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

Significance Dimerization of the RNA genome is a key event in HIV-1 virion assembly and has a strong impact in viral replication and evolution. Packaging the dimeric genome allows frequent recombination to rescue genetic information in damaged RNAs and to generate variants that can evade the host immune response or resist antiviral treatments. Furthermore, genome packaging is regulated by recognition of dimeric RNA. Our studies demonstrate that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane, often early during the assembly process, and that Gag protein is required for maintenance of the RNA dimer. These studies address the timing, location, and partners involved in RNA dimerization, an important process for HIV-1 replication. 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.