The interdomain linker region of HIV-1 capsid protein is a critical determinant of proper core assembly and stability

Section on Viral Gene Regulation, Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health, National Institutes of Health, Bethesda, MD 20892-2780, USA.
Virology (Impact Factor: 3.32). 12/2011; 421(2):253-65. DOI: 10.1016/j.virol.2011.09.012
Source: PubMed


The HIV-1 capsid protein consists of two independently folded domains connected by a flexible peptide linker (residues 146-150), the function of which remains to be defined. To investigate the role of this region in virus replication, we made alanine or leucine substitutions in each linker residue and two flanking residues. Three classes of mutants were identified: (i) S146A and T148A behave like wild type (WT); (ii) Y145A, I150A, and L151A are noninfectious, assemble unstable cores with aberrant morphology, and synthesize almost no viral DNA; and (iii) P147L and S149A display a poorly infectious, attenuated phenotype. Infectivity of P147L and S149A is rescued specifically by pseudotyping with vesicular stomatitis virus envelope glycoprotein. Moreover, despite having unstable cores, these mutants assemble WT-like structures and synthesize viral DNA, although less efficiently than WT. Collectively, these findings demonstrate that the linker region is essential for proper assembly and stability of cores and efficient replication.

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Available from: James A Thomas, Sep 03, 2014
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    • "The CTD is roughly spherical, consisting of four a-helices and a 3 10 helix, and forms dimers in solution [26]. The linker region joining both NTD and CTD is also important for capsid assembly and viral infectivity [27]. Within the mature viral capsid core, CA assembles into both hexamer and pentamer structures [28] [29], and recent cryo-electron microscopy identified cone-shaped structures comprising 12 pentamers with either 216 or 186 hexamers [30], which is in agreement with previous estimation/model of the structure of the fullerene cone capsid core [31] [32]. "
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    ABSTRACT: The HIV-1 Gag precursor protein, Pr55Gag, is a multi-domain polyprotein that drives HIV-1 assembly. The morphological features of HIV-1 suggested Pr55Gag assumes a variety of different conformations during virion assembly and maturation, yet structural determination of HIV-1 Pr55Gag has not been possible due to an inability to express and to isolate large amounts of full-length recombinant Pr55Gag for biophysical and biochemical analyses. This challenge is further complicated by HIV-1 Gag’s natural propensity to multimerize for the formation of viral particle (with ∼2500 Gag molecules per virion), and this has led Pr55Gag to aggregate and be expressed as inclusion bodies in a number of in vitro protein expression systems. This study reported the production of a recombinant form of HIV-1 Pr55Gag using a bacterial heterologous expression system. Recombinant HIV-1 Pr55Gag was expressed with a C-terminal Hisx6 tag, and purified using a combination of immobilized metal affinity chromatography and size exclusion chromatography. This procedure resulted in the production of milligram quantities of high purity HIV-1 Pr55Gag that has a mobility that resembles a trimer in solution using size exclusion chromatography analysis. The high quantity and purity of the full length HIV Gag will be suitable for structural and functional studies to further understand the process of viral assembly, maturation and the development of inhibitors to interfere with the process.
    Protein Expression and Purification 08/2014; 100. DOI:10.1016/j.pep.2014.04.013 · 1.70 Impact Factor
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    • "The relationship between the morphology and stability of the capsid has not been extensively studied. There are examples in the literature, where CA (p24) mutants show aberrant morphology due to altered stability of the capsid [40,41]. There are several reports of the presence of aberrant morphology of capsids when IN protein is mutated (class II IN mutants). "
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    ABSTRACT: Retroviral integrase catalyzes integration of viral DNA into the host genome. Integrase interactor (INI)1/hSNF5 is a host factor that binds to HIV-1 IN within the context of Gag-Pol and is specifically incorporated into HIV-1 virions during assembly. Previous studies have indicated that INI1/hSNF5 is required for late events in vivo and for integration in vitro. To determine the effects of disrupting the IN-INI1 interaction on the assembly and infectivity of HIV-1 particles, we isolated mutants of IN that are defective for binding to INI1/hSNF5 and tested their effects on HIV-1 replication. A reverse yeast two-hybrid system was used to identify INI1-interaction defective IN mutants (IID-IN). Since protein-protein interactions depend on the surface residues, the IID-IN mutants that showed high surface accessibility on IN crystal structures (K71R, K111E, Q137R, D202G, and S147G) were selected for further study. In vitro interaction studies demonstrated that IID-IN mutants exhibit variable degrees of interaction with INI1. The mutations were engineered into HIV-1NL4-3 and HIV-Luc viruses and tested for their effects on virus replication. HIV-1 harboring IID-IN mutations were defective for replication in both multi- and single-round infection assays. The infectivity defects were correlated to the degree of INI1 interaction of the IID-IN mutants. Highly defective IID-IN mutants were blocked at early and late reverse transcription, whereas partially defective IID-IN mutants proceeded through reverse transcription and nuclear localization, but were partially impaired for integration. Electron microscopic analysis of mutant particles indicated that highly interaction-defective IID-IN mutants produced morphologically aberrant virions, whereas the partially defective mutants produced normal virions. All of the IID-IN mutant particles exhibited normal capsid stability and reverse transcriptase activity in vitro. Our results demonstrate that a severe defect in IN-INI1 interaction is associated with production of defective particles and a subsequent defect in post-entry events. A partial defect in IN-INI1 interaction leads to production of normal virions that are partially impaired for early events including integration. Our studies suggest that proper interaction of INI1 with IN within Gag-Pol is necessary for proper HIV-1 morphogenesis and integration.
    Retrovirology 06/2013; 10(1):66. DOI:10.1186/1742-4690-10-66 · 4.19 Impact Factor
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    • "Furthermore, capsid-targeting cellular restriction factors that block infection at an early phase of replication appear to act by inducing premature uncoating in target cells [15-17]. Mutagenesis studies have also shown that CA surfaces are important for both particle assembly and capsid formation, and CA mutants that exhibit abnormal core morphology are poorly infectious [18-22]. Thus, the emerging view is that the capsid is a critical component of the virion whose structure, assembly, and disassembly require a better understanding for antiviral targeting. "
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    ABSTRACT: Background Lentiviruses exhibit a cone-shaped capsid composed of subunits of the viral CA protein. The intrinsic stability of the capsid is critical for HIV-1 infection, since both stabilizing and destabilizing mutations compromise viral infectivity. Structural studies have identified three intersubunit interfaces in the HIV-1 capsid, two of which have been previously studied by mutational analysis. In this present study we analyzed the role of a third interface, that which is formed between the amino terminal domain (NTD) and carboxyl terminal domain (CTD) of adjacent subunits. Results We provided evidence for the presence of the NTD-CTD interface in HIV-1 particles by engineering intersubunit NTD-CTD disulfide crosslinks, resulting in accumulation of disulfide-linked oligomers up to hexamers. We also generated and characterized a panel of HIV-1 mutants containing substitutions at this interface. Some mutants showed processing defects and altered morphology from that of wild type, indicating that the interface is important for capsid assembly. Analysis of these mutants by transmission electron microscopy corroborated the importance of this interface in assembly. Other mutants exhibited quantitative changes in capsid stability, many with unstable capsids, and one mutant with a hyperstable capsid. Analysis of the mutants for their capacity to saturate TRIMCyp-mediated restriction in trans confirmed that the unstable mutants undergo premature uncoating in target cells. All but one of the mutants were markedly attenuated in replication owing to impaired reverse transcription in target cells. Conclusions Our results demonstrate that the NTD-CTD intersubunit interface is present in the mature HIV-1 capsid and is critical for proper capsid assembly and stability.
    Retrovirology 03/2013; 10(1):29. DOI:10.1186/1742-4690-10-29 · 4.19 Impact Factor
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