This research initiates studies to characterize a newly isolated human gene currently designated human fetal lung cDNA A27 (hfI-A27). It is one of three novel human genes isolated in a functional screen of a human fetal lung cDNA library. We looked for genes that transfer susceptibility for HSV entry to porcine cells that lack a receptor for entry of HSV. The nucleotide sequence of hfl-A27 contains an open reading frame that produces in vitro translated proteins of 37kD and 28kD. Its gene product mediates HSV-1 entry when expressed transiently in porcine cells. Goals are to develop reagents and obtain fundamental information about the uncharacterized hfl- A27 gene product required for long term studies of its structure and functions. Priorities are preparation of antibody and development of reliable systems to express full length or truncated proteins.
Specific aims are to: (1) Determine tissue distribution of mRNA and express hfI-A27 in an E.coli or baculovirus system for antibody production and studies of structure. (2) Express the gene stably in porcine or human cell lines under control of constitutive or inducible promoters. (3) Determine protein size, cellular location and oligomeric structure. (4) Initiate studies to determine function of the hfI-A27 gene product in HSV entry and its natural role(s) for human cells. The long-term objective is to understand how HSV infection involves multiple cellular protein receptors and viral envelope proteins for attachments that lead to membrane fusion at neutral pH. Based on results of these initial experiments with hfI-A27 and using cell lines and antibodies developed, the A27 protein will be studied in the context of other human receptor proteins for HSV entry and for its natural human cell function(s). Experimental results combined with computer analyses of the predicted protein sequence should allow formation of hypotheses to drive further studies of A27. The research is significant to understanding viral cell interactions at entry and to defining natural functions of one new human gene and its uncharacterized protein product. ? ?
