This application focuses on the roles of Herpes simplex virus 1 (HSV-1) glycoproteins in the initiation of infection, virus maturation and transport in polarized cells. Preliminary data shows that (1) cells have at least two different receptors for attachment of HSV that interact with different viral glycoproteins, and that are asymmetrically distributed on apical and basal surfaces of polarized MDCK cells, (ii) one glycoprotein (Gc) not required for infection of nonpolarized cells is essential for attachment to apical surfaces of two different polarized cell lines, (iii) three other glycoproteins not required for viral replication in nonpolarized cells (gG, Ge,Gi) are necessary for either post-attachment entry or dissemination from cell to cell in polarized cells, (iv) Ge and particularly Gi play roles in the apical/basal sorting of other viral glycoproteins, and (v) a virus deleted in three of the dispensable glycoproteins is viable in nonpolarized cells in culture. The objectives of the proposed studies are to continue and expand this work. The targets of the investigation are the functions of the """"""""dispensable"""""""" viral glycoproteins in determining infection of polarized cells. The specific objectives are (i) To determine the roles of each of these proteins in various stages of infection of polarized epithelial cells. Assays of attachment, post-attachment entry, glycoprotein processing and cell to cell dissemination will be done using recombinant viruses with deletions in each of the dispensable glycoproteins. (ii) To define the roles of heparan sulfate proteoglycan and other putative receptors in Gc-dependent attachment of the virus to surfaces of polarized cells. A series of experiments will assess the ability of several molecules or enzymatic treatments to prevent infection. (iii) To determine the sorting patterns of the viral glycoproteins and membrane proteins in polarized cells, and whether they are sorted individually or as aggregates of multiple proteins. Apical/basal sorting patterns of viral glycoproteins will be determined after infection of polarized cells with viruses with deletions in the glycoproteins, and in cells transfected or selected for expression of one or multiple glycoproteins. (iv) To determine the sequences of each of the proteins necessary for their functions in polarized cells, by peptide competitions and by mutagenesis of each glycoprotein.
| Tran, L C; Kissner, J M; Westerman, L E et al. (2000) A herpes simplex virus 1 recombinant lacking the glycoprotein G coding sequences is defective in entry through apical surfaces of polarized epithelial cells in culture and in vivo. Proc Natl Acad Sci U S A 97:1818-22 |
