This is one of several projects (in this program) concerned with the variability of surface structures that mediate adhesion of microbial pathogens to genital mucosa. In this project the ultimate objective is to relate structural variations in virion glycoproteins specified by clinical isolates of herpes simplex virus (HSV) to possible functional differences among the isolates that may be of clinical significance. Four glycoproteins of HSV (gB, gC, gD and gH) mediate the attachment of virus to cells and the penetration of virus into the cell. Examination of laboratory strains of HSV has revealed variations in amino acid sequences of these glycoproteins (between strains of the two serotypes and also within each serotype) in domains that may be critical for adsorption and penetration. One of our specific aims in this project is to determine the nucleotide sequences (and inferred amino acid sequences) of these four glycoprotein genes for selected clinical isolates (such as paired pharyngeal and genital isolates from individual patients with primary disease, paired sequential isolates from individuals with recurrent disease, etc.). In the course of this work we will determine whether propagation of clinical isolates in cell culture selects for variant viruses with changes in the sequences of the genes in question. If so, all the sequencing will be done by PCR directly on DNA obtained from the clinical specimens. In addition, the genes will be cloned from the clinical specimens by PCR. The second specific aim is to determine whether interesting structural variations in the glycoproteins can be related to functional differences in virion infectivity. Isogenic virions differing in one or another of the glycoproteins will be produced by standard genetic methods, using clones genes of the variant glycoproteins, or by complementation if it proves to be impossible to propagate the recombinant viruses in cell culture without changes in the genes of interest. The complementation approach requires passage of a glycoprotein deletion mutant once through a transformed cell line that carries the variant glycoprotein gene. Induction of the gene by viral regulatory factors enables the glycoprotein will then be compared with respect to specific adsorption activity, ability to penetrate cells and susceptibility to interference mediated by gD (cells expressing gD are resistant to infection by some strains of HSV and not others). The cell lines used for these tests will include primary foreskin keratinocytes as well as permanent cell lines. We expect to learn from these studies whether there is any pattern to structural and functional variations in selected glycoprotein genes that warrants larger studies of this variability in clinical isolates. We also expect to identify genetic variants of the glycoproteins that will be useful in investigating functions of these proteins.
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