RAPID COMMUNICATION The First Strand Transfer during HIV-1 Reverse Transcription Can Occur either Intramolecularly or Intermolecularly

Reverse transcription is a complicated process that involves at least two cDNA transfer reactions to produce a full-length copy DNA of the retroviral RNA genome. Because one retrovirus particle contains two identical genomic RNA molecules, the transfers can occur in an intramolecular or intermolecular manner. The mechanism of the first transfer step (minus-strand strong-stop cDNA transfer) has been studied previously in detail in transduction experiments with spleen necrosis virus vectors containing genetic markers. Different results have been reported with respect to the type of strand transfer mechanism. In this study, we analyzed the first strand transfer for human immunodeficiency virus type 1 (HIV-1). Two genetically marked genomes were copackaged into virions and reverse transcription was initiated within these particles upon permeabilization by NP-40 and addition of dNTPs. To test whether intrastrand or interstrand transfer had occurred, the cDNA products of this endogenous reverse transcription reaction were extracted from the virions and analyzed for the presence of restriction enzyme recognition sites provided by the genetic markers. The results of this analysis demonstrated that the first DNA transfer reaction occurs in a random manner, with approximately the same contribution of intrastrand and interstrand transfers. The ability to perform intermolecular strand transfer was lost upon extraction of the dimeric RNA template from the virion particle. © 1998 Academic Press Reverse transcription converts a retroviral RNA genome into double-stranded DNA that is termed the provirus (1, 2). Retroviruses are able to synthesize a copy DNA that is larger than the RNA template, thereby reconstituting sequences that are located upstream of the transcriptional start site. This upstream region is critically important for subsequent transcription of the integrated provirus as it encodes the transcriptional promoter and enhancer signals. The production of a DNA copy that is larger than the template RNA is mediated by a complicated process that involves two cDNA transfer reactions ((3), reviewed in (4)). Reverse transcription is initiated by a cellular tRNA primer bound to the complementary primer-binding site (PBS) near the 5Ј end of the viral RNA genome. An intermediate product is synthesized, termed the minus-strand strong-stop cDNA, which includes the R element present at the extreme 5Ј end of the retroviral genome. Upon removal of the template RNA sequences by the RNaseH activity of the RT enzyme, this cDNA intermediate can transfer to the second copy of the R element that is present at the extreme 3Ј end of the genome. Subsequent elongation of minus-strand synthesis, initiation of plus-strand synthesis, and a second DNA transfer step are required to produce a full-length DNA provirus (reviewed in (5)). Because two identical RNA molecules are present as a noncovalently linked dimer in a retrovirus particle, the first DNA transfer reaction (also termed minus-strand or strong-stop cDNA transfer) can occur in an intramolecular or intermolecular manner. This mechanism has been studied in detail in transduction experiments with spleen necrosis virus (SNV) vectors that contain genetic markers. Dissimilar results have been reported with respect to the type of strand transfer. It was initially reported that the first strand transfer reaction occurred exclusively between the two packaged RNA genomes in a pure interstrand manner (6), but another group observed both intrastrand and interstrand transfers (7). Furthermore, there is some evidence that the type of strand transfer may differ among viral subpopulations (8), and a similar observation has been reported for recombination processes in general (9). In this study, we have analyzed the first strand transfer reaction for the human immunodeficiency virus type 1 (HIV-1) by a more direct assay. Two different RNA genomes were copackaged into virion particles and reverse transcription was initiated within the extracellular virions by permeabilization and addition of dNTPs. The cDNA products of this endogenous re-1 To whom correspondence and reprint requests should be addresse