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.

SummaryHuman immunodeficiency virus type 1 (HIV-1) Gag selects for and mediates genomic RNA (vRNA) encapsidation into progeny virus particles. The host protein, Staufen1 interacts directly with Gag and is found in ribonucleoprotein (RNP) complexes containing vRNA, which provides evidence that Staufen1 plays a role in vRNA selection and encapsidation. In this work, we show that Staufen1, vRNA and Gag are found in the same RNP complex. These cellular and viral factors also colocalize in cells and constitute novel Staufen1 RNPs (SHRNPs) whose assembly is strictly dependent on HIV-1 expression. SHRNPs are distinct from stress granules and processing bodies, are preferentially formed during oxidative stress and are found to be in equilibrium with translating polysomes. Moreover, SHRNPs are stable, and the association between Staufen1 and vRNA was found to be evident in these and other types of RNPs. We demonstrate that following Staufen1 depletion, apparent supraphysiologic-sized SHRNP foci are formed in the cytoplasm and in which Gag, vRNA and the residual Staufen1 accumulate. The depletion of Staufen1 resulted in reduced Gag levels and deregulated the assembly of newly synthesized virions, which were found to contain several-fold increases in vRNA, Staufen1 and other cellular proteins. This work provides new evidence that Staufen1-containing HIV-1 RNPs preferentially form over other cellular silencing foci and are involved in assembly, localization and encapsidation of vRNA.

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.