Single Point Mutations in the Zinc Finger Motifs of the Human Immunodeficiency Virus Type 1 Nucleocapsid Alter RNA Binding Specificities of the Gag Protein and Enhance Packaging and Infectivity

Abstract

A specific interaction between the nucleocapsid (NC) domain of the Gag polyprotein and the RNA encapsidation signal (⌿) is required for preferential incorporation of the retroviral genomic RNA into the assembled virion. Using the yeast three-hybrid system, we developed a genetic screen to detect human immunodeficiency virus type 1 (HIV-1) Gag mutants with altered RNA binding specificities. Specifically, we randomly mutated full-length HIV-1 Gag or its NC portion and screened the mutants for an increase in affinity for the Harvey murine sarcoma virus encapsidation signal. These screens identified several NC zinc finger mutants with altered RNA binding specificities. Furthermore, additional zinc finger mutants that also demonstrated this phenotype were made by site-directed mutagenesis. The majority of these mutants were able to produce normal virion-like particles; however, when tested in a single-cycle infection assay, some of the mutants demonstrated higher transduction efficiencies than that of wild-type Gag. In particular, the N17K mutant showed a seven-to ninefold increase in transduction, which correlated with enhanced vector RNA packaging. This mutant also packaged larger amounts of foreign RNA. Our results emphasize the importance of the NC zinc fingers, and not other Gag sequences, in achieving specificity in the genome encapsidation process. In addition, the described mutations may contribute to our understanding of HIV diversity resulting from recombination events between copackaged viral genomes and foreign RNA.The assembly process of retroviruses includes the encapsidation of the genomic RNA inside the nascent virion by Gag proteins in the cytoplasm. Although this RNA accounts for Ͻ1% of the total cytoplasmic RNA, it reaches Ͼ50% of the virion nucleic acids by weight (reviewed in reference 7). This selective enrichment is the result of specific interactions between cis-acting packaging elements in the genomic RNA and trans-acting RNA binding sites in the Gag precursor.For human immunodeficiency virus type 1 (HIV-1), many studies have demonstrated that the major encapsidation signal (⌿) is a structured RNA sequence that lies downstream of the primer binding site and extends into the 5Ј portion of the gag gene (29,30,34,44,45). Within this region, four adjacent stem-loop structures contribute to the encapsidation of the viral RNA, with each stem-loop binding the nucleocapsid (NC) domain in the HIV-1 Gag precursor with a different affinity (2,3,8,16,17,21,46,47). Two copies of a Cys-His box, containing the consensus sequence Cys-X2-Cys-X4-His-X4-Cys, in the HIV-1 NC are crucial for an interaction with RNA. These form two "zinc finger" or "zinc knuckle" motifs (also designated F1 for the N-terminal motif and F2 for the C-terminal motif), since the conserved cysteine and histidine residues bind a zinc ion, which leads to a rigid conformation. The compact structure of each of the zinc fingers forms a direct contact with the RNA (21, 61). This structure is important for the encapsidation of the viral RNA, a...

Transduction of Nondividing Human Macrophages with Gammaretrovirus-Derived Vectors

“…The Friend MLV-derived vector was compared to a Moloney MLV-derived vector produced in parallel. This vector has been previously described elsewhere (23,24), and it is comparable to the Friend MLV-derived vector used here. After normalization of their infectious titers on HeLa cells, equal MOIs were used in the transduction of day 4 macrophages, and infection was scored 3 days later by flow cytometry analysis.…”

“…Retroviral particles were pseudotyped with the vesicular stomatitis virus glycoprotein (VSVg) envelope expressed from the plasmid MD.G to confer on them ample cellular tropism (28). The Moloney MLV (Mo-MLV)-based vector used here has been previously described (23,24). The D1513A IN mutation was introduced in the pTG13077 Friend gag-pro-pol vector by standard molecular biology techniques.…”

The Remarkable Frequency of Human Immunodeficiency Virus Type 1 Genetic Recombination

“…Both MLV and HIV-1 are capable of packaging ⌿-positive (⌿ ϩ ) RNAs more than twice the native length (177,295). Similarly, host and virus factor alterations can modulate RNA packaging, which demonstrates flexibility in packaged RNA composition (200,218). Although some retroviruses may copackage more than two RNAs when gRNAs are short (285), and it remains unclear if most of them contain precisely two RNAs, it seems likely that if variation exists, it is in increments of 2-0, 2, 4, or more copackaged gRNAs.…”

Properties and functions of the nucleocapsid protein in virus assembly

“…42,43 Regarding binding specificity, NC in the form of the Gag polyprotein precursor appears to specifically recognize the Psi encapsidation sequences, which is thought to account for the selection of the genomic RNA among a vast excess of cellular RNAs, during the initial stage of particle assembly (reviewed in refs. 16,[44][45][46].…”