Characterization of the Genome of Equine Herpesvirus 1 Subtype 2

Equine herpesvirus type-1 (EHV-1), a member of the Alphaherpesvirinae. is a major cause of abortion and respiratory disease in horses worldwide. It is also associated with a neurological syndrome, neonatal foal disease and more rarely, coital exanthema. Collectively, these diseases represent a significant economic loss to the thoroughbred industry each year. Two antigenically and genetically distinct subtypes of EHV-1 exist. They can be unequivocally differentiated by restriction endonuclease analysis of their DNAs. Molecular epizootiological studies in America and Australia indicate that both subtypes of EHV-1 are respiratory pathogens with the potential to cause abortion, but only subtype 1 has been associated with abortion storms and the neurological form of the disease. Most of the molecular data published concern the subtype 1 virus. At the onset of this project the genome of EHV-1 subtype 2 was totally uncharacterized. This was probably due in part to the original misconception that it was identical to EHV-1 subtype 1 and in part to the relative difficulty of growing this virus to a high titre in tissue culture. The purpose of this work was to determine whether the two subtypes of EHV-1 shared a common genome structure, to construct the first restriction endonuclease maps for EHV-1 subtype 2 and to investigate the homology between this virus and other members of the Alphaherpesvirinae by molecular hybridization and DNA sequence analysis. Electron microscopy of EHV-1 subtype 2 DNA which had been denatured and self-annealed indicated that a sequence of 11kbp approximately at one genome terminus is repeated in inverse orientation at one internal site. The inverted repeats were shown to be separated by a unique sequence of approximately 13kbp. The presence of repeated sequence within the EHV-1 subtype 2 genome was confirmed by hybridization studies using DNA probes isolated from virion DMA. A library of plasmid clones containing Ram HI fragments representing approximately 75% of the genome was prepared, and the clones were then used to derive Bam HI and Eco RI restriction endonuclease maps for EHV-1 subtype 2 DNA. The results show that the EHV-1 subtype 2 genome consists of two segments, L (111kbp) and S (35kbp). The S component consists of a unique sequence (Us; 9.6kpb -16kbp) flanked by inverted repeats (TRs and 9.5kbp - 12.7kbp). Published data indicate that the EHV-1 subtype 1 genome has a similar structure. However, the maps for the two subtypes are quite different. Eco RI and Bam HI cleave within the TRs/IRs , and so it was not possible to determine whether Us inverts relative to the L region, as it does in subtype 1. HSV-1 DNA fragments containing coding sequences for genes which have been shown previously to be well-conserved in the alphaherpesviruses were hybridized to EHV-1 subtype 2 DMA. Thus, the regions of the EHV-1 subtype 2 genome homologous to probes for the HSV-1 genes encoding the ribonucleotide reductase, the major capsid protein, the major DNA-binding protein and the immediate early protein VmwIE175 were identified. Cloned DNA fragments of EHV-1 subtype 2 were used in comparative hybridization experiments to further determine the extent and distribution of homologous sequences in the genomes of both subtypes of EHV-1 and HSV-1. Regions of detectable homology are arranged colinearly along the genomes suggesting that the three viruses share a common gene arrangement. These results imply that it should be possible to predict the locations of most EHV-1 subtype 2 genes on the basis of our existing knowledge of HSV-1 gene location and function. Published data indicate that the S segment is the least related region in the genomes of several members of the Alphaherpesvirinae and that the TRs /Us and IRs /Us junctions have altered in location, relative to adjacent genes, during evolution. To elucidate the nature of the genes near the EHV-1 subtype 2 TRs/Us junction, the DNA sequence of a 4.57kbp Bam HI fragment was determined using the Sanger chain terminating dideoxynucleotide method. The junction was located within a 100bp region by using several M13 clones in a hybridization study, indicating that TRs/IRs and Us are approximately 10.9kbp and 13.1kbp in size respectively. The G+C content of the TRs portion of the fragment is approximately 17% greater that that of the Us portion. An 8bp sequence is tandemly repeated within the TRs. Analysis of the sequence showed that Bam HI 1 contains two complete open reading frames and the parts of two others. The amino acid sequences of predicted EHV-1 subtype 2 proteins were compared with those coded by the S segments of VZV and HSV-1. Homologues of the four EHV-1 genes were detected in both HSV-1 and VZV. The EHV-1 genes and the TRs/Us junction have an arrangement intermediate between that of their HSV-1 and VZV counterparts. One of the EHV-1 subtype 2 genes apparently encodes a glycoprotein

Nucleotide Sequence Analysis of the NS Genes of Wild-Type and Three Complementation Group E Mutants of Vesicular Stomatitis Virus New Jersey

Vesicular stomatitis virus (VSV) is the prototype of the family Rhabdoviridae. There are two serotypes of VSV -Indiana (IND) and New Jersey (NJ) which can be differentiated from one another on the basis of little or no cross neutralisation using antibody raised against the virion glycoprotein. VSV has a genome consisting of a non-segmented single strand of negative sense RNA approximately 11,000 bases in length. The genome codes for five proteins: L (large), M (matrix), NS (non-structural), N (nucleocapsid) and G (glycoprotein). The L, NS and N proteins associate with the genomic RNA and form the ribonucleoprotein core. N protein is the major structural component of the ribonucleoprotein core, the L and NS proteins function in viral transcription and replication; G and M comprise the viral envelope. M protein is believed to be located on the inner side of the envelope and is the group-specific antigen. G is the transmembrane glycoprotein and forms the spikes that project from the virion

The Regulation of Herpes Simplex Virus Immediate Early Gene Expression

The experiments presented in this thesis deal with two aspects of the transcriptional control of herpes simplex virus type 1 (HSV-1) immediate early (IE) gene expression. Stimulation of the IE gene set is mediated by a protein component of the virion, Vmw65, whose gene has been mapped on the viral genome (Campbell et al, 1984). A specific DNA fragment, BamHI F, containing the entire coding and flanking sequences for Vmw65 has been sequenced using a bacteriophage M13 shot-gun cloning strategy and the dideoxy sequencing technology. The fragment is 8,055 base pairs in size. The mRNA for Vmw65 has been positioned precisely, by nuclease SI mapping. The predicted open reading frame for Vmw65 consists of 490 codons and translates to a protein of molecular weight 54,342. The protein would appear to have no outstanding physical characteristics, such as regions of extreme hydrophobicity or hydrophilicity, but has significantly more acidic residues than the average protein, especially towards the carboxy terminus. The 5' and 3' flanking sequences of the gene exhibit recognisable signals known to be involved in the regulation of transcription and termination of eukaryotic genes. A homologue to Vmw65 has been identified in the genome of varicella-zoster virus (VZV; A. J. Davison, personal communication). The proteins from the two viruses are highly conserved at the level of amino acid sequence and approximately colinear, although the VZV equivalent is 80 amino acids shorter than Vmw65 at the carboxy terminus. Three other genes can be detected in the sequence of BamHI F, consistent with the mRNA mapping of Hall et al (1982). The functions of the products of these genes are unknown. All three genes have homology to genes on the VZV genome at the level of amino acid sequence, but the degree of conservation is variable. The region of VZV which is analogous to the BamHI F fragment shows an identical arrangement of genes to HSV-1. The second aspect of IE gene control which has been investigated is the reported autoregulation of IE gene 3 expression by its product Vmw175. This is based on experiments which showed Vmw175 to be present in lower abundance at early and late times, when compared to IE times, and the observation that a temperature sensitive mutant in Vmw175 (tsK) overproduces IE gene products. The investigation involved the use of plasmid constructs in which the HSV-1 thymidine kinase (TK) was placed under the control of IE promoters. These plasmids were analysed for expression of TK activity after introduction into tissue culture cells, together with the cloned Vmw175 gene from wild type or tsK virus, by calcium-phosphate transfection. The results presented confirm that autoregulation does occur. Polypeptide Vmw175 is able to stimulate expression from the promoters of IE genes 2 and 4/5, but not from its own promoter. This finding suggests that autoregulation may occur indirectly via competitive exclusion of the IE gene 3 promoter from the transcription machinery at post-immediate early times. The reason why Vmw175 cannot activate its own promoter is unclear, however some evidence is presented implicating the enhancer region of IE gene 3. The observation that Vmw175 activates other IE promoters, presumably in an analogous manner to the activation of early and late gene expression, may provide an insight into the role of these IE gene products in the viral lytic cycle

DNA Sequence Analysis of the Repeat and Adjoining Unique Region of the Long Segment of Herpes Simplex Virus Type 1

The DNA sequences of the BamHI b fragment and of a Smal-BamHI subfragment of BamHI e from the herpes simplex virus type 1 (HSV-1) genome have been determined. These restriction fragments are located at each end of the UL sequence and span the RL/UL junctions. The BamHI b fragment contains over 6 kb of the Rl element. The DNA sequence of the two restriction fragments was determined by chain terminator sequencing reactions of recombinant M13 clones generated from sonicated DNA or restriction enzyme digested DNA

Herpes Simplex Virus Ribonucleotide Reductase: Structural Features and Transcriptional Regulation

Ribonucleotide reductase (EC 1.17.4.1. ) catalyses the direct reduction of all four ribonucleotides to the corresponding deoxyribonucleotides, this reaction being the first unique step in the de novo pathway of DNA biosynthesis. The herpes simplex virus type 1 (HSV-1)-induced enzyme is composed of two non-identical subunits, termed large (RR1) and small (RR2), which are dimers of the Vmw136 (RR1) and Vmw38 (RR2) polypeptides respectively. These polypeptides are specified by two early, unspliced and 3' co-terminal mRNAs with sizes of 5.0kb (RR1 mRNA) and 1.2kb (RR2 mRNA). The work presented in this thesis has been primarily directed at obtaining the predicted amino acid sequence of the HSV-1 RR1 polypeptide. The HSV-1 RR1 and RR2 amino acid sequences were analysed for conserved structural and functional features by comparisons to equivalent polypeptides of herpesviral and cellular origin. Other studies have identified the nucleotide changes in a portion of the RR1 gene of the HSV-1 temperature-sensitive (ts) mutant tsl207 and have examined the transcriptional regulation of RR1 and RR2 mRNA expression. The Nucleotide and Predicted Amino Acid Sequence of the HSV-1 RR1 Polypeptide. The nucleotide sequence of the HSV-1 DNA region encoding the RR1 polypeptide was obtained with the M13 dideoxy/chain termination method in combination with a 'shotgun' cloning approach. The sequencing data predicted that the RR1 DNA coding region is an open reading frame (ORF) of 3414 nucleotides which encodes a polypeptide of 1137 amino acids in length. In contrast to the remainder of the RR1 polypeptide, the N-terminal region contains unique amino acid composition features and seven sets of tandemly repeated amino acid sequences. A hypothetical scheme of evolutionary events leading to the formation of this region has been postulated. Further, as this region appears not be directly involved in enzymatic activity, a possible function has been suggested on the basis of two potential nuclear localisation signals. Amino Acid Conservation between Herpesvirus and Cellular Ribonucleotide Reductases. Analysis of amino acid conservation between the HSV-1 RR1 and RR2 polypeptides with identified or proposed large (RR L) and small (RRS) subunit polypeptides of herpesviral or cellular origin was performed using computer programs. a) Comparisons of the HSV-1 RR1 polypeptide with homologue RRL polypeptides. Comparison of the HSV-1 RR1 polypeptide with the equivalent herpes simplex virus type 2 (HSV-2) polypeptide revealed that they are essentially colinear with the exception of the N-terminal regions where number of insertions or deletions were predicted. Other analyses revealed that the RR1 N-terminal region was absent from other RRL polypeptides while the colinear parts exhibited clustered homology. b) Comparisons of the HSV-1 RR2 polypeptide with homologue herpesviral RRS polypeptides. Comparisons of the HSV-1 RR2 polypeptide with homologue herpesviral RRS polypeptides revealed the existence of clustered homology. The Escherichia coli (E. coli) tyrosine residue, on which the (essential for function) stable free radical has been localised, is conserved in all the RRS polypeptides examined These comparisons strongly indicate that the herpesviral RRL and RRS polypeptides examined are the constituents of the ribonucleotide reductase activities specified by these viruses. Conserved Structural and Potential Functional Features of the Herpesviral and Cellular Ribonucleotide Reductases. To identify more precisely regions of clustered homology and to determine potential functional features of the RRL and RRS polypeptide sequences, these were aligned with the consensus template alignment program and secondary structure predictions were obtained. (Abstract shortened by ProQuest.)

A fast homology program for aligning biological sequences.

The algorithm of Gotoh computes in two passes of MN steps the alignment of a pair of sequences of lengths M and N, subject to a constraint on the form of the gap weighting function. This compares with the previous algorithm of Waterman et al. which runs in M2N steps. Gotoh also gave a method using two passes of (L+2)MN steps in the case where gap weights remain constant for gaps of length greater than L. Here we describe a procedure for computing the alignment (evolutionary distance and optimal path) in a single pass of MN steps for both cases

Sequence analysis of enzymes of the shikimate pathway: Development of a novel multiple sequence alignment algorithm

The possibility of homology modelling the shikimate pathway enzymes, 3-dehydroquinate synthase (el), 3-dehydroquinase (e2), shikimate dehydrogenase (e3), shikimate kinase (e4) and 5-enolpyruvylshikimate 3 -phosphate (EPSP) synthase (e5) is investigated. The sequences of these enzymes are analysed and the results found indicate that for four of these proteins, el, e2, e3, and e5, no structural homologues exist. Developing a model structure by homology modelling is therefore not possible. For shikimate kinase, statistically significant alignments are found to two proteins with known structures, adenylate kinase and H-ras p21 protein. These are also judged to be biologically significant alignments. However, the alignments obtained show too little sequence identity to permit homology modelling based on primary sequence data alone. An ab initio based methodology is next applied, with the initial step being careful evaluation of multiple sequence alignments of the shikimate pathway enzymes. Altering the parameters of the available multiple sequence alignment algorithms, produces a large range of differing alignments, with no objective way to choose a single alignment or construct a composite from the many produced for each shikimate pathway enzyme. This problem with obtaining a reliable alignment for the shikimate pathway enzyme will occur in other low sequence identity protein families, and is addressed by the development of a novel multiple sequence alignment method, Mix'n'Match. Mix'n'Match is based on finding alternating Strongly Conserved Regions (SCRs) and Loosely Conserved Regions (LCRs) in the protein sequences. The SCRs are used as 'anchors' in the alignment and are calculated from analysis of several different multiple alignments, made using varying criteria. After divided the sequences into Strongly Conserved Regions (SCRs) and Loosely Conserved Regions (LCRs), the 'best' alignment for each LCR is chosen, independently of the other LCRs, from a selection of possibilities in the multiple alignments. To help make this choice for each LCR, the secondary structure is predicted and sliown alongside each different possible alignment. One advantage of this method over automatic, non-interactive, methods, is that the final alignment is not dependent on the choice of a single set of scoring parameters. Another is that, by allowing interactive choice and by taking account of secondary structural information, the final alignment is based more on biological, rather than mathematical factors. This method can produce better alignments than any of the initial automatic multiple alignment methods used. The SCRs identified by Mix'n'Match, are found to show good correlation with the actual secondary structural elements present in the enzyme families used to test the method. Analysis of the Mix'n'Match alignment and consensus secondary structure predictions for shikimate kinase, suggest a closer match with the actual secondary structure of adenylate kinase, than is found between their amino acid sequences. These proteins appear to share functional, sequence and secondary structural homology. The proposal is made that a model structure of shikimate kinase, based on the structure of adenylate kinase, could be constructed using homology modelling techniques