Transmission Electron Microscopy Reveals an Optimal HIV-1 Nucleocapsid Aggregation with Single-stranded Nucleic Acids and the Mature HIV-1 Nucleocapsid Protein

Human immunodeficiency virus type 1 (HIV-1) Gag is expressed as a polyprotein that is cleaved into six proteins by the viral protease in a maturation process that begins during assembly and budding. While processing of the N terminus of Gag is strictly required for virion maturation and infectivity, the necessity for the C-terminal cleavages of Gag is less well defined. To examine the importance of this process, we introduced a series of mutations into the C terminus of Gag that interrupted the cleavage sites that normally produce in the nucleocapsid (NC), spacer 2 (SP2), or p6Gag proteins. Protein analysis showed that all of the mutant constructs produced virions efficiently upon transfection of cells and appropriately processed Gag polyprotein at the nonmutated sites. Mutants that produced a p9 NC/SP2 protein exhibited only minor effects on HIV-1 infectivity and replication. In contrast, mutants that produced only the p8 SP2/p6 or p15 NC/SP2/p6 protein had severe defects in infectivity and replication. To identify the key defective step, we quantified reverse transcription and integration products isolated from infected cells by PCR. All mutants tested produced levels of reverse transcription products either similar to or only somewhat lower than that of wild type. In contrast, mutants that failed to cleave the SP2-p6Gag site produced drastically less provirus than the wild type. Together, our results show that processing of the SP2-p6Gag and not the NC-SP2 cleavage site is important for efficient viral DNA integration during infection in vitro. In turn, this finding suggests an important role for the p9 NC/SP2

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“…Recent reports of in vitro experiments have implicated p9 NC/SP2 in the maturation process (31,32). Theoretically, p9…”

Section: Discussionmentioning

confidence: 99%

“…In vitro, p7 NC and especially p9 NC/SP2 have been found to increase the efficiency of concerted integration (7,14,38). Interestingly, p9 NC/SP2 can condense double-stranded DNA into dense protein/DNA complexes much more efficiently than either p7 NC or p15 NC/SP2/p6 (32), suggesting that p9…”

Section: Discussionmentioning

confidence: 99%

Mutational Analysis of the C-Terminal Gag Cleavage Sites in Human Immunodeficiency Virus Type 1

Coren

Thomas

Chertova

et al. 2007

J Virol

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“…Nucleic acid aggregation is thought to be dependent on the N-terminal basic part of NC, whereas duplex destabilization is associated with the zinc fingers (Beltz et al 2005;Levin et al 2005Levin et al , 2010Vo et al 2009a,b;Godet and Mély 2010). However, some studies have suggested a role of zinc fingers in nucleic acid aggregation (Krishnamoorthy et al 2003;Levin et al 2005;Mirambeau et al 2006;Mitra et al 2013) and others have implicated basic residues in nucleic acid destabilization .…”

Section: Introductionmentioning

confidence: 99%

Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein

Belfetmi¹,

Zargarian²,

Tisné³

et al. 2016

RNA

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The mature HIV-1 nucleocapsid protein NCp7 (NC) plays a key role in reverse transcription facilitating the two obligatory strand transfers. Several properties contribute to its efficient chaperon activity: preferential binding to single-stranded regions, nucleic acid aggregation, helix destabilization, and rapid dissociation from nucleic acids. However, little is known about the relationships between these different properties, which are complicated by the ability of the protein to recognize particular HIV-1 stem-loops, such as SL1, SL2, and SL3, with high affinity and without destabilizing them. These latter properties are important in the context of genome packaging, during which NC is part of the Gag precursor. We used NMR to investigate destabilization of the full-length TAR (trans activating response element) RNA by NC, which is involved in the first strand transfer step of reverse transcription. NC was used at a low protein:nucleotide (nt) ratio of 1:59 in these experiments. NMR data for the imino protons of TAR identified most of the base pairs destabilized by NC. These base pairs were adjacent to the loops in the upper part of the TAR hairpin rather than randomly distributed. Gel retardation assays showed that conversion from the initial TAR-cTAR complex to the fully annealed form occurred much more slowly at the 1:59 ratio than at the higher ratios classically used. Nevertheless, NC significantly accelerated the formation of the initial complex at a ratio of 1:59.

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“…This difference may be due to the specific change introduced and to the viral background, but the results of Coren et al (10) clearly indicate that separation of SP2 from NC is not essential for HIV-1 infection in tissue culture. In vitro studies have shown that both NC and NC-SP2 function as nucleic acid chaperones, while uncleaved NC-p6 is poorly active in this regard (11,12,27,32,33,43). Nucleic acid chaperone activity contributes to genomic RNA dimerization and condensation, to tRNA primer placement, and to efficient reverse transcription and thus plays an important role in HIV replication (13,29,42).…”

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confidence: 99%

Role of the SP2 Domain and Its Proteolytic Cleavage in HIV-1 Structural Maturation and Infectivity

Marco

Heuser

Glass

et al. 2012

J Virol

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b HIV-1 buds as an immature, noninfectious virion. Proteolysis of its main structural component, Gag, is required for morphological maturation and infectivity and leads to release of four functional domains and the spacer peptides SP1 and SP2. The Nterminal cleavages of Gag and the separation of SP1 from CA are all essential for viral infectivity, while the roles of the two Cterminal cleavages and the role of SP2, separating the NC and p6 domains, are less well defined. We have analyzed HIV-1 variants with defective cleavage at either or both sites flanking SP2, or largely lacking SP2, regarding virus production, infectivity, and structural maturation. Neither the presence nor the proteolytic processing of SP2 was required for particle release. Viral infectivity was almost abolished when both cleavage sites were defective and severely reduced when the fast cleavage site between SP2 and p6 was defective. This correlated with an increased proportion of irregular core structures observed by cryo-electron tomography, although processing of CA was unaffected. Mutation of the slow cleavage site between NC and SP2 or deletion of most of SP2 had only a minor effect on infectivity and did not induce major alterations in mature core morphology. We speculate that not only separation of NC and p6 but also the processing kinetics in this region are essential for successful maturation, while SP2 itself is dispensable.

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Transmission Electron Microscopy Reveals an Optimal HIV-1 Nucleocapsid Aggregation with Single-stranded Nucleic Acids and the Mature HIV-1 Nucleocapsid Protein

Cited by 44 publications

References 82 publications

Mutational Analysis of the C-Terminal Gag Cleavage Sites in Human Immunodeficiency Virus Type 1

Mutational Analysis of the C-Terminal Gag Cleavage Sites in Human Immunodeficiency Virus Type 1

Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein

Role of the SP2 Domain and Its Proteolytic Cleavage in HIV-1 Structural Maturation and Infectivity

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