FURTHER CHARACTERIZATION OF HIV RNA-SYNTHESIS EARLY AFTER CELL-TO-CELL TRANSMISSION INFECTION

Using a one-step model for cell-to-cell transmission of HIV infection we have identified two distinct phases of HIV RNA. synthesis. The firs t phase (4 h-12 h p.i.) was marked by an increase in only the full-len gth 9 kb RNA, while the second phase (24 h p.i. onwards) comprised a s ignificant increase in the levers of all three species of viral RNA. W e now report that while the continual presence of actinomycin D at 50 mu g/ml abolished all detectable viral nucleic acid synthesis when vir us donor H3B cells were pre-treated with 50 mu g/ml of actinomycin D ( AmD), washed free of unbound drug (a procedure which inhibited >99% of total cellular RNA transcription) then mixed with untreated recipient Hut78 cells, normal amounts of full length linear unintegrated viral DNA were produced and the first phase of viral RNA transcription was u naffected. Similarly, when both the virus donor cells and recipient ce lls were arrested in the late G(1) phase of the cell cycle by aphidico lin and then mixed, linear unintegrated viral DNA was the major viral DNA species produced. The appearance of circular viral DNA and progeny virus was inhibited, but the first phase of induced viral RNA synthes is was unaffected. When AZT was added at 2 h or 4 h after cell-cell mi xing, the level of 9 kb RNA detected was significantly lower, correspo nding to reduction in the level of viral DNA. These and previous resul ts indicate that the template for the first phase of viral RNA synthes is was likely to be newly synthesized, linear unintegrated viral DNA a nd not pre-existing proviral DNA present in the donor cells. Taken tog ether, our results suggest that there exists a yet to be fully charact erized pathway of concurrent viral DNA and RNA synthesis early after c ell to cell transmission of HIV infection.