The 41-kDa Protein of Human Herpesvirus 6 Specifically Binds to Viral DNA Polymerase and Greatly Increases DNA Synthesis

AbstractWe previously isolated a 41-kDa early antigen of human herpesvirus 6 (HHV-6), which exhibited nuclear localization and DNA-binding activity (Agulnicket al.,1993). In this study, we observed that a 110-kDa protein was coimmunoprecipitated with p41 from HHV-6-infected cells by an anti-p41 antibody. This 110-kDa protein was identified as the HHV-6 DNA polymerase (Pol-6) by an antibody raised against the N terminus of Pol-6. Reciprocal immunoprecipitation and Western blot analyses confirmed that p41 complexes with Pol-6 in HHV-6-infected cells. In addition, both p41 and Pol-6 were expressedin vitroand shown to form a specific complex. Anin vitroDNA synthesis assay using primed M13 single-stranded DNA template demonstrated that p41 not only increased the DNA synthesis activity of Pol-6 but also allowed Pol-6 to synthesize DNA products corresponding to full-length M13 template (7249 nucleotides). By contrast, Pol-6 alone could only synthesize DNA of <100 nucleotides. The functional interaction between Pol-6 and p41 appears to be specific because they could not be physically or functionally substitutedin vitroby their herpes simplex virus 1 homologues. Moreover, as revealed by mutational analysis, both the N and C termini of Pol-6 contribute to its binding to p41. In the case of p41, the N terminus is required for increasing DNA synthesis but not binding to Pol-6, whereas the C terminus is totally dispensable

Analysis of the DNA Binding Domain of the Herpes Simplex Virus Type 1 UL9 Protein

The UL9 gene is one of seven genes which map within the long unique region of the HSV-1 genome and are both necessary and sufficient for viral origin-dependent DNA replication in transfected tissue culture cells. The UL9 gene encodes a polypepetide of 851 amino acids which binds specifically to the HSV-1 replication origins ori3 and oriL. From DNase I footprinting and gel retardation analyses, two binding sites for the origin binding protein (OBP) have been identified within both of these origins. Previous work (Weir et al., 1989) demonstrated that the sequence-specific recognition and binding activities resided within the C-terminal 317 amino acids of the UL9 protein. My work has involved a mutational analysis of the UL9 DNA binding domain in an attempt to define the regions involved in interaction with its target sequence. Using an E.coli expression system, a series of C- terminal deletion (with 16 - 62 amino acids deleted) and small in-frame insertion mutants of the DNA binding domain were constructed and expressed as fusions linked to the N-terminal one third of the Staphylococcus aureus protein A. This system enabled easy detection of mutant proteins in Western blot assays by virtue of interaction of the Fc portions of the antibody conjugates with the protein A moieties. Fusions proteins were tested for sequence-specific DNA binding activity using gel retardation assays. Protein extracts were incubated with radio-labelled oligonucleotides containing ori3 binding sites I or II or a 100 bp fragment containing a functional ori3 (i.e. both sites) at 2

Purification and Characterisation of the Herpes Simplex Virus Type 1 DNA Replication Protein UL8

The genome of herpes simplex virus type 1 (HSV-1) contains seven genes whose products are directly involved in viral DNA replication. These are UL5, UL8, UL9, UL29, UL30, UL42 and UL52. I investigated the products of genes UL5, UL8, UL9, and UL52, as these were the least characterised. Peptides were synthesised with sequences corresponding to portions of the amino acid sequence of these proteins and the peptides were used to generate antisera in rabbits. The peptides produced sera reacting with two of the four proteins - UL8 and UL9. Sera were produced that reacted with both the N and C termini of UL8. The project now focused on UL8 protein which was purified to homogeneity to allow its further characterisation. An existing recombinant baculovirus/Spodoptera frugiperda expression system was used as a source of the protein. The protein was extracted from the cells in a soluble form and was stable in the extraction buffer (neither denatured nor proteolysed) for at least 4 hours at room temperature. The protein was also stable during freezing and thawing, dilution, and incubation at

Importance of the Pre-NH2NH_2-Terminal Domain of HSV-1 DNA Polymerase for Viral Replication

The catalytic subunit of the herpes simplex virus 1 DNA polymerase (HSV-1 Pol) has been extensively studied; however, its full complement of functional domains has yet to be characterized. The previously uncharacterized pre-NH2-terminal domain (residues 1-140) within HSV-1 Pol is unique to the herpesvirus Pol family. We sought to investigate the importance of this domain for viral replication in cell culture and an animal model of infection. We evaluated the enzymatic activity of purified pre-NH2-terminal Pol mutant proteins in which conserved residues had been deleted or substituted. Subsequently, the corresponding pol mutant viruses were engineered for viral genetic analyses. We found that the extreme N-terminal 51 residues were not required for wild type 5’-3’ polymerase activity in vitro. Interestingly, the extreme N-terminal 42 residues were dispensable for viral replication in cell culture while a conserved motif at residues 44-49 was necessary for efficient viral DNA synthesis and production of infectious virus. Viral replication proteins have proven to be particularly important in the context of acute and latent infections in animals. Characterization of pol mutant virus replication in a mouse ocular model of infection revealed that the extreme N-terminal 42 residues were not required for viral replication and reactivation from latency. The conserved motif, however, was shown to be required for robust acute ganglionic replication and efficient latency establishment. We hypothesized that the conserved motif at residues 44-49 mediates a protein- protein interaction that positively impacts viral DNA synthesis during infection. Specific protein candidates were evaluated using purified proteins in vitro, and proteins that coprecipitated with wild type and mutant polymerases from infected cell lysates were analyzed. To date, we have yet to identify a protein whose binding was disrupted as a result of the mutation. Ultimately, we have established a role for the pre-NH2-terminal domain of HSV-1 Pol during viral replication that is distinct from 5’-3’ polymerase activity. The conserved motif mediates a function that is required for efficient viral DNA synthesis in cell culture and is of even greater importance for acute ganglionic replication in mice. The mechanism of action more than likely reflects a conserved mechanism for herpesvirus replication

The role of DNA polymerase fidelity on genetic variation and pathogenicity of Marek’s disease virus

Gallid herpesvirus 2, also known as Marek’s disease virus, is the causative agent of Marek’s disease in chickens that can cause up to 100 % mortality in unvaccinated hosts. Vaccination against MDV is one of the most successful vaccination campaigns in the history of veterinary medicine, reducing disease incidence by more than 99%. Despite this success, MDV is still prevalent in chicken flocks worldwide and has shown a remarkable increase in virulence over the past decades. A major reason for the persistence of MDV could be the fact that vaccination against MD is not inducing sterilizing immunity but is permissive for (reduced) viral replication and shedding. It is argued that the imperfection of vaccination drives viral evolution towards higher virulence by selecting for viral phenotypes that maintain lytic replication and thereby the ability to be shed and transmitted in the presence of vaccine-induced immune response. The phenotypes selected in this way could ultimately benefit from vaccination, as vaccinated chickens which survive the infection shed the most replication competent viruses for a prolonged time, and thus contribute to the spread and evolution of particularly virulent virus strains. As a result, the development of MDV vaccines is caught in a vicious circle – vaccination drives selection of rapidly replicating escape mutants, which requires development of new vaccines based on viral strains that can replicate in the vaccinated host. This scenario has indeed been observed with vaccines of the first and second generation. In the light of these possibilities, the increase in virulence observed during the last decades is undoubtedly alarming. In the context of selection for higher virulence, genetic variation of MDV in vaccinated hosts could provide a selective advantage similar to what is known for some RNA viruses, which have evolved error-prone genome replication and form highly diverse quasispecies. As large DNA virus, MDV is believed to be genetically relatively stable, employing a proofreading DNA polymerase for genome replication. There is, however, evidence for remarkable genetic variation among several large DNA viruses, including herpes viruses such as HCMV and HSV-1. The objectives of this study were 1) to develop a NGS sequencing strategy for this highly cell associated virus 2) to determine, if genetic variation in MDV is a function of the fidelity of its DNA polymerase and 3) to examine replicative fitness and pathogenicity of proofreading-deficient viruses in vivo. Following the development of a tiling array for highly specific capture of viral sequences from infected chicken cell extracts, we were able to sequence whole viral genomes from a variety of samples ranging from infected chicken embryonic cells to dust collected from chicken farms. Next, we constructed MDV mutants with point mutations, in the exonuclease and finger domain of Pol (UL30), that could enhance or reduce replication fidelity. The observed level of residual exonuclease activity correlated with the capacity of mutated viruses to replicate in cell culture. Viruses that encoded a DNA Pol which lacked the majority of its inherent exonuclease activity proved to be suicidal in cell culture, losing their replication fitness within a few passages after reconstitution from BAC DNA. Sequencing of clonal genomes obtained from virus propagated in chicken cells revealed that Pol mutants indeed exhibited higher mutation rates than wild type virus. Following in vitro characterization, three Pol mutants – a hypermutator (Y567F, mutation rate approximately 80-fold higher than WT), a weak mutator (Y547S, mutation rate approximately 3-fold higher than WT) and a putative hypomutator (L755F, mutation rate possibly slightly lower than WT) were examined in vivo. The survival of chickens indicates that a hypermutator phenotype (Y567F) is detrimental for viral pathogenicity while no significant difference between Y547S, L755F and WT was observed. Sequencing of MDV DNA enriched from different chicken tissues showed that this difference in virulence correlates with a higher mutation rate in the Y567F virus. Increasing the mutation rate through reducing MDV Pol fidelity seems to be deleterious for the replicative capacity and fitness of MDV in vitro as well as in vivo through generation of an excessive number of mutations. Nevertheless, the potential of escaping this “error catastrophe” by partial repair of exonuclease function and establishment of a highly diverse viral population with WT like fitness was observed in cell culture for one of the hypermutator mutants (Y567F). The formation of functional and hyperdiverse populations by Pol mutant herpesviruses should be further investigated with special respect to potential quasispecies population dynamics

Characterisation of UL102, the Helicase-Primase Associated Protein of Human Cytomegalovirus

Amongst the set of 11 proteins which are required for human cytomegalovirus (HCMV) origin-dependent DNA synthesis, are six which are conserved amongst the herpesvirus family and which perform the essential functions required for viral DNA synthesis. In HCMV these functions, a processive DNA polymerase, ss DNA-binding and helicase-primase activities are provided by the UL54, UL44, UL57, UL70, UL102 and UL105 proteins which are referred to as the core replication fork proteins. UL54 and UL44 comprise the catalytic and processivity subunits, respectively, of the DNA polymerase holoenzyme, UL29 functions as the ss DNA-binding protein whilst UL70, UL102 and UL105 associate to form the heterotrimeric helicase-primase complex. Current models of herpesvirus DNA synthesis are based mostly upon knowledge regarding the herpes simplex virus type 1 (HSV-1) replication fork proteins which have been more extensively studied than any of their counterparts in other herpesviruses. In HSV-1 the replication fork proteins are UL30/UL42 (DNA polymerase), UL29 (ss DNA- binding protein) and UL5/UL8/UL52 (helicase-primase complex). Multiple interactions between the HSV-1 replication fork proteins have been demonstrated and it is thought that these interactions serve to co-ordinate the functions of the HSV-1 replication proteins for efficient viral DNA synthesis. Unlike the HSV-1 replication proteins, the HCMV replication proteins are not well characterised and therefore their roles in HCMV DNA synthesis have been largely predicted by analogy with their HSV-1 counterparts. The aim of this project was to investigate the properties of UL102, one of the essential HCMV replication fork proteins which forms part of the helicase-primase complex, and compare its characteristics with that of its HSV-1 homologue, UL8, to assess whether it may perform similar functions to UL8 during HCMV DNA synthesis. The following specific properties of UL102 were investigated to allow direct comparison with HSV-1 UL8: 1) Its ability to interact with the HCMV DNA polymerase catalytic subunit, UL54. 2) Its ability to bind to DNA and DNA/RNA hybrid oligonucleotides representing the forms of nucleic acid present at the replication fork. 3) Its intracellular localisation when expressed atone in mammalian cells and also its ability to alter the intracellular localisations of the other HCMV helicase-primase subunits, UL70 and UL105, when it is co-expressed with these proteins in mammalian cells. In order to address these objectives and to enable subsequent structure-function analysis, a variety of UL102 reagents were produced. These included 1) a panel of 51 UL102-specific monoclonal antibodies 2) constructs to express, in E. coli, full-length and truncated UL102 GST-fusion proteins 3) constructs to express, in mammalian cells, full-length UL102, UL70 and UL105 proteins and truncated UL102 proteins and 4) constructs which express UL102 and UL54 that permit 2-hybrid analysis in mammalian cells. Using three different methods, ELISA, co-immunoprecipitation and mammalian-2- hybrid analysis, no evidence for a specific interaction between UL102 and UL54 could be demonstrated. In this respect UL102 may differ from its HSV-1 counterpart, UL8. In immunofluorescence studies, UL102 efficiently translocated to the nucleus of mammalian transfected cells. In this respect it differs from UL8, which localises to the cytoplasm when expressed alone. Both UL70 and UL105 displayed a cytoplasmic localisation when expressed on their own. UL102 did not influence their cytoplasmic localisations when co-expressed with either protein. Preliminary results indicate that nuclear localisation of the helicase-primase proteins occurs only when all three are coexpressed. In this respect, UL102 behaves similarly to UL8, which is required for efficient nuclear localisation of the HSV-1 helicase-primase proteins but does not influence their intracellular localisation when it is expressed with either protein individually. Nucleic acid binding experiments were performed either in the presence of 50 mM NaCI or in the absence of any salt. Under both conditions, UL102 did not detectably bind to ss DNA, ds DNA or DNA-RNA hybrids representing the structures of nucleic acid present at the replication fork. Similarly, UL8 did not detectably bind DNA. The UL102 characterisation studies presented in this thesis indicate that it is unlikely that UL102 performs the same precise set of functions which have been attributed to HSV-1 UL8. As UL102 shares some characteristics in common but also possesses differing characteristics to UL8, it is probable that it performs a subset of the functions carried out by UL8 including a likely role in nuclear translocation of the HCMV helicase-primase complex

Characterization of the Genome of Baboon Cytomegalovirus Strain (Ocom4-37) Isolated from the Olive Baboon, Papio Cynocephalus Anubis

This project involved cloning, sequencing, and analyzing the genome of baboon cytomegalovirus (BaCMV) strain OCOM4-37. After isolation, cloning and sequencing the coding sequence of the BaCMV genome, comparisons were made with other CMV genomes. These analyses showed that the OCOM4-37 strain is most closely related to CMVs isolated from primates most closely related to baboons.Department of Biochemistry and Molecular Biolog

Étude des mécanismes biochimiques et moléculaires de la résistance du cytomégalovirus humain et du virus herpès simplex 1 au foscarnet

La structure des ADN polymérases (pol) du cytomégalovirus humain (CMV) et du virus herpès simplex 1 (VHS-1), appartenant tous deux à la famille des Herpesviridae, est associée à la forme d’une main droite comportant entre autres les domaines de la paume, du pouce et des doigts. L’ADN pol adopte différentes conformations (ouverte et fermée) impliquant un mouvement du domaine des doigts dans le but de faciliter l’intéraction entre le nucléotide et l’ADN en cours d’élongation. Il a été montré que l’antiviral foscarnet (FOS) qui cible l’ADN pol du CMV et du VHS-1 se lierait à l’enzyme quand elle est dans sa conformation fermée et que des mutations dans le domaine des doigts conférant la résistance à cet antiviral favoriseraient une conformation plus ouverte de l’enzyme pour laquelle le FOS a une affinité plus faible. Nous avons voulu analyser si cette hypothèse s’appliquait également à des mutations qui sont localisées dans des régions du domaine NH2-terminal et de la paume qui interagissent avec le domaine des doigts lors des changements de conformation de l’enzyme au cours de la réaction de polymérisation. Notre hypothèse est que des mutations localisées dans l’hélice K (domaine NH2-terminal) et la région II (domaine de la paume) qui participent aux changements de conformation de l’enzyme pourraient favoriser une conformation plus ouverte des ADN pol virales, et par conséquent, réduire la sensibilité des virus au FOS. Nous avons donc sélectionné des substitutions théoriques en utilisant une stratégie basée sur l’alignement des séquences en acides aminés des ADN pol du CMV et du VHS-1 (sensibles au FOS) avec celles des bactériophages RB69 et T4 (résistantes au FOS). Nous avons tenté de générer les virus recombinants CMV et VHS-1 possédant les différentes substitutions théoriques sélectionnées. Cependant, l’introduction de certaines substitutions [Q578P, R581T, L587F (hélice K), P712Y, F718L (région II) pour le CMV et Q617P, R620T, L626F (hélice K), F718L (région II) pour le VHS-1] étaient délétères pour les ADN pol, ce qui empêchait les virus recombinants de se répliquer en culture cellulaire. Parmi les substitutions sélectionnées dans l’hélice K, la substitution I619K confère une résistance du VHS-1 au FOS. Au sein de l’hélice K, nous avons également caractérisé la substitution théorique Q579I qui conférait une hypersensibilité du CMV au FOS. Nous avons caractérisé cette substitution en la comparant à la mutation K805Q localisée dans l’hélice P (domaine des doigts) déjà connue pour induire une hypersensibilité du CMV au FOS. Dans la région II, les substitutions V715S et A719T confèrent une résistance des deux virus au FOS. La substitution Q697P du CMV confère une résistance du CMV au FOS mais pas pour le VHS-1. Les profils de sensibilité des virus recombinants au FOS ont également été confirmés par des tests enzymatiques dans lesquels nous avons déterminé l’inhibition de l’activité des ADN pol recombinantes mutées par cet antiviral. Nous avons également constaté une diminution des capacités réplicatives des CMV et VHS-1 recombinants possédant ces substitutions par rapport à celle des virus sauvages correspondants. Des analyses tri-dimensionnelles ont été réalisées et ont suggéré que les substitutions conférant une résistance au FOS seraient associées à une déstabilisation de la conformation fermée des ADN pol et favoriseraient une conformation plus ouverte pour laquelle l’antiviral a une plus faible affinité. D’autre part, les modélisations tri-dimensionnelles des protéines possédant les substitutions conférant une hypersensibilité au FOS ont montré que les ADN pol favorisaient une conformation plus fermée pour laquelle le FOS a une plus grande affinité. La caractérisation de la substitution théorique V715S du CMV et du VHS-1 (FOSR/GCVR et FOSR/ACVR, respectivement) a été comparée aux substitutions V715G du VHS-1 (FOSR/ACVR), V715M du CMV et du VHS-1 (FOSR/GCVS et FOSS/ACVR, respectivement), déjà décrites dans la littérature. Brièvement, nous avons montré que l’introduction de ces différentes substitutions induisaient des changements au niveau de l’environnement hydrophobe de la valine à la position 715 influençant ainsi les phénotypes de sensibilité aux antiviraux observés. L’ensemble de ces résultats nous ont permis d’appuyer notre hypothèse selon laquelle les mutations localisées dans l’hélice K et la région II peuvent influencer la sensibilité des virus au FOS en modifiant la structure de la protéine et en interférant avec les changements de conformation de l’enzyme.The structure of the human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1) DNA polymerase (pol), belonging to the Herpesviridae family, is associated to a right hand with palm, thumb and fingers domains. The viral DNA pol adopts different conformations (open and closed) that implies a move of the fingers domain to facilitate the interaction between the nucleotide and the elongating DNA. It has been shown that the antiviral foscarnet (FOS) which targets the HCMV and HSV-1 DNA pol binds to the enzyme in its closed conformation and mutations confering resistance to this antiviral and localised in the fingers domain would favor a more open conformation of the enzyme for which FOS has a lower affinity. The aim of this thesis was to analyse whether this hypothesis could be extended to mutations localised in the NH2-terminal and the palm domains which interact with the fingers domain during the conformational changes of the enzyme during the polymerization process. Our hypothesis is that mutations localized in the helix K (NH2-terminal domain) and region II (palm domain) that participate in the conformational changes of the enzyme could favor a more open conformation of the viral DNA pol, and thus, decrease the susceptibility of viruses to FOS. We selected theoretical substitutions using a strategy based on amino acid sequences alignement of the DNA pol of HCMV and HSV-1 (susceptible to FOS) with those of RB69 and T4 bacteriophages (naturally resistants to this antiviral). We tried to generate recombinant HCMV and HSV-1 containing the different theoretical substitutions that we selected. However, the introduction of some theoretical substitutions [Q578P, R581T, L587F (helix K), P712Y, F718L (region II) for HCMV and Q617P, R620T, L626F (helix K), F718L (region II) for HSV-1] was so detrimental for the DNA pol that recombinant viruses were not able to grow in cell culture. Among the substitutions selected in the helix K, the substitution I619K confers resistance of HSV-1 to FOS. In the helix K, we also characterized the theoretical Q579I substitution that confers hypersusceptibility of HCMV to FOS. We compared this substitution with the K805Q substitution located in the helix P (fingers domain), already known to induce hypersusceptibility of HCMV to FOS. In region II, substitutions V715S and A719T confer resistance of both viruses to FOS whereas the Q697P substitution was associated with resistance of HCMV to FOS but not for HSV-1. The susceptibility profiles of recombinant viruses to FOS were confirmed by enzymatic assays that allowed us to determine the inhibition of the recombinant mutated DNA pol activity by this antiviral compound. We observed a decrease of the replicative capacities of recombinant HCMV and HSV-1 harboring these mutations compared to their wild-type counterparts. Tri-dimensional modeling was also performed to better understand the impact of these substitutions on the DNA pol of HCMV and HSV-1. On the one hand, substitutions confering resistance to FOS were associated to a destabilization of the closed conformation of the DNA pol and would favor a more open conformation for which the antiviral has a lower affinity. On the other hand, substitutions associated to a hypersusceptibility profile would favor a more closed conformation of the DNA pol for which FOS has a higher affinity. The characterization of the theoretical substitution V715S of HCMV and HSV-1 (FOSR/GCVR and FOSR/ACVR, respectively) was compared to the substitutions V715G of HSV-1 (FOSR/ACVR), V715M of HCMV and HSV-1 (FOSR/GCVS and FOSS/ACVR, respectively), already described in the literature and that were, thus, associated with different antiviral susceptibility phenotypes compared to those of V715S. Briefly, we showed that the introduction of these different substitutions could induce varying changes of the hydrophobic environment of the valine at position 715 influencing the antiviral susceptibility profile. Altogether, these results support our hypothesis that substitutions in helix K and region II could influence the susceptibility of HCMV and HSV-1 to FOS by modifiying the protein structure and impacting the correct conformational changes of the enzyme

Characterisation of the single-stranded DNA binding protein encoded by Kaposi's sarcoma herpesvirus

The work presented in this thesis is on the characterisation of the single stranded DNA (ssDNA) binding protein of the human herpesvirus, Kaposi's sarcoma herpesvirus (KSHV), encoded by the ORF6 gene. There is a core set of six proteins conserved throughout the herpesvirus family that are required for viral DNA replication and are active at the replication fork. These proteins function as a DNA polymerase and its processivity factor, a trimeric helicase-primase complex and a single-stranded DNA binding protein. In KSHV these functions are carried out by the proteins expressed by the following genes: 0RF9 (DNA polymerase), ORF59 (processivity factor), ORF56 (helicase), ORF44 (primase), ORF40/41 (helicase-primase associated factor) and ORF6 (single-stranded DNA binding protein). Current models of herpesvirus DNA synthesis are based mostly upon knowledge of the herpes simplex virus type 1 (HSV-1) replication fork proteins, which have been more extensively studied than any of their homologues in other herpesviruses. The DNA replication proteins in KSHV have not been well studied and therefore their roles in KSHV DNA synthesis have been largely predicted by analogy with their HSV-1 counterparts.To date there has been no published characterisation of the KSHV ssDNA binding protein (pORF6). The work completed for this thesis describes the over-expression and purification of pORF6 and characterisation of its binding to ssDNA. A recombinant baculovirus expressing pORF6 was constructed and used to infect Spodoptera frugiperda cells. The protein was purified from these cells using heparin sulphate and mono-Q columns. Following successful purification of pORF6 it was established that it could bind to ssDNA, using an electrophoretic mobility shift assay (EMSA) and surface plasmon resonance measurements on a BIAcore instrument. The number of nucleotides required for pORF6 to bind to ssDNA and the effect of NaCl on binding were also investigated, revealing that optimal binding occurred at 150 mM NaCl. Fourteen nucleotides were required for pORF6 to bind, however, binding became more efficient as the length was increased to twenty. ICP8, the HSV-1 homologue of pORF6 has been shown to bind to ssDNA in a cooperative manner. The binding mechanism of pORF6 to a ssDNA of thirty-two nucleotides was investigated, again using an EMSA and surface plasmon resonance. This revealed that pORF6 may also bind to single-stranded DNA cooperatively. A comparison of ICP8 and pORF6 binding to ssDNA using the BIAcore indicated that they bind to single-stranded DNA with a similar affinity. A panel of monoclonal antibodies was generated against pORF6 and was tested by western blotting, immune-fluorescence and immune-precipitation, using Spodoptera frugiperda cells infected with a recombinant baculovirus expressing pORF6. Human B-cells infected with KSHV that had been induced into their lytic cycle were also used in immune-fluorescence assays, which revealed pORF6 to be present in globular areas within the cell, reminiscent of herpesvirus DNA replication compartments

Identification of a 40KD Protein Increased by HSV-2 Infection

Herpes simplex virus (HSV) has been implicated in the etiology of human cancer, but its role in the transformation process is not well understood. A set of cellular polypeptides of 200KD, 90KD (a doublet U90 and L90) and 40KD (TBS:40) was previously detected by immunoprecipitation (i.p.) with tumour bearing serum in the Bn5T cell line. The Bn5T cell line is derived from rat embryo fibroblast transformed by a fragment of the HSV type 2 (HSV-2). These polypeptides were detected in cell lines transformed by other agents and were not detectable in control rat embryo (RE) cells (Macnab et al., 1985). The aim of the project was to purify and obtain amino acid sequence for the TBS:40. The 40KD polypeptide was characterized by its digestion pattern with the enzyme Staph, aureus V8 protease. The U90 and the TBS: 40 were increased upon infection with HSV (Macnab et al., 1992) but this was not known at the start of this thesis. The 40KD protein was purified from Bn5T cells and notfrom infected cells. Attempts to raise antibodies in mice and rabbits were carried out. In rabbit there was no immunological response. To raise monoclonal antibodies (Mab) twelve mice were immunized against Bn5T tumour cells. The 90KD polypeptide i.p. by mouse antisera and the U90 i.p. by TBS were similar, in respect of the Staph, aureus V8 protease peptide map. By contrast the 40KD polypeptide i.p. by the serum of the mice and the TBS:40 had different peptide maps. Therefore, in this instance, the immune system of the rat and the mouse recognized different 40KD proteins. Unfortunately attempts to raise Mabs against the 90KD and the 40KD polypeptides failed. Although the 40KD polypeptide did initially raise Mabs, these were unfortunately subsequently lost. The TBS:40 was purified by biochemical methods. In these experiments TBS was found to i.p. more than one polypeptide. It was decided to purify and identify each polypeptide in turn arid lastly to test the effect of HSV-2 infection on its expression. Ammonium sulphate fractionation separated two 40KD proteins i.p. by TBS. One was mainly insoluble, the other was soluble in a 70% saturated ammonium sulphate solution. The Staph, aureus V8 peptide map of the TBS:40 and the 40KD protein soluble in a 70% saturated ammonium sulphate solution were undistinguishable. Therefore it was decided to purify the soluble 40KD protein. The 40KD protein was further purified by anion exchange chromatography at pH. 8. The 40KD protein eluted in the void volume. The pH. of the void volume was increased to pH.9.5 and anion exchange chromatography at pH. 9.5 separated two 40KD polypeptides, one eluted in the void volume and was called the "VOID VOLUME 40"; the other was eluted from the columm and was called the "COLUMN 40". Both had a Stapli. aureus V8 peptide maps different from the TBS:40 peptide map suggesting that the TBS:40 peptide map was produced when both proteins interact with each other. The "COLUMN 40" was i.p. by TBS but not the "VOID VOLUME 40", these results suggested that the "VOID VOLUME 40" is i.p. as part of a complex. The amino acid sequences obtained from the "VOID VOLUME 40" matched the sequence of the mitochondrial aspartate aminotransferase (mAspAT). The "VOID VOLUME 40" was also immunologically related to the mAspAT in a Western blotting experiment. Expression of polypeptides immunologically related to the mAspAT were increased upon infection with HSV-2. Two peptides obtained by digestion of the "COLUMN 40" were successfully sequenced. The sequence of one matched the sequence of the rat fructose 1-6 diphosphate aldolase, and the sequence of the second peptide matched the sequence of the rat phosphoglycerate kinase-1. The experiment was repeated and the sequence data obtained suggested that the "COLUMN 40" was also related to the mAspAT were obtained in the second experiment. Further experiments must be set up to conform this result