Distribution and Redistribution of HIV-1 Nucleocapsid Protein in Immature, Mature, and Integrase-Inhibited Virions: a Role for Integrase in Maturation

被引:64
作者
Fontana, Juan [1 ]
Jurado, Kellie A. [2 ]
Cheng, Naiqian [1 ]
Ly, Ngoc L. [2 ]
Fuchs, James R. [3 ]
Gorelick, Robert J. [4 ]
Engelman, Alan N. [2 ]
Steven, Alasdair C. [1 ]
机构
[1] NIAMSD, Struct Biol Res Lab, NIH, Bethesda, MD 20892 USA
[2] Dana Farber Canc Inst, Dept Canc Immunol & AIDS, Boston, MA 02115 USA
[3] Ohio State Univ, Coll Pharm, Div Med Chem & Pharmacognosy, Columbus, OH 43210 USA
[4] Leidos Biomed Res Inc, AIDS & Canc Virus Program, Frederick Natl Lab Canc Res, Frederick, MD USA
关键词
IMMUNODEFICIENCY-VIRUS TYPE-1; SMALL-MOLECULE INHIBITORS; RADIATION-DAMAGE; CRYOELECTRON TOMOGRAPHY; VIRAL-REPLICATION; ELECTRON CRYOTOMOGRAPHY; 3-DIMENSIONAL STRUCTURE; NUCLEAR-LOCALIZATION; STRUCTURAL-ANALYSIS; IN-VITRO;
D O I
10.1128/JVI.01522-15
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
During virion maturation, HIV-1 capsid protein assembles into a conical core containing the viral ribonucleoprotein (vRNP) complex, thought to be composed mainly of the viral RNA and nucleocapsid protein (NC). After infection, the viral RNA is reverse transcribed into double-stranded DNA, which is then incorporated into host chromosomes by integrase (IN) catalysis. Certain IN mutations (class II) and antiviral drugs (allosteric IN inhibitors [ALLINIs]) adversely affect maturation, resulting in virions that contain "eccentric condensates," electron-dense aggregates located outside seemingly empty capsids. Here we demonstrate that in addition to this mislocalization of electron density, a class II IN mutation and ALLINIs each increase the fraction of virions with malformed capsids (from similar to 12% to similar to 53%). Eccentric condensates have a high NC content, as demonstrated by "tomo-bubblegram" imaging, a novel labeling technique that exploits the susceptibility of NC to radiation damage. Tomo-bubblegrams also localized NC inside wild-type cores and lining the spherical Gag shell in immature virions. We conclude that eccentric condensates represent nonpackaged vRNPs and that either genetic or pharmacological inhibition of IN can impair vRNP incorporation into mature cores. Supplying IN in trans as part of a Vpr-IN fusion protein partially restored the formation of conical cores with internal electron density and the infectivity of a class II IN deletion mutant virus. Moreover, the ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose a role for IN in initiating core morphogenesis and vRNP incorporation into the mature core during HIV-1 maturation. IMPORTANCE Maturation, a process essential for HIV-1 infectivity, involves core assembly, whereby the viral ribonucleoprotein (vRNP, composed of vRNA and nucleocapsid protein [NC]) is packaged into a conical capsid. Allosteric integrase inhibitors (ALLINIs) affect multiple viral processes. We have characterized ALLINIs and integrase mutants that have the same phenotype. First, by comparing the effects of ALLINIs on several steps of the viral cycle, we show that inhibition of maturation accounts for compound potency. Second, by using cryoelectron tomography, we find that ALLINIs impair conical capsid assembly. Third, by developing tomo-bubblegram imaging, which specifically labels NC protein, we find that ALLINIs block vRNP packaging; instead, vRNPs form "eccentric condensates" outside the core. Fourth, malformed cores, typical of integrase-deleted virus, are partially replaced by conical cores when integrase is supplied in trans. Fifth, vRNA is necessary for ALLINI-induced eccentric condensate formation. These observations suggest that integrase is involved in capsid morphogenesis and vRNP packaging.
引用
收藏
页码:9765 / 9780
页数:16
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