Distinct functions and requirements for the Cys-His boxes of the human immunodeficiency virus type 1 nucleocapsid protein during RNA encapsidation and replication

Abstract

The process of retroviral RNA encapsidation involves interaction between transacting viral proteins and cis-acting RNA elements. The encapsidation signal on human immunodeficiency virus type 1 (HIV-1) RNA is a multipartite structure composed of functional stem-loop structures. The nucleocapsid (NC) domain of the Gag polyprotein precursor contains two copies of a Cys-His box motif that have been demonstrated to be important in RNA encapsidation. To further characterize the role of the Cys-His boxes of the HIV-1 NC protein in RNA encapsidation, the relative efficiency of RNA encapsidation for virus particles that contained mutations within the Cys-His boxes was measured. Mutations that disrupted the first Cys-His box of the NC protein resulted in virus particles that encapsidated genomic RNA less efficiently and subgenomic RNA more efficiently than did wild-type virus. Mutations within the second Cys-His box did not significantly affect RNA encapsidation. In addition, a full complement of wild-type NC protein in virus particles is not required for efficient RNA encapsidation or virus replication. Finally, both Cys-His boxes of the NC protein play additional roles in virus replication.

HIV-1 Gag Proteins: Diverse Functions in the Virus Life Cycle

“…The process of encapsidation strongly favors viral RNA over cellular RNA and full-length, unspliced viral RNA over spliced viral RNA. Mutations in the highly conserved Cys and His residues of the HIV-1 NC zinc-finger domains caused major defects in the specific encapsidation of full-length viral RNA and in vitro RNA binding activity (Aldovini and Young, 1990;Gorelick et al, 1990;Dorfman et al, 1993;Barklis, 1995, 1997;Schwartz et al, 1997). In some cases, these mutations reduced the specificity of encapsidation, resulting in an increased ratio of spliced to unspliced RNA in virus particles (Zhang and Barklis, 1995;Schwartz et al, 1997).…”

“…Mutations in the highly conserved Cys and His residues of the HIV-1 NC zinc-finger domains caused major defects in the specific encapsidation of full-length viral RNA and in vitro RNA binding activity (Aldovini and Young, 1990;Gorelick et al, 1990;Dorfman et al, 1993;Barklis, 1995, 1997;Schwartz et al, 1997). In some cases, these mutations reduced the specificity of encapsidation, resulting in an increased ratio of spliced to unspliced RNA in virus particles (Zhang and Barklis, 1995;Schwartz et al, 1997). Mutations in basic residues flanking the zinc fingers also reduced RNA binding in vitro (Schmalzbauer et al, 1996) and RNA encapsidation into virions (Poon et al, 1996).…”

The cooperative function of arginine residues in the Prototype Foamy Virus Gag C-terminus mediates viral and cellular RNA encapsidation

“…The highest Gag signal intensity in each gradient was set to 100%. (B) Western blot analysis of the Gag content in individual gradient fractions (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), pelleted virus particles (L) and cell lysates of transfected 293T cells (CL) using polyclonal antibodies specific for PFV Gag (α-Gag) (see above). (C) Cryo-electron microscopy analysis of wild type PFV (left) and of GR R/A mutant (right) particles.…”

“…Orthoretroviral Gag proteins mediate the interaction with nucleic acids largely but not entirely by the Cterminal nucleocapsid (NC) domain (reviewed in [5]). They contain conserved motifs of regularly spaced cysteine and histidine residues (Cis-His), which are important for recognition of the complex structured packaging signal (called Psi) in the vgRNA [6]. This specific mechanism allows selective vgRNA encapsidation in orthoretroviral particles (reviewed in [2,7]).…”

Binding of the Human Immunodeficiency Virus Type 1 Gag Protein to the Viral RNA Encapsidation Signal in the Yeast Three-Hybrid System

“…The major protein component of the interaction is the nucleocapsid (NC) region of Gag, a basic domain containing two copies of a motif known as a Cys-His box (consensus sequence Cys-X 2 -Cys-X 4 -His-X 4 -Cys) or a zinc knuckle because the four conserved residues coordinate a zinc ion. Mutational analysis of the HIV-1 Cys-His boxes has demonstrated their importance in the binding and encapsidation of the HIV-1 RNA (2,11,18,25,26,43,45,54,55). Recently, the three-dimensional structure of an HIV-1 NC bound to RNA stem-loop 3 (SL3) was determined (17).…”