Nucleocapsid and Matrix Protein Contributions to Selective Human Immunodeficiency Virus Type 1 Genomic RNA Packaging

Abstract

The nucleocapsid protein (NC) of retroviruses plays a major role in genomic RNA packaging, and some evidence has implicated the matrix protein (MA) of certain retroviruses in viral RNA binding. To further investigate the role of NC in the selective recognition of genomic viral RNA and to address the potential contribution of MA in this process, we constructed chimeric and deletion human immunodeficiency virus type 1 (HIV-1) mutants that alter the NC or MA protein. Both HIV and mouse mammary tumor virus (MMTV) NC proteins have two zinc-binding domains and similar basic amino acid compositions but differ substantially in total length, amino acid sequence, and spacing of the zinc-binding motifs. When the entire NC coding sequence of HIV was replaced with the MMTV NC coding sequence, we found that the HIV genome was incorporated into virions at 50% of wild-type levels. Viruses produced from chimeric HIV genomes with complete NC replacements, or with the two NC zinc-binding domains replaced with MMTV sequences, preferentially incorporated HIV genomes when both HIV and MMTV genomes were simultaneously present in the cell. Viruses produced from chimeric MMTV genomes in which the MMTV NC had been replaced with HIV NC preferentially incorporated MMTV genomes when both HIV and MMTV genomes were simultaneously present in the cell. In contrast, viruses produced from chimeric HIV genomes containing the Moloney NC, which contains a single zinc-binding motif, were previously shown to preferentially incorporate Moloney genomic RNA. Taken together, these results indicate that an NC protein with two zinc-binding motifs is required for specific HIV RNA packaging and that the amino acid context of these motifs, while contributing to the process, is less crucial for specificity. The data also suggest that HIV NC may not be the exclusive determinant of RNA selectivity. Analysis of an HIV MA mutant revealed that specific RNA packaging does not require MA protein.

Analysis of a human immunodeficiency virus type 1 gag mutant with an engineered 110-amino-acid insertion in the matrix protein domain

“…As shown in Figure 6, the level of MAII particle-associated RNA was roughly comparable to that of WT when normalized for the Gag protein level. Results from RT-PCR experiments (data not shown) by using gagspecific primers [Poon et al, 1998] also support the conclusion that the MAII mutation has no significant effects on RNA incorporation.…”

“…Although a number of studies have shown that the HIV MA domain is not required for incorporating viral RNA into particles Reicin et al, 1995;Poon et al, 1998], we examined genomic viral RNA of MAII particles by the slot-blot experiment as described in Materials and Methods. Aliquots of the identical medium pelleted samples were also analyzed for Gag proteins by Western immunoblotting.…”

The Nucleocapsid Domain Is Responsible for the Ability of Spleen Necrosis Virus (SNV) Gag Polyprotein To Package both SNV and Murine Leukemia Virus RNA

“…The NC domain is responsible for nonreciprocal RNA packaging of MLV and SNV. Several chimeras of Gag containing NC from different viruses have been previously tested, and mixed results were obtained (2,14,28,45,58). For example, chimeric HIV-1 Gag with MLV NC preferentially packaged MLV RNA (2, 58), but chimeric HIV-1 Gag with MMTV NC still preferentially packaged HIV-1 RNA (45).…”

“…These studies indicated that NC is, at least in part, responsible for RNA packaging specificity. In contrast, the chimeric HIV-1 Gag containing NC derived from mouse mammary tumor virus (MMTV) still preferentially packaged HIV-1 RNA (45). This observation indicated that replacement of the NC was not sufficient to alter the packaging specificity and that other Gag domains were involved.…”

Severe acute respiratory syndrome coronavirus nucleocapsid protein confers ability to efficiently produce virus-like particles when substituted for the human immunodeficiency virus nucleocapsid domain

“…Retroviral gag apparently possesses all required signals for VLP assembly and budding, and functional domains involved in coronavirus VLP assembly and budding are allocated throughout the M, E, and N proteins. Despite being completely unrelated, the SARS-CoV N protein and HIV-1 NC have similar properties: both contain putative proteinprotein interaction domains and both play roles in viral RNA packaging [4,5,18,32,33,53]. A domain responsible for Gag-Gag interactions (referred to as an I domain) has been mapped to HIV-1 NC [3,7].…”