Efficient packaging of HIV-1 genomes via recognition of its adenosine-rich content by a heterologous RNA-binding domain

The HIV-1 genome (gRNA) has an unusually biased nucleotide content and is rich in adenosines. Selective packaging of the gRNA is thought to be driven by specific binding of the nucleocapsid (NC) domain of the viral Gag protein to the packaging signal ({Psi}) in the host cell cytosol. However, deletion of regions within {Psi} reduces--but does not completely abolish--genome packaging. To probe whether another feature of the gRNA may contribute to the selective gRNA packaging process, we replaced NC with heterologous RNA-binding domains (RBDs) with distinct RNA-binding properties. Surprisingly, despite disparate RNA binding specificities, all Gag-RBD chimeras successfully recruited the gRNA to the plasma membrane, suggesting that the initial gRNA recognition in the cytosol is not rate limiting. Notwithstanding, many chimeras exhibiting G/C binding specificity were arrested at the assembly stage. Only the Gag-SRSF5 chimera, which multimerized efficiently on adenosine-rich sequences on the gRNA, assembled efficiently and packaged gRNA at near wild-type levels. Importantly, rationally designed mutations that altered the A/G-rich binding specificity of Gag-SRSF5 decreased genome encapsidation efficiency. Furthermore, many Gag chimeras displayed potent dominant negative activities, highlighting NC functions as a targetable step in virus replication. Together, our findings reveal an unexpected aspect of the HIV-1 gRNA, its biased nucleotide content, as a key driver of selective genome packaging. SIGNIFICANCEHow HIV-1 selectively packages its genome (gRNA) into virions is poorly understood. To probe this, we replaced the viral nucleocapsid (NC) protein with heterologous RNA-binding domains (RBDs) from cellular hnRNP and SR protein families. Remarkably, despite their distinct RNA-binding specificities, all Gag-RBD chimeras successfully recruited the gRNA to the plasma membrane, suggesting that the initial gRNA recognition is not rate limiting. Only the Gag-SRSF5 chimera packaged gRNA efficiently which correlated with its capacity to multimerize on adenosine-rich sequences on the gRNA. Notably, several Gag chimeras exhibited strong dominant negative effects, underscoring NC functions as a targetable step in virus replication. Together, these findings uncover that the biased nucleotide content of the HIV-1 gRNA facilitates its selective packaging into virus particles.