Plasma Membrane Anchoring and Gag:Gag Multimerization on Viral RNA Are Critical Properties of HIV-1 Gag Required To Mediate Efficient Genome Packaging

• Published in: mBio 2021-12, Vol.12 (6), p.e0325421-e0325421
• Main Authors: Duchon, Alice, Santos, Steven, Chen, Jianbo, Brown, Matthew, Nikolaitchik, Olga A, Tai, Sheldon, Chao, Jeffrey A, Freed, Eric O, Pathak, Vinay K, Hu, Wei-Shau
• Format: Article
• Language: English
• Subjects: Cell Membrane - virology; Gag; gag Gene Products, Human Immunodeficiency Virus - chemistry; gag Gene Products, Human Immunodeficiency Virus - metabolism; Gag:RNA interactions; genome packaging; Genome, Viral; HIV Infections - virology; HIV-1 - genetics; HIV-1 - physiology; human immunodeficiency virus; Humans; membrane targeting; Research Article; RNA; RNA, Viral - chemistry; RNA, Viral - genetics; RNA, Viral - metabolism; Virion - genetics; Virion - physiology; Virology; Virus Assembly
• ISSN: 2150-7511; 2150-7511
• DOI: 10.1128/mbio.03254-21

Human immunodeficiency virus type 1 (HIV-1) Gag selects and packages the HIV RNA genome during virus assembly. However, HIV-1 RNA constitutes only a small fraction of the cellular RNA. Although Gag exhibits a slight preference to viral RNA, most of the cytoplasmic Gag proteins are associated with cellular RNAs. Thus, it is not understood how HIV-1 achieves highly efficient genome packaging. We hypothesize that besides RNA binding, other properties of Gag are important for genome packaging. Many Gag mutants have assembly defects that preclude analysis of their effects on genome packaging. To bypass this challenge, we established complementation systems that separate the particle-assembling and RNA-binding functions of Gag: we used a set of Gag proteins to drive particle assembly and an RNA-binding Gag to package HIV-1 RNA. We have developed two types of RNA-binding Gag in which packaging is mediated by the authentic nucleocapsid (NC) domain or by a nonviral RNA-binding domain. We found that in both cases, mutations that affect the multimerization or plasma membrane anchoring properties of Gag reduce or abolish RNA packaging. These mutant Gag can coassemble into particles but cannot package the RNA genome efficiently. Our findings indicate that HIV-1 RNA packaging occurs at the plasma membrane and RNA-binding Gag needs to multimerize on RNA to encapsidate the viral genome. To generate infectious virions, HIV-1 must package its full-length RNA as the genome during particle assembly. HIV-1 Gag:RNA interactions mediate genome packaging, but the mechanism remains unclear. Only a minor portion of the cellular RNA is HIV-1 RNA, and most of the RNAs associated with cytoplasmic Gag are cellular RNAs. However, >94% of the HIV-1 virions contain viral RNA genome. We posited that, besides RNA binding, other properties of Gag contribute to genome packaging. Using two complementation systems, we examined features of Gag that are important for genome packaging. We found that the capacities for Gag to multimerize and to anchor at the plasma membrane are critical for genome packaging. Our results revealed that Gag needs to multimerize on viral RNA at the plasma membrane in order to package RNA genome.