination in any purified mRNA. An examination of the sequence did not locate without ambiguity the limit of polypeptide coding RNA. The hexanucleotide AAUAAA, previously found in all poly(A)-containing eukaryote mRNAs, is not present, although the sequence immediately adjacent to the 3'-terminal poly(A) has a high content of A+U.

Sequence of 200 nucleotides at the 3'-terminus of the genome RNA of vesicular stomatitis virus.

The sequence of 200 nucleotides at the 3'-terminus of the genome RNA of vesicular stomatitis virus, Indiana serotype, was determined by adding a poly(A) tract to the 3'-terminus of genome RNA, then using the poly(A) as a binding site for a primer to initiate reverse transcription of the RNA, and analysing the complementary DNA sequence by the dideoxynucleoside triphosphate chain termination method. Proceeding 3' to 5', the genome RNA sequence consisted of a sequence complementary to the leader RNA, followed by the sequence AAA, followed by a sequence complementary to the 5'-extremity of N protein mRNA. These results are discussed in terms of leader RNA function, mechanism of transcript processing at the junction between leader RNA and N mRNA, and N mRNA structure.

Identification and mapping of two polypeptides encoded within the herpes simplex virus type 1 thymidine kinase gene sequences.

mRNA's homologous to the herpes simplex virus type 1 DNA restriction endonuclease fragment BamHI p, which contains the thymidine kinase gene, have been identified and mapped by hybrid-arrested translation and mRNA selection. Such mRNA's, when translated in vitro, directed the synthesis of polypeptides of apparent molecular weights 43,000 (VI43) and 39,000 (VI39). mRNA for enzymatically active thymidine kinase was enriched by more than 20-fold after selection. Mapping was carried out with restriction endonuclease fragments of BamHI p, and locations of the 5' and 3' termini of VI43 mRNA were deduced. Analysis of nucleotide sequences around the 5' terminus revealed several consensus sequences commonly found at the start of eucaryotic mRNA's and which are presumably involved in initiation of transcription by RNA polymerase II. Translation of mRNA's for VI43, VI39, and the thymidine kinase enzyme was arrested only by a 1,170-base-pair region of BamHI p. Since this region is insufficient for adjacent genes, coding sequences for VI43 and VI39 must overlap; the possible relationship of these two polypeptides is discussed. A virus-induced product equivalent to VI39 was detected in infected cells.

DNA sequence analysis of an immediate-early gene region of the herpes simplex virus type 1 genome (map coordinates 0.950 to 0.978).

The nucleotide sequence of 4 kilobases of DNA from within the short region of the genome of herpes simplex virus type 1 has been determined. This portion of DNA contains the junctions of the terminal and inverted repeat sequence components with the unique sequence component and the 5'-regions of the genes which encode the Vmw 12, Vmw 68 and Vmw 175 immediate-early polypeptides. The transcription and translation initiation sites of all three genes and the 5' and 3' boundaries of the Vmw 12 and Vmw 68 gene introns have been localized on the DNA sequence and shown to be flanked by sequences which resemble those found in similar positions in other eukaryotic genes. For the Vmw 12 and Vmw 68 genes the promoters, the 5'-non-coding regions of the mRNAs, and the introns lie within the terminal and internal inverted repeats respectively while the polypeptide-coding regions lie in the short unique component. The introns consist largely of tandemly reiterated copies of a 22-nucleotide sequence: the Vmw 12 gene intron contains seven of these and the Vmw 68 gene intron five. The Vmw 175 gene is located entirely within the short repeats, of which those regions sequenced here have the extremely high G + C content of 78%, in marked contrast to the value of 66% obtained for the adjacent region of the unique sequence component. Prediction of the complete amino acid sequence of the Vmw 12 polypeptide, accounting for a mol. wt. of 9830, and of the first 523 amino-terminal amino acids of the Vmw 175 polypeptide has been made from the DNA sequence. The polypeptide-coding region of the Vmw 175 gene has an average G + C content of 80% but nevertheless specifies a wide range of amino acid types because of the maximal assignment of G and C residues to colon third base positions.

A 3' co-terminal family of mRNAs from the herpes simplex virus type 1 short region: two overlapping reading frames encode unrelated polypeptide one of which has highly reiterated amino acid sequence.

We have used DNA sequencing, mRNA mapping and in vitro translation to characterise three partially overlapping genes in the genome of herpes simplex virus (HSV) type 1. These genes specify three mRNAs with distinct 5' termini but a common 3' terminus, the longest of which is immediate-early (IE) mRNA-5. The 12,000 MW (12K) IE polypeptide encoded by IEmRNA-5 is translated from an 88 codon open reading frame, leaving a 1200 base 3' non-translated region. The second mRNA (mRNA-B) is initiated within the coding sequence of IEmRNA-5, and encodes a 21K polypeptide. The 12K and 21K polypeptide coding regions do not overlap. The third mRNA (mRNA-C) is initiated within the coding region of mRNA-B, and encodes a 33K polypeptide. The reading frame for 33K has a 110 codon out-of-frame overlap with the 21K reading frame. This is the first instance of overlapping genes described for HSV. The 21K polypeptide is thought to be a DNA binding protein and is remarkable for an array of 24 tandem repeats of the sequence X/Pro/Arg (where X represents predominantly Glu, Asp, Thr, Ser or Val) in its C-terminal portion. This array, which occupies most of the region of overlap with 33K, can vary in repeat number between virus strains.

Detailed analysis of the mRNAs mapping in the short unique region of herpes simplex virus type 1.

We have analysed the mRNAs which map within the short unique (US) region of the herpes simplex virus type 1 (HSV-1) genome. US has a total length of 12979 base pairs (1) and is extensively transcribed with approximately 94% of the total sequence present in cytoplasmic mRNAs and 79% of the total sequence considered to be protein coding. There are several examples of overlapping functions and multiple use of DNA sequence within this region. US contains 12 genes (1) which are expressed as 13 mRNAs. Two of these mRNAs are thought to arise from the same gene since they differ only slightly in the positions of their 5' ends and probably specify the same polypeptide. 11 of the 13 mRNAs are arranged into four nested families with unique 5' ends and common 3' co-termini. The other two mRNAs have unique 5' and 3' ends.

DNA sequence of the region in the genome of herpes simplex virus type 1 containing the genes for DNA polymerase and the major DNA binding protein.

In the long unique region of the genome of herpes simplex virus type 1 (HSV-1), the genes for DNA polymerase and the major DNA binding protein are arranged in a head to head manner, with an origin of DNA replication (termed OriL) located between them. This paper reports an 8400 base pair DNA sequence containing both genes and the origin, obtained mostly by M13/dideoxy analysis of plasmid cloned fragments. Amino acid sequences of the two proteins were deduced. Homologues of both genes were detected in the genome sequence of the distantly related Epstein-Barr virus (EBV). Arrangement of these HSV-1 and EBV genes differs in genome location and in relative orientation. A part of HSV-1 DNA polymerase was found to be similar to a sequence in adenovirus 2 DNA polymerase, but the significance of this is unclear. Since a DNA sequence in the locality of OriL deletes on plasmid cloning, this region was analysed using virus DNA. A palindrome with 72-residue arms was found, which shows great similarity to the better characterized origin, OriS.

Isolation and characterization of conditional lethal amber nonsense mutants of vesicular stomatitis virus.

We describe the isolation and characterization of conditional lethal amber nonsense mutants of vesicular stomatitis virus (VSV), Indiana serotype. The mutants were isolated from a chemically mutagenized stock of wild-type virus by their ability to grow on genetically engineered cells which express a Xenopus laevis amber suppressor tyrosine tRNA gene (su+ cells) but not on the non-suppressor parental cells (su- cells). Five mutations were assigned to complementation group I (the L gene) and one to complementation group V (the G gene) by complementation analysis using temperature-sensitive mutants representing each of the five VSV cistrons. Four of the group I mutants were observed to synthesize a novel polypeptide species in su+ cells. Immunoprecipitation and immunoblotting studies using monospecific antisera directed against the N and C termini of the VSV L protein showed that the novel polypeptide species contain N terminal- but not C terminal-specific sequences and can thus be considered to be truncated versions of the L protein. In addition a protein which again contained N terminal- but not C terminal-specific sequences could be identified for the fifth group I mutant. Revertants of four of the group I mutants were isolated on the su- cells. The revertants all synthesized normal L protein but not the putative truncated version.