Two obligatory DNA strand transfers take place during reverse transcription of a retroviral RNA genome. The first strand transfer is facilitated by terminal repeat (R) elements in the viral genome. This strand-transfer reaction depends on base pairing between the cDNA of the 59R and the 39R. There is accumulating evidence that retroviral R regions contain features other than sequence complementarity that stimulate this critical nucleic acid hybridization step. The R region of the human immunodeficiency virus type 1 (HIV-1) is relatively extended (97 nt) and encodes two wellconserved stem-loop structures, the TAR and poly(A) hairpins. The role of these motifs was studied in an in vitro strand-transfer assay with two separate templates, the 59R donor and the 39R acceptor, and mutants thereof. The results indicate that the upper part of the TAR hairpin structure in the 59R donor is critical for efficient strand transfer. This seems to pose a paradox, as the 59R template is degraded by RNase H before strand transfer occurs. We propose that it is not the RNA hairpin motif in the 59R donor, but rather the antisense motif in the ssDNA copy, which can also fold a hairpin structure, that is critical for strand transfer. Mutation of the loop sequence in the TAR hairpin of the donor RNA, which is copied in the loop of the cDNA hairpin, reduces the transfer efficiency more than fivefold. It is proposed that the natural strand-transfer reaction is enhanced by interaction of the anti-TAR ssDNA hairpin with the TAR hairpin in the 39R acceptor. Base pairing can occur between the complementary loops ("loop-loop kissing"), and strand transfer is completed by the subsequent formation of an extended RNA-cDNA duplex.

The 5' and 3' ends of HIV-1 transcripts are identical in sequence. This repeat region (R) folds a stem-loop structure that is termed the poly(A) hairpin because it contains polyadenylation or poly(A) signals: the AAUAAA hexamer motif, the cleavage site and part of the GU-rich downstream element. Obviously, HIV-1 gene expression necessitates differential regulation of the two poly(A) sites. Previous transfection experiments indicated that the wild-type poly(A) hairpin is slightly inhibitory to the process of polyadenylation, and further stabilization of the hairpin inhibited polyadenylation completely. In this study, we tested wild-type and mutant transcripts with poly(A) hairpin structures of differing thermodynamic stabilities for the in vitro binding of polyadenylation factors. Mutant transcripts with a destabilized hairpin efficiently bound the polyadenylation factors, which were provided either as purified proteins or as nuclear extract. The RNA mutant with a stabilized hairpin did not form this 'poly(A) complex'. Additional mutations that repair the stability of this hairpin restored the binding capacity. Thus, an inverse correlation was measured between the stability of the poly(A) hairpin and its ability to interact with polyadenylation factors. The wild-type HIV-1 transcript bound the polyadenylation factors suboptimally, but full activity was obtained in the presence of the USE enhancer element that is uniquely present upstream of the 3' poly(A) site. We also found that sequences of the HIV-1 leader, which are uniquely present downstream of the 5' poly(A) site, inhibit formation of the poly(A) complex. This inhibition could not be ascribed to a specific leader sequence, as we measured a gradual loss of complex formation with increasing leader length. We will discuss the regulatory role of RNA structure and the repressive effect of leader sequences in the context of differential HIV-1 polyadenylation.

The untranslated leader region of the human immunodeficiency virus (HIV) RNA genome contains multiple hairpin motifs. The repeat region of the leader, which is reiterated at the 3 end of the RNA molecule, encodes the well-known TAR hairpin and a second hairpin structure with the polyadenylation signal AAUAAA in the single-stranded loop [the poly(A) hairpin]. The fact that this poly(A) stem-loop structure and its thermodynamic stability are well conserved among HIV and simian immunodeficiency virus isolates, despite considerable divergence in sequence, suggests a biological function for this RNA motif in viral replication. Consistent with this idea, we demonstrate that mutations that alter the stability of the stem region or delete the upper part of the hairpin do severely inhibit replication of HIV type 1. Whereas destabilizing mutations in either the leftor right-hand side of the base-paired stem interfere with virus replication, the double mutant, which allows the formation of new base pairs, replicates more rapidly than the two individual virus mutants. Upon prolonged culturing of viruses with an altered hairpin stability, revertant viruses were obtained with additional mutations that restore the thermodynamic stability of the poly(A) hairpin. Transient transfection experiments demonstrated that transcription of the proviral genomes, translation of the viral mRNAs, and reverse transcription of the genomic RNAs are not affected by mutation of the 5 poly(A) hairpin. We show that the genomic RNA content of the virions is reduced by destabilization of this poly(A) hairpin but not by stabilization or truncation of this structure. These results suggest that the formation of the poly(A) hairpin structure at the 5 end of the genomic RNA molecule is necessary for packaging of viral genomes into virions and/or stability of the virion RNA.