The primary goal of this project is the analysis of the strategies of gene expression utilized by human retroviral genomes and of the functions of the different regions of these genomes. Our studies have focused on two classes of human retroviral genomes; the exogenous human retrovirus associated with the Acquired Immunodeficiency Syndrome (AIDS RV) and endogenous human type-C retroviral sequences related to murine leukemia viruses. The pattern of gene expression of the AIDS RV during acute infection has been examined by Northern blot hybridization. Viral specific RNAs were detected early in the course of infection, three days prior to the appearances of reverse transcriptase activity in culture supernatant. We have identified five major species of viral RNA of 9.1, 5.5, 5.0, 4.3 and 1.8-2.0 kb. The 9.1 kb RNA hybridized to probes from all regions of the virus and thus represents the full-length viral transcript as well as putative gag-pol mRNAs. The 5.5 and 5.0 kb RNAs hybridized to LTR and """"""""A"""""""" region probes as well as probes 3' to """"""""A"""""""". The 4.3 kb RNA contained env sequences and the 1.8-2.0 kb family of mRNAs hybridized to the """"""""B"""""""" region. Interestingly, the 5.5 kb RNA was not detected in cells producing defective virions. The expression of human endogenous retroviral sequences has been observed in a variety of human cell types. Human pol sequences are expressed in hematopoietic cells and appear to be inducible in an erythroleukemia line following TPA treatment. Human melanoma cells contain abundant amounts of 3.6 and 2.2 kb """"""""LTR-only"""""""" RNAs. Size-fractionation of the melanoma RNA has been carried out in preparation for cDNA cloning. The function of a cloned human U3 LTR (derived from a cDNA clone) is being assessed by ligation of the human LTR to human ras genes. These plasmids will be tested for their transforming potential in NIH 3T3 cells.
