Basic Residues of the Retroviral Nucleocapsid Play Different Roles in Gag-Gag and Gag-Ψ RNA Interactions

Abstract

The Orthoretrovirus Gag interaction (I) domain maps to the nucleocapsid (NC) domain in the Gag polyprotein. We used the yeast two-hybrid system to analyze the role of Alpharetrovirus NC in Gag-Gag interactions and also examined the efficiency of viral assembly and release in vivo. We could delete either or both of the two Cys-His (CH) boxes without abrogating Gag-Gag interactions. We found that as few as eight clustered basic residues, attached to the C terminus of the spacer peptide separating the capsid (CA) and NC domains in the absence of NC, was sufficient for Gag-Gag interactions. Our results support the idea that a sufficient number of basic residues, rather than the CH boxes, play the important role in Gag multimerization. We also examined the requirement for basic residues in Gag for packaging of specific packaging signal (⌿)-containing RNA. Using a yeast three-hybrid RNA-protein interaction assay, second-site suppressors of a packaging-defective Gag mutant were isolated, which restored ⌿ RNA binding. These suppressors mapped to the p10 or CA domains in Gag and resulted in either introduction of a positively charged residue or elimination of a negatively charged one. These results imply that the structural interactions of NC with other domains of Gag are necessary for ⌿ RNA binding. Taken together, our results show that while Gag assembly only requires a certain number of positively charged amino acids, Gag binding to genomic RNA for packaging requires more complex interactions inherent in the protein tertiary structure.The Alpharetrovirus Gag protein, exemplified by Rous sarcoma virus (RSV), is synthesized as a precursor protein consisting of matrix (MA); two small peptides, p2 and p10; capsid (CA), nucleocapsid (NC), and protease (PR). Cleavage of the precursor by PR occurs after, or concomitant with, viral budding from the plasma membrane. Three Gag domains (M [membrane association], I [interaction], and L [late]) are necessary for viral assembly (reviewed in references 18 and 40). The interaction, or I, domain is required for Gag multimerization and virus particle assembly. Deletion of the NC domain has been shown to result in drastic reduction of viral assembly and release, suggesting that I domain function resides in NC. In Orthoretrovirinae, NC has two structural characteristics: one or two copies of the conserved sequence Cys-X 2 -Cys-X 4 -His-X 4 -Cys (Cys-His [CH] box), which coordinates Zn 2ϩ ion binding, and clustered basic residues (BRs) flanking the CH box or boxes. NC plays important roles in essential steps of virus replication, including reverse transcription, integration, RNA packaging, viral assembly, and infectivity (reviewed in reference 15). Some of these functions have been shown to require distinct structural motifs. A previous and extensive yeast twohybrid study (43) found that the minimal domain required for Gag-Gag interactions mapped to human immunodeficiency virus (HIV) NC but did not characterize the specific residues required within the minimal domain. Gag multimerization ...

Properties and functions of the nucleocapsid protein in virus assembly

“…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. 19,[75][76][77][78] These NC motifs are involved in specific GaggRNA interactions via the Psi signal located in the 5' leader region of the gRNA (Fig. 3).…”

“…Indeed, NC mutants of these motifs are viruses defective in the efficacy of Gag assembly, budding and particle morphogenesis for many retroviruses (HIV, SIV, MLV, RSV, BLV, MPMV). 19,[74][75][76][77][78][81][82][83]85 However, in most cases, some mutant Gag are still targeted to the assembly sites, i.e., at the plasma membrane, but Gag oligomer formation or viral particle budding is, at least in part, prevented and aberrant budding particles are observed in many instances. 76,[82][83][84] In the case of HIV-1, the situation is rather complex due to the fact that NC is located between sp1 and sp2 with the p6 domain at the extreme C-terminus (Fig.…”

“…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.…”

Solution Structure of the Rous Sarcoma Virus Nucleocapsid Protein: μΨ RNA Packaging Signal Complex

“…These findings are also consistent with mutagenesis studies showing that Arg61 is important for the high affinity Gag-Ψ RNA binding in vitro and efficient genome packaging in vivo. 40,44,45 The nucleobase of A168 packs against the side chains of Leu49, Cys60 and Lys58, and appears to be stabilized by a hydrogen bond between the A168 phosphate oxygen and Lys58 side chain amino group, Figure 8.…”

Cell biology of retroviral RNA packaging

“…Mutation of the functional domains of NC has shown that the ZFs and basic residues of the HIV and MLV GagNC are involved in gRNA selection and dimerization through tight interactions with the Psi packaging signal. 12,[31][32][33][34][35][36][37][38] Strikingly, loosening the interactions between GagNC and the gRNA by mutating either the NC ZFs or its 5' basic residues, not only reduces the gRNA content of mutant virus but turns HIV-1 into a DNA-containing virus. [38][39][40] These mutant HIVs activate viral DNA synthesis by the viral reverse transcriptase (called "late RT") in HIV-1 producer cells before virus release leading to the packaging of viral DNA (up to 100-fold increase of DNA level as compared with wild-type virions).…”