Deletions that eliminated the 5'(HIVDel-5'), middle (HIVDel- MID) and 3'(HIVDel-3') thirds of the U5 region of the HIV (LTR) functioning in the context of an infectious molecular clone were studied for their effects on virus expression (transient transfection) and infectivity (T cell lines and PBLs). All three U5 mutants directed the synthesis of progeny virus particles as monitored by reverse transcriptase activity released from transfected HeLa cells. The HIVDel-MID had virtually no effect on viral replication. When human PBL and CEM cells were infected with HIVDel-5' virus, no viral replication was observed throughout 50 days of culture. Studies of HIVDel-5' particles revealed a packaging defect characterized by a 10-fold reduction in virion-associated RNA. The HIVDel-3' was also not infectious; no unintegrated viral DNA was detected in cells infected with this mutant, suggesting a defect in reverse transcription. However, following infection of CEM cells with HIVDel-3, virus replication was detected 30 days post infection in 1 of 4 independent experiments. This virus exhibited infection kinetics indistinguishable from wild type (WT) virus. PCR analysis revealed that the original 26 nucleotides (nt) deletion associated with HIVDel-3' had been extended an additional 19 nts in the 5' direction (towards the R region). The enlargement of the original deletion in HIVDel-3' restored infectivity. The location(s) of the HIVDel-3' mutation and its revertant will be discussed in the context of the initiation of reverse transcription and the adjacent tRNALys-3 primer binding site.

Cell Line Hela Cells Human *HIV Long Terminal Repeat Kinetics RNA-Directed DNA Polymerase/GENETICS *Sequence Deletion Transcription, Genetic/*GENETICS Virus Replication/GENETICS ABSTRACT

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