Vuong, H. R.; Zhou, Q.; Lesko, S.; Tenneti, K.; Davis, K.; Scott, S.; Boodwa-Ko, D.; Eschbach, J. E.; Gopal, K.; Porter, J.; Wang, Y.; Mohammed, S.; Lee, N.; Telesnitsky, A.; Sherer, N. M.; Kutluay, S. B.

Selective packaging of a dimeric HIV-1 genome (gRNA) is thought to be driven by specific binding of the nucleocapsid (NC) domain of viral Gag protein to the packaging signal (psi) in the host cell cytosol. Through replacement of NC with heterologous RNA-binding domains (RBDs) with distinct RNA-binding properties, we show that the biased adenosine-rich nucleotide content of the gRNA facilitates its selective packaging. Despite disparate RNA binding specificities, all Gag-RBD chimeras successfully recruited the gRNA to the plasma membrane, but many were arrested at the assembly stage. Only the Gag-SRSF5 chimera, which multimerized efficiently on adenosine-rich sequences on the gRNA, packaged gRNA at near wild-type levels. 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.