Anti-herpesviral agents and assays therefor

Details Anti-herpesviral agents and assays therefor Anti-herpesviral agents and assays therefor

1999-01-25

2001-03-13

Salimi, Ali R. (Department: 1648)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Virus or component thereof

C424S204100, C424S231100, C435S005000, C435S975000, C435S007930, C530S300000, C536S023720

Reexamination Certificate

active

06200577

ABSTRACT:

The present invention relates to an anti-viral agent effective against herpesviruses and to an assay for screening for other suitable anti-viral agents.
Herpesviruses are a large family of viruses which infect a wide range of organisms. The term “Herpesvirus” is used herein to refer to any virus of the Herpes family, including viruses in the a group (e.g. HSV, PrV), the &bgr; group (eg HCMV) and in the &ggr; group (eg EBV). Seven herpesviruses are known to infect humans and there is evidence for an eighth human herpesvirus. The most highly characterised human herpesvirus is herpes simplex type 1 (HSV-1) which is associated with causing lesions around the mouth (cold sores). HSV-2, which is closely related to HSV-1, is a primary cause of genital infections. A common feature of herpesviruses is their ability to establish latent infections and recurrences of HSV-1 and HSV-2 infections are common among infected individuals. For a sizeable proportion of these individuals, recurrences are highly debilitating and impact upon quality of life. In other situations, HSV-1 and HSV-2 infection can be life-threatening. A third related virus, varicella zoster virus (VZV), is the causative agent of chickenpox in children and shingles in adults.
Herpesvirus virions consist of four morphologically distinct components, the core, capsid, tegument and envelope (reviewed in Rixon, 1993).
In virions made by HSV-1, the prototype a-herpesvirus, there are about 29 viral polypeptides in the tegument and envelope (15 to 18 polypeptides in the tegument and 11 glycoproteins in the envelope). Thus these two regions of virus particles account for more than 30% of the genes encoded by the virus genome. From studies on L-particles, which are virus-related particles that lack a nucleocapsid and are made by HSV-1, it has been demonstrated that the tegument and envelope can combine to assemble mature particles whose properties are indistinguishable from those of virions during the early events after infection (Szilágyi and Cunningham, 1991; McLauchlan et al., 1992; Rixon et al., 1992). The compositions of the tegument and envelope in virions and L-particles are also very similar (Szilágy! and Cunningham, 1991; McLauchlan and Rixon, 1992), hence, interaction with the capsid is not a primary determinant for incorporation into either of these sub-structures of virions. It follows that interactions between the tegument and envelope components play a critical role in particle assembly and maturation.
Three of the most abundant structural proteins are glycoprotein B (gB), VP16 and VP22. gB is located in the envelope while VP16 and VP22 are tegument proteins.
VP16 is the product of the UL48 gene and is 490 amino acid residues in length with an apparent molecular weight of 65 KDa on denaturing polyacrylamide gels. This protein plays an essential role in both activation of transcription of immediate early (IE) genes and the assembly of the progeny virions (Weinheimer et al., 1992; reviewed in O'Hare, 1993). Hence, deletion of this gene abrogates virus growth and, to date, it is the only tegument protein known to be essential for virus growth. Mutagenesis of the UL48 gene demonstrated that distinct regions of the VP16 protein are involved in transactivation and assembly (Ace et al., 1988). The sequences involved in transactivation can be separated into two domains. One domain, within the N-terminal portion of the protein, is specific for protein interactions with cellular transcription factors. Another domain is located within the C-terminal tail region of the polypeptide; this region is rich in acidic residues, however, apart from HSV-2, it is not conserved in homologues of VP16.
The function of the other major tegument protein, VP22, has not been well characterised. The protein is encoded by the UL49 gene (Elliott and Meredith, 1992) and the open reading frame (ORF) consists of 301 amino acid residues. On denaturing polyacrylamide gels, the protein has an apparent molecular weight of approximately 38 KDa. In infected cells, it is extensively modified post-translationally by phosphorylation, poly(ADP)ribosylation and nucleotidylylation (Blaho et al., 1994). Immunofluorescence studies have shown that, in infected cells, VP22 is located in the cytoplasm with high concentrations around the nuclear membrane (Elliott and Meredith, 1992). It also associates with the nuclear matrix and therefore may have DNA-binding ability (Knopf and Kaerner, 1980). Recent evidence has revealed that VP22 has the ability to exit and re-enter cells although the mechanism which mediates this property is unknown (Elliott and O'Hare, 1997). Within the tegument, VP22 is the most abundant structural protein and recent evidence has shown that its abundance in the tegument can be further enhanced by altering the amount of VP22 produced during infection (Leslie et al., 1996). we have evidence that mutations within this protein significantly reduce virus growth (J. McLaughlan and Y. Sun, unpublished data). In a related bovine herpesvirus, the removal of the gene that encodes the protein homologous to VP22 severely impairs virus growth (Liang et al., 1995).
gB is the most abundant of the envelope components. It is encoded by gene UL27 and is the most highly conserved gene among those encoding herpesvirus glycoproteins. Along with three other glycoproteins (gD, gH and gL), it is essential for virus replication in tissue culture and is required for virus penetration and cell to cell spread. The unprocessed polypeptide consists of 904 residues and, on denaturing polyacrylamide gels, the mature species has an apparent molecular weight of about 120 KDa. The encoded polypeptide can be separated into four domains: a cleavable signal sequence of 30 residues, an ectodomain (external domain) of 697 residues, a hydrophobic transmembrane domain of 68 amino acids and an extensive endodomain (cytoplasmic region) of 109 amino acids (Cai et al., 1988). The cytoplasmic domain is reported to have a role in cell-cell fusion and this is supported by the mapping of syn mutations to this region (Bond et al., 1982; Gage et al., 1993). The biologically active form of gB is an oligomer. Two discontinuous sites for oligomer formation have been characterised, a non-essential region in the N-terminal portion of the mature polypeptide and an essential site proximal to the membrane-spanning domain (Highlander et al., 1991; La Querre et al., 1996). Defective forms of gB, which retain the ability to form hetero-oligomers, inhibit complementation of gB null mutants by the wild-type gB molecule and thus exhibit negative transdominance (Cai et al., 1988). Among the mutants which display this property are C-terminally truncated forms which retain the transmembrane domain and the regions required for oligomerisation but lack the cytoplasmic tail.
Following treatment of virus particles with a cross-linking reagent, four structured proteins, which were not present on the virus envelope, were co-precipitated with gB using a gB-specific polyclonal antiserum (Zhu and Courtney, 1994); this suggested that, in the virus particle, gB is in close proximity to these proteins. One of these proteins was immunologically characterised to be VP16, two were tentatively identified as VP11/12 (encoded by gene UL46) and VP13/14 (encoded by gene UL47) and the fourth was not classified but did have the same apparent molecular weight as VP22. From the topography of gB, it is reasonable to speculate that the cytoplasmic domain of the protein may interact with tegument proteins underlying the envelope. Blocking any interaction of the C-terminal domain of gB with tegument proteins may inhibit incorporation of the protein into virions, thus generating virus with either no or reduced infectivity. This could be achieved through binding of a peptide or a peptide derivative to the intracellular domain of wild type gB.
Recent studies have shown that VP16 and VP22 also interact (Elliott et al., 1995). This interaction is detected in infected cells by immunoprecipitation of the complex by a VP16-specific antib

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