In vitro identification and characterization of an early complex linking HIV-1 genomic RNA recognition and Pr55Gag multimerization

The minimal protein requirements that drive virus-like particle formation of human immunodeficiency virus type 1 (HIV-1) have been established. The C-terminal domain of capsid (CTD-CA) and nucleocapsid (NC) are the most important domains in a so-called minimal Gag protein (mGag). The CTD is essential for Gag oligomerization. NC is known to bind and encapsidate HIV-1 genomic RNA. The spacer peptide, SP1, located between CA and NC is important for the multimerization process, viral maturation and recognition of HIV-1 genomic RNA by NC. In this study, we show that NC in the context of an mGag protein binds HIV-1 genomic RNA with almost 10-fold higher affinity. The protein region encompassing the 11th alpha-helix of CA and the proposed alpha-helix in the CA/SP1 boundary region play important roles in this increased binding capacity. Furthermore, sequences downstream from stem loop 4 of the HIV-1 genomic RNA are also important for this RNA-protein interaction. In gel shift assays using purified mGag and a model RNA spanning the region from +223 to +506 of HIV-1 genomic RNA, we have identified an early complex (EC) formation between 2 proteins and 1 RNA molecule. This EC was not present in experiments performed with a mutant mGag protein, which contains a CTD dimerization mutation (M318A). These data suggest that the dimerization interface of the CTD plays an important role in EC formation, and, as a consequence, in RNA-protein association and multimerization. We propose a model for the RNA-protein interaction, based on previous results and those presented in this study.     

Matrix domain modulates HIV-1 Gag's nucleic acid chaperone activity via inositol phosphate binding

Retroviruses replicate by reverse transcribing their single-stranded RNA genomes into double-stranded DNA using specific cellular tRNAs to prime cDNA synthesis. In HIV-1, human tRNA(3)(Lys) serves as the primer and is packaged into virions during assembly. The viral Gag protein is believed to chaperone tRNA(3)(Lys) placement onto the genomic RNA primer binding site; however, the timing and possible regulation of this event are currently unknown. Composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains, the multifunctional HIV-1 Gag polyprotein orchestrates the highly coordinated process of virion assembly, but the contribution of these domains to tRNA(3)(Lys) annealing is unclear. Here, we show that NC is absolutely essential for annealing and that the MA domain inhibits Gag's tRNA annealing capability. During assembly, MA specifically interacts with inositol phosphate (IP)-containing lipids in the plasma membrane (PM). Surprisingly, we find that IPs stimulate Gag-facilitated tRNA annealing but do not stimulate annealing in Gag variants lacking the MA domain or containing point mutations involved in PM binding. Moreover, we find that IPs prevent MA from binding to nucleic acids but have little effect on NC or Gag. We propose that Gag binds to RNA either with both NC and MA domains or with NC alone and that MA-IP interactions alter Gag's binding mode. We propose that MA's interactions with the PM trigger the switch between these two binding modes and stimulate Gag's chaperone function, which may be important for the regulation of events such as tRNA primer annealing.     

Charged amino acid residues of human immunodeficiency virus type 1 nucleocapsid p7 protein involved in RNA packaging and infectivity.

Interaction of the human immunodeficiency virus type 1 (HIV-1) Gag precursor polyprotein (Pr55Gag) with the viral genomic RNA is required for retroviral replication. Mutations that reduce RNA packaging efficiency have been localized to the highly basic nucleocapsid (NC) p7 domain of Pr55Gag, but the importance of the basic amino acid residues in specific viral RNA encapsidation and infectivity has not been thoroughly investigated in vivo. We have systematically substituted the positively charged residues of the NC domain of Pr55Gag in an HIV-1 viral clone by using alanine scanning mutagenesis and have assayed the effects of these mutations on virus replication, particle formation, and RNA packaging in vivo. Analysis of viral clones with single substitutions revealed that certain charged amino acid residues are more critical for RNA packaging efficiency and infectivity than others. Analysis of viral clones with multiple substitutions indicates that the presence of positive charge in each of three independent domains--the zinc-binding domains, the basic region that links them, and the residues that Hank the two zinc-binding domains--is necessary for efficient HIV-1 RNA packaging. Finally, we note that some mutations affect virus replication more drastically than RNA incorporation, providing in vivo evidence for the hypothesis that NC p7 may be involved in aspects of the HIV life cycle in addition to RNA packaging.     

Effects of nucleocapsid mutations on human immunodeficiency virus assembly and RNA encapsidation.

The human immunodeficiency virus (HIV) Pr55Gag precursor proteins direct virus particle assembly. While Gag-Gag protein interactions which affect HIV assembly occur in the capsid (CA) domain of Pr55Gag, the nucleocapsid (NC) domain, which functions in viral RNA encapsidation, also appears to participate in virus assembly. In order to dissect the roles of the NC domain and the p6 domain, the C-terminal Gag protein domain, we examined the effects of NC and p6 mutations on virus assembly and RNA encapsidation. In our experimental system, the p6 domain did not appear to affect virus release efficiency but p6 deletions and truncations reduced the specificity of genomic HIV-1 RNA encapsidation. Mutations in the nucleocapsid region reduced particle release, especially when the p2 interdomain peptide or the amino-terminal portion of the NC region was mutated, and NC mutations also reduced both the specificity and the efficiency of HIV-1 RNA encapsidation. These results implicated a linkage between RNA encapsidation and virus particle assembly or release. However, we found that the mutant ApoMTRB, in which the nucleocapsid and p6 domains of HIV-1 Pr55Gag were replaced with the Bacillus subtilis MtrB protein domain, released particles efficiently but packaged no detectable RNA. These results suggest that, for the purposes of virus-like particle assembly and release, NC can be replaced by a protein that does not appear to encapsidate RNA.