Role of Human Immunodeficiency Virus Type 1 Integrase in Uncoating of the Viral Core

Briones, Marisa; Dobard, Charles; Chow, Samson A.

doi:10.1128/jvi.02382-09

Cited by 48 publications

(52 citation statements)

References 70 publications

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“…ing does not occur randomly in the cytoplasm and is critical for infection; but, the exact timing and location of uncoating has been debated (35)(36)(37)(38). Although biochemical analyses of intracellular HIV initially concluded that capsids were lost immediately postfusion (39,40), EM data revealed intact HIV capsids at or near nuclear pores (33) indicating that capsid uncoating does not necessarily precede viral trafficking to the NE.…”

Section: Stochastic Fluorescence Of Single Flash-labeled Tetracysteinementioning

confidence: 99%

Superresolution imaging of HIV in infected cells with FlAsH-PALM

Lelek

Nunzio

Henriques

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

140

135

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Imaging protein assemblies at molecular resolution without affecting biological function is a long-standing goal. The diffractionlimited resolution of conventional light microscopy (∼200-300 nm) has been overcome by recent superresolution (SR) methods including techniques based on accurate localization of molecules exhibiting stochastic fluorescence; however, SR methods still suffer important restrictions inherent to the protein labeling strategies. Antibody labels are encumbered by variable specificity, limited commercial availability and affinity, and are mostly restricted to fixed cells. Fluorescent protein fusions, though compatible with live cell imaging, substantially increase protein size and can interfere with their biological activity. We demonstrate SR imaging of proteins tagged with small tetracysteine motifs and the fluorescein arsenical helix binder (FlAsH-PALM). We applied FlAsH-PALM to image the integrase enzyme (IN) of HIV in fixed and living cells under experimental conditions that fully preserved HIV infectivity. The obtained resolution (∼30 nm) allowed us to characterize the distribution of IN within virions and intracellular complexes and to distinguish different HIV structural populations based on their morphology. We could thus discriminate ∼100 nm long mature conical cores from immature Gag shells and observe that in infected cells cytoplasmic (but not nuclear) IN complexes display a morphology similar to the conical capsid. Together with the presence of capsid proteins, our data suggest that cytoplasmic IN is largely present in intact capsids and that these can be found deep within the cytoplasm. FlAsH-PALM opens the door to in vivo SR studies of microbial complexes within host cells and may help achieve truly molecular resolution.

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Section: Stochastic Fluorescence Of Single Flash-labeled Tetracysteinementioning

confidence: 99%

Superresolution imaging of HIV in infected cells with FlAsH-PALM

Lelek

Nunzio

Henriques

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

140

135

View full text Add to dashboard Cite

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“…IN also influences stages of viral replication other than integration, as is evident from the study of IN mutants. While class I IN mutants with substitutions in the catalytic residues block proviral DNA integration, class II IN mutants are defective for viral assembly, particle production, postentry uncoating, reverse transcription, and/or nuclear import (3,14,21,44,45). The exact mechanism by which IN mutants exert these pleiotropic effects is unknown.…”

mentioning

confidence: 99%

Inhibition of Early Stages of HIV-1 Assembly by INI1/hSNF5 Transdominant Negative Mutant S6

Cano

Kalpana

2011

J Virol

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INI1/hSNF5 is an HIV-1 integrase (IN) binding protein specifically incorporated into virions.A truncated mutant of INI1 (S6, amino acids 183 to 294) harboring the minimal IN binding Rpt1 domain potently inhibits HIV-1 particle production in a transdominant manner. The inhibition requires interaction of S6 with IN within Gag-Pol. While INI1 is a nuclear protein and harbors a masked nuclear export signal (NES), the transdominant negative mutant S6 is cytoplasmic, due to the unmasking of NES. Here, we examined the effects of subcellular localization of S6 on HIV-1 inhibition and further investigated the stages of assembly that are affected. We found that targeting a nuclear localization signal-containing S6 variant [NLS-S6(Rpt1)] to the nucleoplasm (but not to the nucleolus) resulted in complete reversal of inhibition of particle production. Electron microscopy indicated that although no electron-dense particles at any stage of assembly were seen in cells expressing S6, virions were produced in cells expressing the rescue mutant NLS-S6(Rpt1) to wild-type levels. Immunofluorescence analysis revealed that p24 exhibited a diffuse pattern of localization within the cytoplasm in cells expressing S6 in contrast to accumulation along the membrane in controls. Pulse-chase analysis indicated that in S6-expressing cells, although Gag(Pr55 gag ) protein translation was unaffected, processing and release of p24 were defective. Together, these results indicate that expression of S6 in the cytoplasm interferes with trafficking of Gag-Pol/Gag to the membrane and causes a defective processing leading to inhibition of assembly at an early stage prior to particle formation and budding.

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“…demonstrated that mutations in the IN gene, including deletion mutants, influence many other stages of viral replication in addition to integration. This pleiotropic effect of IN is characterized by defects in uncoating, reverse transcription, nuclear import, viral gene expression, virion precursor protein processing, and virion morphology (Shin et al, 1994;Engelman et al, 1995;Masuda et al, 1995;Bukovsky & Gottlinger, 1996;Leavitt et al, 1996;Nakamura et al, 1997;Engelman, 1999;Tsurutani et al, 2000;Lu et al, 2004;Dar et al, 2009;Briones et al, 2010). However, the mechanisms for these pleiotropic effects of the IN gene are still poorly understood.…”

Section: Integration Reactionmentioning

confidence: 99%