RNA Incorporation Is Critical for Retroviral Particle Integrity after Cell Membrane Assembly of Gag Complexes

Abstract

The nucleocapsid (NC) domain of retroviruses plays a critical role in specific viral RNA packaging and virus assembly. RNA is thought to facilitate viral particle assembly, but the results described here with NC mutants indicate that it also plays a critical role in particle integrity. We investigated the assembly and integrity of particles produced by the human immunodeficiency virus type 1 M1-2/BR mutant virus, in which 10 of the 13 positive residues of NC have been replaced with alanines and incorporation of viral genomic RNA is virtually abolished. We found that the mutations in the basic residues of NC did not disrupt Gag assembly at the cell membrane. The mutant Gag protein can assemble efficiently at the cell membrane, and viral proteins are detected outside the cell as efficiently as they are for the wild type. However, only ϳ10% of the Gag molecules present in the supernatant of this mutant sediment at the correct density for a retroviral particle. The reduction of positive charge in the NC basic domain of the M1-2/BR virus adversely affects both the specific and nonspecific RNA binding properties of NC, and thus the assembled Gag polyprotein does not bind significant amounts of viral or cellular RNA. We found a direct correlation between the percentage of Gag associated with sedimented particles and the amount of incorporated RNA. We conclude that RNA binding by Gag, whether the RNA is viral or not, is critical to retroviral particle integrity after cell membrane assembly and is less important for Gag-Gag interactions during particle assembly and release.

Distinct Roles for Nucleic Acid in In Vitro Assembly of Purified Mason-Pfizer Monkey Virus CANC Proteins

“…This study shows that assembly of ⌬ProCANC occurs even in the absence of nucleic acids, which is in agreement with previously published data for HIV-1 Gag (78,79). Calculations based on experimental data show that the RSV NC binding site for nucleic acids spans eight nucleotides and therefore a 16-mer should be sufficient for bridging together two NC molecules (53,54).…”

Detection of HIV-1 P24 Gag in Plasma by a Nanoparticle-Based Bio-Barcode-Amplification Method

“…Occasional expansion of an HIV-1-infected cell by antigenic stimulation may provide an opportunity for the virus to replicate for a short number of generations. HIV-1 protease inhibitors prevent cleavage of the Gag and GagPol protein precursors in infected cells and thereby affect virus-particle assembly and RNA packaging [37,38]. Thus, assembly and release of virus particles occur in the presence of protease inhibitors, although the particles produced are immature and not infectious.…”

Properties and functions of the nucleocapsid protein in virus assembly

“…46,85 In the absence of NC, MLV Gag is still targeted and associated to the plasma membrane but no Gag-RNA refs. 19,44,[74][75][76][77][78][79][80][81][82][83][84][85][86]. Mutations in the polybasic region of NC or the zinc fingers impair and even abrogate genomic RNA packaging with the notion that the ZnF and hydrophobic plateau are driving the specificity of gRNA encapsidation, while the basic residues are more involved in Gag-Gag multimerization and assembly.…”