The diploid genome of all retroviruses is made of two homologous copies of RNA intimately associated near their 5' end, in a region called the dimer linkage structure. Dimerizatlon of genomic RNA is thought to be important for crucial functions of the retroviral life cycle (reverse transcription, translation, encapsidation). Previous in vitro studies mapped the dimer linkage structure of human immunodeficiency virus type 1 (HIV-1) in a region downstream of the splice donor site, containing conserved purine tracts that were postulated to mediate dimerization, through purine quartets. However, we recently showed that dimerization of HIV-1 RNA also involves sequences upstream of the splice donor site. Here, we used chemical modification interference to identify nucleotides that are required in unmodified form for dimerization of a RNA fragment containing nucleotides A ubiquitous property of retroviruses is that their genonme consists of two homologous copies of single-stranded RNA (1). Electron microscopy showed that these two RNAs are joined together in an apparent parallel orientation by a structure called the dimer linkage structure (DLS) located near their 5' end (2-5). Dimerization of genomic RNA is considered to control several essential steps of the retroviral life cycle. First, it was proposed to act as a positive signal for encapsidation (6,7). Second, it was suggested to downregulate the translation ofthe gag gene in Rous sarcoma virus (RSV) and human immunodeficiency virus type 1 (HIV-1) (8, 9). Third, the dimeric nature of the retroviral genome is thought to be of importance in the process of reverse transcription and recombination since it may account for the first strand transfer and template switching during proviral DNA synthesis (10-14). Therefore, dimerization of genomic RNA most likely represents a potential target for the design of antiviral drugs against HIV and other retroviruses.However, the process of dimerization is still poorly understood. Dimerization of synthetic fragments containing the (29). Again, probing data indicate that the same structure is found in synthetic RNA fragments (9,30) and in genomic RNA extracted from infected cells (29).In HIV-1, several reports showed that a RNA region of -100 nucleotides located downstream of the splice donor (SD) site is able to dimerize in vitro (6,(21)(22)(23)(24). This region that includes important components of the packaging signal (29,31,32) was assumed to contain the putative DLS. However, the mechanism of dimerization is still a subject of controversy. In an initial study, we proposed that polypurine tracts, the only common motifs found in the putative dimerization-encapsidation region of most retroviral RNAs, may be involved in the dimerization process through the formation of quartets involving both guanines and adenines (21). This concept has been disputed by recent reports suggesting that dimers formed with short RNA restricted to the postulated DLS are stabilized by quartets containing only guanines (23,24). However, the di...

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Initiation of RNA‐dependent DNA synthesis by retroviral reverse transcriptases is generally considered as unspecific. In the case of human immunodeficiency virus type 1 (HIV‐1), the natural primer is tRNA3Lys. We recently found evidence of complex interactions between tRNA3Lys and HIV‐1 RNA that may be involved in the priming process. In this study, we compare the ability of natural and unmodified synthetic tRNA3Lys and 18mer oligoribo‐ and oligodeoxyribonucleotides complementary to the viral primer binding site to initiate replication of HIV‐1 RNA using either homologous or heterologous reverse transcriptases. We show that HIV‐1 RNA, HIV‐1 reverse transcriptase and primer tRNA3Lys form a specific initiation complex that differs from the unspecific elongation complex formed when an oligodeoxyribonucleotide is used as primer. Modified nucleosides of tRNA3Lys are required for efficient initiation and transition to elongation. Transition from initiation to elongation, but not initiation of reverse transcription itself, is facilitated by extended primer‐template interactions. Elongation, but not initiation of reverse transcription, is inhibited by Mn2+, which further differentiates these two different functional states of reverse transcriptase. These results define initiation of reverse transcription as a target to block viral replication.