EFFECTS OF THE TAT AND NEF GENE-PRODUCTS OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 (HIV-1) ON TRANSCRIPTION CONTROLLED BY THE HIV-1 LONG TERMINAL REPEAT AND ON CELL-GROWTH IN MACROPHAGES

The RAW264 murine macrophage cell line was used as a model to examine the role of the tat and nef gene products in the transcription regulat ion of the human immunodeficiency virus type 1 (HIV-1) long terminal r epeat (LTR) in macrophages. Contrary to claims that the activity of th e HIV-1 LTR responds poorly in rodent cells to trans activation by the viral tat gene product, cotransfection of RAW264 cells with a tat exp ression plasmid in transient transfection assays caused a >20-fold inc rease in reporter gene expression that was inhibited by mutations in t he TAR region. RAW264 cells stably transfected with the tat plasmid di splayed similarly elevated HIV-1 LTR-driven reporter gene activity. By contrast to previous reports indicating a negative role for nef in HI V transcription, cotransfection of RAW264 cells with a nef expression plasmid trans activated the HIV-1 LTR driving either a chloramphenicol acetyltransferase or a luciferase reporter gene. The action of nef wa s specific to the LTR, as expression of nef had no effect on the activ ity of the simian virus 40, c-fms, urokinase plasminogen activator, or type 5 acid phosphatase promoter. trans-activating activity was also manifested by a frameshift mutant expressing only the first 35 amino a cids of the protein. The effects of nef were multiplicative with those of tat gene product and occurred even in the presence of bacterial li popolysaccharide, which itself activated LTR-directed transcription. E xamination of the effects of selected mutations in the LTR revealed th at neither the KB sites in the direct repeat enhancer nor the TAR regi on was required as a cis-acting element in nef action. The action of n ef was not species restricted; it was able to trans activate in the hu man monocyte-like cell line Mono Mac 6. The presence of a nef expressi on cassette in a neomycin phosphotransferase gene expression plasmid g reatly reduced the number of G418-resistant colonies generated in stab le transfection of RAW264 cells, and many of the colonies that were fo rmed exhibited very slow growth. The frameshift mutant was also active in reducing colony generation. Given the absence of any effect of the frameshift mutation on nef function, its actions on macrophage growth and HIV transcription are discussed in terms of the role of the N-ter minal 30 amino acids and of stable secondary structures in the mRNA.