In vivo gene therapy applications will be enhanced if viral vectors can be targeted to cells where transgene expression is desired. Herpes simplex virus type 1 (HSV-1) has significant potential utility as a powerful gene vector system, but its broad host range limits its general clinical applicability. Accordingly, the overall goal of this research is to develop suitable methods for targeting infection by directing virus attachment to novel receptors and contribute to a fundamental understanding of virus attachment and entry. To this end, we will attempt to (i) alter the natural binding functions of glycoproteins C, B, and D with N-terminal addition of novel receptor-binding ligands, and (ii) explore further the utility of endosomal release of vector using internalizing receptors in combination with chloroquine. Specifically we propose to: (I) target infection via gC by evaluating the ability of an HS/HveA (HVEM) binding deficient (rid1) gC:EPO (erythropoietin) recombinant virus to infect CHO cells bearing the EPO receptor alone or in combination with either chloroquine or the gD-specific HveC receptor; (II) target infection via gB by evaluating the ability of an HS/HveA binding deficient virus carrying a gB:bungarotoxin (BTX) chimeric glycoprotein to infect CHO cells expressing the a7 subunit of the acetylcholine receptor either alone or in combination with HveC; (III) target infection via gD by constructing HveA-binding deficient viruses in which (i) the BTX sequence replaces N-terminal residues 6-24 and evaluating the ability of the gD:BTX viruses to infect CHO cells expressing the a7 receptor or in which (ii) a random set of peptide sequences replace residues 6-24 (created in an infectious HSV-BAC, bacterial artificial chromosome) and selecting from the library of progeny viruses produced by transfection those that are infectious for refractory CHO cells; and (IV) target infection via gD defective for recognition of HveA and HveC by constructing gD mutants which can mediate infection via HveA but not HveC using two strategies: (i) saturation mutagenesis of residues 216-234 or 28-70 using the HSV:BAC system and selection of recombinants on CHO-HveA cells following neutralization with soluble HveC receptor, and (ii) replacement of residues 28-70 with random peptide sequences and selection of mutants which rescue entry into CHO-HveA but not CHO-HveC cells. HveA binding will then be disrupted by replacing N-terminal residues 6-24 with BTX or novel CHO binding ligands identified in Aim III and recombinant virus mutants selected for infection of CHO-a7 or CHO cells; and (V) study the mechanism of infection using VSV-G and chloroquine by evaluating the infectivity of gD deficient VSV-G recombinant vectors carrying other altered or deleted HSV glycoproteins.