The human immunodeficiency virus type 1 (HIV-1) RNA genome is flanked by a repeated sequence (R) that is required for HIV-1 replication. The first 57 nucleotides of R form a stable stem-loop structure called the transactivation response element (TAR) that can interact with the virally encoded transcription activator protein, Tat, to promote high levels of gene expression. Recently, we demonstrated that TAR is also important for efficient HIV-1 reverse transcription, since HIV-1 mutated in the upper stem-loop of TAR showed a reduced ability both to initiate and to complete reverse transcription. We have analyzed a series of HIV-1 mutant viruses to better defined the structural or sequence elements required for natural endogenous reverse transcription and packaging of virion RNA. Our results indicate that the requirement for TAR in reverse transcription is conformation dependent, since mutants with mutations that alter the upper stem-loop orientation are defective for reverse transcription initiation and have minor defects in RNA packaging. In contrast, TAR mutations that allowed the formation of alternative upper stem-loop structure greatly reduced RNA packaging but did not affect reverse transcription efficiency. These results are consistent with direct involvement of the upper stem-loop structure in packaging of genomic RNA and suggest that the TAR RNA stem-loop from nucleotide ؉18 to ؉42 interacts with other components of the reverse transcription initiation complex to promote efficient reverse transcription.The human immunodeficiency virus type 1 (HIV-1) RNA genome can form numerous RNA structures, many of which regulate virus replication. Viral RNA structures are required for many processes, including transcription by RNA polymerase II, polyadenylation of viral mRNA, transport of singly spliced and unspliced viral mRNA, viral genomic RNA dimerization and packaging, and reverse transcription. For example, the TAR element, which comprises the first 57 nucleotides (nt) of the viral transcript, can form a stable stem-loop structure. The Tat protein and cellular kinases, which are recruited by Tat, can bind to the TAR stem-loop to enable efficient transcription by RNA polymerase II (for reviews, see references 12a and 24a). Another example is the HIV-1 RNA packaging signal, which includes a series of RNA stem-loop structures (designated SL1, SL3, and SL4) which flank the 5Ј major splice donor site (3,9,19,27) and bind to the nucleocapsid domain of the pr55 Gag protein to promote packaging of unspliced HIV-1 transcripts into virions (2, 5, 12, 29). Another regulatory viral RNA structure is formed by sequences flanking the primer binding site in conjunction with cellular tRNA 3Lys . Interactions between an A-rich loop upstream of the virus primer binding site and the anticodon loop of tRNA 3 Lys are reported to be required for the formation of an efficient reverse transcription initiation complex (21,23,24).It has long been appreciated that a single viral RNA structure can have effects on multiple steps in the repl...

Tat expression is required for efficient human immunodeficiency virus type 1 (HIV-1) reverse transcription. In the present study, we generated a series of 293 cell lines that contained a provirus with atat gene deletion (Δtat). Cell lines that contained Δtat and stably transfected vectors containing either wild-type tat or a number of tat mutants were obtained so that the abilities of these tat genes to stimulate HIV-1 gene expression and reverse transcription could be compared. tat genes with mutations in the amino terminus did not stimulate either viral gene expression or HIV-1 reverse transcription. In contrast, tat mutants in the activation, core, and basic domains of Tat did not stimulate HIV-1 gene expression but markedly stimulated HIV-1 reverse transcription. No differences in the levels of virion genomic RNA or tRNA3 Lys were seen in the HIV-1 Δtat viruses complemented with either mutant or wild-type tat. Finally, overexpression of the Tat-associated kinases CDK7 and CDK9, which are involved in Tat activation of HIV-1 transcription, was not able to complement the reverse transcription defects associated with the lack of a functionaltat gene. These results indicate that the mechanism by which tat modulates HIV-1 reverse transcription is distinct from its ability to activate HIV-1 gene expression.

The tat gene is required by HIV-1 for efficient reverse transcription and this function of Tat can be distinguished from its role in transcription by RNA polymerase II using tat point mutations that abrogate each function independently. The mechanism of Tat's role in reverse transcription, however, is not known, nor is it known whether this role is conserved among trans-activating factors in other retroviruses. Here we examine the abilities of heterologous viral trans-activating proteins from jembrana disease virus (jTat), HIV-2 (Tat2), and equine infectious anemia virus (eTat) to substitute for HIV-1 Tat (Tat1) and restore reverse transcription in HIV-1 carrying an inactivated tat gene. Natural endogenous reverse transcription assays showed that trans-activators from some retroviruses (Tat2 and jTat, but not eTat) could substitute for Tat1 in complementation of HIV-1 reverse transcription. Finally, we show that Y47 is critical for Tat1 to function in reverse transcription, but not HIV-1 gene expression. We mutated the homologous position in jTat to H62Y and found it did not improve its ability to stimulate reverse transcription, but an H62A mutation did inhibit jTat complementation. These data highlight the finding that the role of Tat in reverse transcription is not related to trans-activation and demonstrate that other tat genes conserve this function.