HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein

Retroviruses package a dimeric genome comprising two copies of the viral RNA. Each RNA contains all of the genetic information for viral replication. Packaging a dimeric genome allows the recovery of genetic information from damaged RNA genomes during DNA synthesis and promotes frequent recombination to increase diversity in the viral population. Therefore, the strategy of packaging dimeric RNA affects viral replication and viral evolution. Although its biological importance is appreciated, very little is known about the genome dimerization process. HIV-1 RNA genomes dimerize before packaging into virions, and RNA interacts with the viral structural protein Gag in the cytoplasm. Thus, it is often hypothesized that RNAs dimerize in the cytoplasm and the RNA–Gag complex is transported to the plasma membrane for virus assembly. In this report, we tagged HIV-1 RNAs with fluorescent proteins, via interactions of RNA-binding proteins and motifs in the RNA genomes, and studied their behavior at the plasma membrane by using total internal reflection fluorescence microscopy. We showed that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane. Dynamic interactions occur among HIV-1 RNAs, and stabilization of the RNA dimer requires Gag protein. Dimerization often occurs at an early stage of the virus assembly process. Furthermore, the dimerization process is probably mediated by the interactions of two RNA–Gag complexes, rather than two RNAs. These findings advance the current understanding of HIV-1 assembly and reveal important insights into viral replication mechanisms.All viruses must encapsidate their genomes into virions to ensure that their genetic information is transferred to the new target cells. In most, if not all, retroviruses, the virion RNA genomes are dimeric, although each RNA encodes all of the genetic information required for replication. Most HIV-1 particles contain two copies of genomes (1), indicating that RNA encapsidation is a highly regulated process. This regulation is achieved by recognizing a dimeric RNA, and not by packaging a certain mass of viral genome (2).Our previous studies showed that HIV-1 RNA dimerization is a critical step in viral RNA genome packaging and virus assembly and that the two copies of copackaged RNA genomes are dimerized before encapsidation (1, 3, 4). The dimerization initiation signal (DIS), a 6-nt palindromic sequence located at the 5′ UTR of the HIV-1 RNA genome (5), most likely initiates the interaction between two HIV-1 RNA genomes (3, 4). When two HIV-1 RNAs contain similar sequences including the same DIS, they are copackaged efficiently at a rate similar to that predicted from random distribution (1, 2). In contrast, when two HIV-1 RNAs contain discordant palindromic sequences that cannot form perfect base pairing, they are not copackaged efficiently into the same viral particle (1, 2). The ability of RNA genomes from different HIV-1 variants to dimerize has important biological consequences. For example, inefficient copackaging is known to be a major barrier for intersubtype HIV-1 recombination (4). Although DIS plays a key role in RNA dimerization, virion RNAs isolated from mutants with DIS deletions remained dimeric, suggesting that other cis-acting element(s) are also involved in the dimerization (6).Despite the importance of RNA dimerization for HIV-1 replication, many aspects of this process are unknown, including the location at which dimerization occurs. Previously, we showed that RNA dimerization leading to HIV-1 genome packaging occurs after viral RNA is exported from the nucleus (7). The viral protein Gag is known to have chaperone activity (8). Additionally, biochemical experiments showed that HIV-1 Gag can interact with viral RNAs in the cytoplasm (9, 10). Thus, it is often hypothesized that two copies of HIV-1 RNAs dimerize in the cytoplasm and that this dimeric RNA is complexed with Gag and travels to the plasma membrane (7, 11–15), the major assembly site for virus assembly. The assembly of HIV-1 RNA and Gag was demonstrated in an elegant study using total internal reflection fluorescence (TIRF) microscopy (14), which illuminates a shallow volume near the glass/cell interface and is ideal for studying events near the plasma membrane (16). However, it was difficult to address the monomeric/dimeric state of the viral RNA in this previous study because the RNA was labeled with a single type of fluorescent protein.In the present study, we sought to delineate the location at which HIV-1 RNA dimerization occurs, which leads to genome encapsidation, and whether Gag is required for RNA dimerization. We used a previously described method to label HIV-1 RNA with fluorescent proteins through interactions of sequence-specific RNA-binding proteins. We engineered HIV-1 genomes to contain RNA stem-loops that are recognized by the Escherichia coli BglG protein or the bacteriophage MS2 coat protein; because these sequences are located in the pol gene, they are present only in full-length unspliced HIV-1 RNAs. When introduced into human cells, these constructs express full-length RNAs that can serve as templates for the translation of Gag proteins and as genomes in the viral particles. Most (>90%) of the particles contain RNA genomes, indicating that the full-length viral RNAs derived from these constructs are efficiently packaged. Furthermore, RNAs derived from different constructs can dimerize and copackage at a rate close to random distribution (1), consistent with the genetic analyses from recombination studies (4, 17, 18). By using this method, we were able to detect HIV-1 RNA with single-RNA-molecule sensitivity (1) and tracked HIV-1 RNA movement in the cytoplasm by using live-cell imaging (19). In this report, we tagged two HIV-1 RNAs and Gag, each with a different fluorescent protein, and studied the RNA:RNA and RNA:Gag interactions on the plasma membrane. We found that HIV-1 RNA dimerizes on the plasma membrane and that Gag protein is required for stabilization of the dimer.