ABSTRACT
The HIV-1 nucleocapsid is formed during protease (PR)-directed viral maturation, and is transformed into pre-integration complexes following reverse transcription in the cytoplasm of the infected cell. Here, we report a detailed transmission electron microscopy analysis of the impact of HIV-1 PR and reverse transcriptase (RT) on nucleocapsid plasticity, using in vitro reconstitutions. After binding to nucleic acids, NCp15, a proteolytic intermediate of nucleocapsid protein (NC), was processed at its C-terminus by PR, yielding premature NC (NCp9) followed by mature NC (NCp7), through the consecutive removal of p6 and p1. This allowed NC co-aggregation with its single-stranded nucleic-acid substrate. Examination of these co-aggregates for the ability of RT to catalyse reverse transcription showed an effective synthesis of double-stranded DNA that, remarkably, escaped from the aggregates more efficiently with NCp7 than with NCp9. These data offer a compelling explanation for results from previous virological studies that focused on i) Gag processing leading to nucleocapsid condensation, and ii) the disappearance of NCp7 from the HIV-1 pre-integration complexes. We propose that HIV-1 PR and RT, by controlling the nucleocapsid architecture during the steps of condensation and dismantling, engage in a successive nucleoprotein-remodelling process that spatiotemporally coordinates the pre-integration steps of HIV-1. Finally we suggest that nucleoprotein remodelling mechanisms are common features developed by mobile genetic elements to ensure successful replication.
Subject(s)
HIV Protease/metabolism , HIV Reverse Transcriptase/metabolism , Nucleocapsid/ultrastructure , Binding Sites , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , DNA, Viral/chemistry , DNA, Viral/genetics , Genome, Viral , HIV Protease/genetics , HIV Reverse Transcriptase/genetics , HIV-1/enzymology , HIV-1/genetics , Kinetics , Models, Molecular , Nucleocapsid/chemistry , RNA, Viral/chemistry , RNA, Viral/geneticsABSTRACT
HIV-1 nucleocapsid protein (NCp7) condenses the viral RNA within the mature capsid. In a capsid-free system, NCp7 promotes an efficient mechanism of aggregation with both RNA and DNA. Here, we show an analysis of these macromolecular complexes by dark-field imaging using transmission electron microscopy. Thousands of mature NCp7 proteins co-aggregate with hundreds of single-stranded circular DNA molecules (ssDNA) within minutes, as observed with poly(rA). These co-aggregates are highly stable but dynamic structures, as they dissociate under harsh conditions, and after addition of potent ssDNA or NCp7 competitive ligands. The N-terminal domain and zinc fingers of NCp7 are both required for efficient association. Addition of magnesium slightly increases the avidity of NCp7 for ssDNA, while it strongly inhibits co-aggregation with relaxed circular double-stranded DNA (dsDNA). This DNA selectivity is restricted to mature NCp7, compared to its precursors NCp15 and NCp9. Moreover, for NCp15, the linkage of NCp7 with the Gag C-terminal p6-peptide provokes a deficiency in ssDNA aggregation, but results in DNA spreading similar to prototypical SSB proteins. Finally, this co-aggregation is discussed in a dynamic architectural context with regard to the mature HIV-1 nucleocapsid. On the basis of the present data, we propose that condensation of encapsidated RNA requires the C-terminal processing of NCp. Subsequently, disassembly of the nucleocapsid should be favoured once dsDNA is produced by HIV-1 reverse transcriptase.
