RNA-Binding Domains of Heterologous Viral Proteins Substituted for Basic Residues in the RSV Gag NC Domain Restore Specific Packaging of Genomic RNA.

Breanna L Rice,Timothy L Lochmann,Leslie J Parent

doi:10.3390/v12040370

Breanna L Rice, Timothy L Lochmann + Show 1 more

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https://doi.org/10.3390/v12040370

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Abstract

The Rous sarcoma virus Gag polyprotein transiently traffics through the nucleus, which is required for efficient incorporation of the viral genomic RNA (gRNA) into virus particles. Packaging of gRNA is mediated by two zinc knuckles and basic residues located in the nucleocapsid (NC) domain in Gag. To further examine the role of basic residues located downstream of the zinc knuckles in gRNA encapsidation, we used a gain-of-function approach. We replaced a basic residue cluster essential for gRNA packaging with heterologous basic residue motif (BR) with RNA-binding activity from either the HIV-1 Rev protein (Rev BR) or the HSV ICP27 protein (ICP27 BR). Compared to wild-type Gag, the mutant ICP27 BR and Rev BR Gag proteins were much more strongly localized to the nucleus and released significantly lower levels of virus particles. Surprisingly, both the ICP27 BR and Rev BR mutants packaged normal levels of gRNA per virus particle when examined in the context of a proviral vector, yet both mutants were noninfectious. These results support the hypothesis that basic residues located in the C-terminal region of NC are required for selective gRNA packaging, potentially by binding non-specifically to RNA via electrostatic interactions.

Highlights

The retroviral Gag protein directs the assembly of new virus particles at the plasma membrane, selecting the viral genomic RNA from the milieu of cellular and viral RNAs.The mechanism by which selective packaging occurs within the cell remains incompletely understood.The Gag NC domain is essential for encapsidation of gRNA and plays an important role in the subcellular trafficking of the Gag protein [1,2,3,4,5,6]
To examine whether the basic residues after the second Cys-His box in Rous sarcoma sarcoma virus virus (RSV) Gag NC were important for virus assembly, we deleted residues 61–73 in the context of the proviral construct pRS.V8 (Figure 1A), and a quantitative radioimmunoprecipitation assay was performed
After detection of metabolically labeled Gag proteins released into the supernatant, quantitation of virus release was performed by dividing the amount of CA in the media by the amount of Gag in the cell lysates [37]

Summary

Introduction

The retroviral Gag protein directs the assembly of new virus particles at the plasma membrane, selecting the viral genomic RNA (gRNA) from the milieu of cellular and viral RNAs.The mechanism by which selective packaging occurs within the cell remains incompletely understood.The Gag NC (nucleocapsid) domain is essential for encapsidation of gRNA and plays an important role in the subcellular trafficking of the Gag protein [1,2,3,4,5,6]. The retroviral Gag protein directs the assembly of new virus particles at the plasma membrane, selecting the viral genomic RNA (gRNA) from the milieu of cellular and viral RNAs. The mechanism by which selective packaging occurs within the cell remains incompletely understood. RSV Gag is initially synthesized in the cytoplasm and undergoes transient nuclear trafficking, a step that is required for efficient gRNA packaging [7,8]. Mutants of Gag with reduced nuclear trafficking have lower levels of gRNA incorporation, whereas enhancing nuclear localization restores gRNA packaging [8]. RSV Gag is cleaved into MA (matrix), p2, p10, CA (capsid), NC, and small spacer peptides

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