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.

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.

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.

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.

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.

The synthesis in vitro of RNA polymerase subunits of Escherichia coli.

DNA from transducing bacteriophage that carry the structural gene for the beta subunit of RNA polymerase has been used to direct protein synthesis in a cell-free transcription-translation system. A polypeptide species has been detected which appears to be beta-subunit material made in vitro.

A mutant transcription factor that is activated by 3':5'-cyclic guanosine monophosphate.

We report here the isolation of an extragenic suppressor of a promoter mutation in the lactose operon of Escherichia coli. Genetic data have indicated that the suppressor of the P(-) mutation maps in the structural gene for the transcription factor catabolite activator protein. Biochemical studies suggest that the mutant molecules of catabolite activator protein are altered in such a way that they are activated by 3':5'-cyclic GMP in addition to their normal effector, 3':5'-cyclic AMP.

Influenza virion RNA-dependent RNA polymerase: stimulation by guanosine and related compounds.

The activity of RNA-dependent RNA polymerase of several influenza viruses is stimulated by guanosine. Depending upon the virus strain used, the stimulation of initial reaction rate is up to 10-fold. 5'-GMP, 3',5'-cyclic GMP, and 5'-GDP show lesser stimulation effects. No other nucleosides of 5'-NMPs stimulate, but the dinucleoside monophosphates GpG and GpC show large stimulations. We present evidence that the stimulation represents preferential initiation of genome complementary RNA chains with guanosine: (i) [3-H] guanosine is incorporated specifically at the 5'terminus of RNA in polymerase reaction mixes in vitro. (ii) This incorporation reaction has several properties similar to those of the virion polymerase elongation reaction. (iii) RNA made in the stimulated reaction behaves as complementary RNA in annealing kinetic studies, as does RNA labeled with [3-H]guanosine.

Influenza virus genome consists of eight distinct RNA species.

The genomic RNA of the avian influenza A virus, fowl plague, was fractionated into eight species by electrophoresis in polyacrylamide-agarose gels containing 6 M urea. The separated 32P-labeled RNA species were characterized by digestion with RNase T1 and fractionation of the resulting oligonucleotides by two-dimensional gel electrophoresis; this demonstrated that each species has a distinct nucleotide sequence. A tentative correlation of each genome RNA species with the virus protein that it encodes was made.

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.

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.

DNA sequence of the herpes simplex virus type 1 gene encoding glycoprotein gH, and identification of homologues in the genomes of varicella-zoster virus and Epstein-Barr virus.

We have determined the sequence of herpes simplex virus type 1 DNA around the previously mapped location of sequences encoding an epitope of glycoprotein gH, and have deduced the structure of the gH gene and the amino acid sequence of gH. The unprocessed polypeptide is predicted to contain 838 amino acids, and to possess an N-terminal signal sequence and a C-terminal transmembrane sequence. Temperature-sensitive mutant tsQ26 maps within the predicted gH coding sequence. Homologous genes were identified in the genomes of two other herpesviruses, namely varicella-zoster virus and Epstein-Barr virus.

Evolutionary comparisons of the S segments in the genomes of herpes simplex virus type 1 and varicella-zoster virus.

The genomes of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) consist of two covalently joined segments, L and S. Each segment comprises an unique sequence flanked by inverted repeats. We have reported previously the DNA sequences of the S segments in these two genomes, and have identified protein-coding regions therein. In HSV-1, the unique sequence of S contains ten entire genes plus the major parts of two more, and each inverted repeat contains one entire gene; in VZV, the unique sequence of S contains two entire genes plus the major parts of two more, and each inverted repeat contains three entire genes. In this report, an examination of polypeptide sequence homology has shown that each VZV gene has an HSV-1 counterpart, but that six of the HSV-1 genes have no VZV homologues. Thus, although these regions of the two genomes differ in gene layout, they are related to a significant degree. The analysis indicates that the inverted repeats are evidently capable of large-scale expansion or contraction during evolution. The differences in gene layout can be understood as resulting from a small number of recombinational events during the descent of HSV-1 and VZV from a common ancestor.

Alphaherpesviruses possess a gene homologous to the protein kinase gene family of eukaryotes and retroviruses.

The US3 genes of herpes simplex virus serotypes 1 and 2, and the corresponding gene of varicella-zoster virus, encode proteins whose sequences are clearly homologous to members of the protein kinase family of eukaryotes and retroviruses. Similarity is most characteristic, and strongest, in an 80 residue region comprising part of the catalytic structure of the kinases. In this region the herpesvirus proteins are most like a yeast cell division control protein, and least like the retrovirus protein-tyrosine kinases. We consider that the herpesvirus proteins are probably involved in modulation of cellular processes during lytic infection, although other roles are also possible, for example in latent infection.