The genome of all retroviruses, including human immunodeficiency virus type 1 (HIV-1), consists of two identical RNAs noncovalently linked near their 5' end. Dimerization of genomic RNA is thought to modulate several steps in the retroviral life cycle, such as recombination, translation, and encapsidation. We report the results of experiments designed to identify the 5' and 3' boundaries of the dimerization domain of the HIV-1 genome: (1) An HIV-1 RNA starting at nucleotide 252 or at other downstream positions (four tested) does not dimerize despite the inclusion of the whole of a previously proposed dimerization domain (nucleotides 295-401); (2) an RNA starting between nucleotides 242 and 249 (five positions tested) dimerizes to a variable extent depending on the starting position; (3) an RNA starting at nucleotide 233 or at other upstream positions (five tested) is fully or > 80% dimeric; (4) an RNA starting at nucleotide 1 but lacking the 233-251 or the 242-251 region is, respectively, fully monomeric or about 50% monomeric; (5) the 343-401 region contains two strings of G's (GGGGG367 and GGG384) that had been postulated to promote genome dimerization through the formation of guanine quartets. We have deleted the 379-401, 358-401, and 343-401 regions from otherwise dimeric RNAs without changing their ability to dimerize. We reach three conclusions: (1) a dimerization signal exists upstream of the major 5' splice donor (nucleotide 290); (2) the previously proposed downstream dimerization domain is insufficient to promote dimerization and has a 3' half that is not necessary to obtain fully dimeric RNAs; (3) the 5' boundary of the HIV-1 dimerization domain is located somewhere between nucleotides 233 and 242, and the 3' boundary is located no farther than at nucleotide 342, making it possible that the 5' and 3' boundaries of the HIV-1 dimerization domain are both located within the leader sequence. We speculate that the 248-270 or 233-285 region forms a hairpin that is the core dimerization domain of HIV-1 RNA.

The genome of all retroviruses consists of two identical RNAs noncovalently linked near their 5' end. Dimerization of genomic RNA is thought to modulate several steps in the retroviral life cycle, such as recombination, translation, and encapsidation. The kissing-loop model of HIV-1 genome dimerization [Laughrea, M., & Jetté, L. (1994) Biochemistry 33, 13464-13474; Skripkin et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 4945-4949] posits that the 248-270 region of the HIV-1 genome, by forming a hairpin and initiating dimerization through a loop-loop interaction, is the full or at least the core dimerization domain of HIV-1 RNA. Here, we show by nested deletion analysis that the 3' boundary of the HIV-1 dimerization domain is immediately downstream of hairpin 248-270 and that the isolated region 248-271 dimerizes at least as readily as longer RNAs. Among various HIV-1Lai RNA transcripts containing hairpin 248-270, all form two types of dimer, as is implicit in the kissing-loop model. The high-stability dimer resists semidenaturing conditions and the low-stability dimer cannot, which is consistent with the model. At physiological temperatures, low-stability dimers are usually formed, as if dimerization without nucleocapsid proteins corresponded to loop-loop interaction without switching from intra- to interstrand hydrogen bonding. Our results show that the 3' DLS (a sequence immediately 3' from the 5' splice junction and originally thought to be the dimerization domain of the HIV-1 genome) and adjacent nucleotides are not necessary for efficient dimerization of HIV-1Lai RNA at low and high ionic strength. Upstream of hairpin 248-270 exists another "DLS-like" sequence that we name 5' DLS: like the isolated 3' DLS, the isolated 5' DLS forms an apparently nonphysiological structure that can become substantially dimeric at high ionic strength.

A stem-loop termed the kissing-loop hairpin is one of the most highly conserved structures within the leader of human immunodeficiency virus type 1 (HIV-1) and chimpanzee immunodeficiency virus genomic RNA. Because it plays a key role in the in vitro dimerization of short HIV-1 RNA transcripts (M. Laughrea and L.

The kissing-loop domain (KLD) encompasses a stem-loop, named kissing-loop or dimerization initiation site (DIS) hairpin (nucleotides [nt] 248 to 270 in the human immunodeficiency virus type 1 strains HIV-1 Lai and HIV-1 Hxb2 ), seated on top of a 12-nt stem-internal loop called stem-loop B (nt 243 to 247 and 271 to 277). Destroying stem-loop B reduced genome dimerization by ϳ50% and proviral DNA synthesis by ϳ85% and left unchanged the dissociation temperature of dimeric genomic RNA. The most affected step of reverse transcription was plus-strand DNA transfer, which was reduced by ϳ80%. Deleting nt 241 to 256 or 200 to 256 did not reduce genome dimerization significantly more than the destruction of stem-loop B or the DIS hairpin. We conclude that the KLD is nonmodular: mutations in stem-loop B and in the DIS hairpin have similar effects on genome dimerization, reverse transcription, and encapsidation and are also "nonadditive"; i.e., a larger deletion spanning both of these structures has the same effects on genome dimerization and encapsidation as if stem-loop B strongly impacted DIS hairpin function and vice versa. A C258G transversion in the palindrome of the kissing-loop reduced genome dimerization by ϳ50% and viral infectivity by ϳ1.4 log. Two mutations, CGCG2613UUAA261 (creating a weaker palindrome) and a ⌬241-256 suppressor mutation, were each able to reduce genome dimerization but leave genome packaging unaffected.The kissing-loop domain (KLD) encompasses a stem-loop, named kissing-loop hairpin (nucleotides [nt] 248 to 270 in human immunodeficiency virus type 1 strain HIV-1 Lai and HIV-1 Hxb2 genomic RNA), seated on top of a short steminternal loop called stem-loop B (nt 243 to 247 and 271 to 277) (18). The apical loop of the kissing-loop hairpin contains an almost invariant hexameric autocomplementary sequence (ACS) (see reference 17 and references therein), also called a palindrome. The palindrome is seen as the dimerization initiation site (DIS) of genomic RNA (13, 15, 31); thus, the kissingloop hairpin is also called the DIS hairpin. The level of genomic RNA dimerization within isolated HIV-1 viruses is influenced by the DIS hairpin (6, 9, 17) and p55Gag processing (8).In the kissing-loop model of HIV-1 genome dimerization (13, 15, 31), stem-loop B has ill-defined roles (15, 17); one might be to properly orient the DIS hairpin within the covalently linked 9,000-nt-long tangle of secondary and tertiary structure (18). Experimentally, substantial deletions within stem-loop B or the DIS hairpin have identical impacts on viral infectivity and genomic RNA encapsidation (18). This raises the possibility that the KLD might be nonmodular, i.e., a highly integrated structure whereby stem-loop B and the DIS hairpin may have similar, if not identical, physiological impacts. To establish this, it is necessary to show that stem-loop B mutations inhibit genomic RNA dimerization and proviral DNA synthesis, two processes affected by the DIS hairpin (6,9,17,25).In this paper, we identify a crucial role of stem-loo...