The Nucleocapsid Region of HIV-1 Gag Cooperates with the PTAP and LYPXnL Late Domains to Recruit the Cellular Machinery Necessary for Viral Budding

Dussupt, Vincent; Javid, Melodi P.; Abou-Jaoudé, Georges; Jadwin, Joshua A.; Cruz, M. Jason de la; Nagashima, Kunio; Bouamr, Fadila

doi:10.1371/journal.ppat.1000339

Cited by 133 publications

(192 citation statements)

References 82 publications

(175 reference statements)

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“…2E and column 5 of Fig. 2G), indicating that the reported interaction between the NC domain of Gag and Bro1 domain of ALIX (45,46) was not sufficient to recruit detectable levels of ALIX in this system. The ALIX PRD contains a P(S/T)AP motif that is capable of binding to the TSG101 subunit of ESCRT-I (18), although the motif is not essential for ALIX function in HIV-1 budding (19,20).…”

Section: Resultsmentioning

confidence: 92%

In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters

Carlson

Hurley

2012

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

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Most membrane-enveloped viruses depend on host proteins of the endosomal sorting complex required for transport (ESCRT) machinery for their release. HIV-1 is the prototypic ESCRT-dependent virus. The direct interactions between HIV-1 and the early ESCRT factors TSG101 and ALIX have been mapped in detail. However, the full pathway of ESCRT recruitment to HIV-1 budding sites, which culminates with the assembly of the late-acting CHMP4, CHMP3, CHMP2, and CHMP1 subunits, is less completely understood. Here, we report the biochemical reconstitution of ESCRT recruitment to viral assembly sites, using purified proteins and giant unilamellar vesicles. The myristylated full-length Gag protein of HIV-1 was purified to monodispersity. Myr-Gag forms clusters on giant unilamellar vesicle membranes containing the plasma membrane lipid PIð4,5ÞP 2 . These Gag clusters package a fluorescent oligonucleotide, and recruit early ESCRT complexes ESCRT-I or ALIX with the appropriate dependence on the Gag PTAP and LYPðXÞ n L motifs. ALIX directly recruits the key ESCRT-III subunit CHMP4. ESCRT-I can only recruit CHMP4 when ESCRT-II and CHMP6 are present as intermediary factors. Downstream of CHMP4, CHMP3 and CHMP2 assemble synergistically, with the presence of both subunits required for efficient recruitment. The very late-acting factor CHMP1 is not recruited unless the pathway is completed through CHMP3 and CHMP2. These findings define the minimal sets of components needed to complete ESCRT assembly at HIV-1 budding sites, and provide a starting point for in vitro structural and biophysical dissection of the system. confocal microscopy | host-pathogen interaction | membrane traffic | virus budding M ost membrane-enveloped viruses utilize host proteins of the endosomal sorting complex required for transport (ESCRT) machinery for their release (1-3). This dependence was first described for HIV-1, where mutation of an ESCRTbinding motif (late domain) in the viral protein Gag was shown to stall virus bud release at a late stage in virus assembly (4, 5). Late domains have subsequently been found in other retroviruses and numerous other virus families including, e.g., flavi-, filo-and rhabdoviruses (6). Indeed, the only well-characterized case of ESCRT-independent release of a membrane-enveloped virus is influenza virus (7). In normal cell physiology, ESCRTs function in cytokinesis, formation of intralumenal vesicles in multivesicular endosomes, and vesicle release from the plasma membrane (8-10). These seemingly disparate processes all involve membrane budding away from the cytoplasm, and ESCRTs are the only described protein machinery to perform vesicle formation with this topology, which is analogous to virus release.The initial events in HIV-1 ESCRT recruitment are well characterized. The structural protein of HIV-1, Gag, binds earlyacting ESCRT factors through two late-domain motifs in its C-terminal p6 domain: a PTAP sequence that interacts with the TSG101 subunit of ESCRT-I (11-16) and a LYPðXÞ n L motif that interacts with the ESCRT...

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Section: Resultsmentioning

confidence: 92%

In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters

Carlson

Hurley

2012

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

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“…61,68 ALIX and Tsg101 are two components necessary for the MVB biogenesis and for retroviral budding that associate with p6 for Tsg101 and for p6 in cooperation with NC for ALIX. 87,88 As mentioned before, the Bro1 domain of Alix interacts with the NC domain of HIV-1 Gag and in that manner cooperates to recruit the ESCRT via the p6 domain for retroviral budding. [87][88][89] Actin and actin-binding proteins are found inside HIV-1 virions 15,121 raising the question of a potential role of actin cytoskeleton dynamic in either Gag trafficking or in virion budding.…”

mentioning

confidence: 87%

“…87,88 As mentioned before, the Bro1 domain of Alix interacts with the NC domain of HIV-1 Gag and in that manner cooperates to recruit the ESCRT via the p6 domain for retroviral budding. [87][88][89] Actin and actin-binding proteins are found inside HIV-1 virions 15,121 raising the question of a potential role of actin cytoskeleton dynamic in either Gag trafficking or in virion budding. 20,[122][123][124][125] The Gag NC domain of several retroviruses (HIV, MLV, EIAV) is known to interact with actin.…”

mentioning

confidence: 87%

Properties and functions of the nucleocapsid protein in virus assembly

2010

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“…6C). Moreover, it has been shown by others that overexpression of full-length ALIX (57), as well as a fragment thereof (58), has a negative effect on the release of wt HIV-1. However, ALIX overexpression failed to increase the presentation of the SL epitope derived from GagSL-GFP wt (Fig.…”

Section: The Cellular Budding Factors Tsg101 or Alix Do Not Influencementioning

confidence: 99%

The PTAP Sequence within the p6 Domain of Human Immunodeficiency Virus Type 1 Gag Regulates Its Ubiquitination and MHC Class I Antigen Presentation

Hahn

Setz

Wild

et al. 2011

The Journal of Immunology

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Endogenous peptides presented by MHC class I (MHC-I) molecules are mostly derived from de novo synthesized, erroneous proteins, so-called defective ribosomal products (DRiPs), which are rapidly degraded via the ubiquitin–proteasome pathway. We have previously shown that the HIV-1 Gag protein represents a bona fide substrate for the DRiP pathway and that the amount of Gag-DRiPs can be enhanced by the introduction of an N-end rule degradation signal, leading to increased MHC-I presentation and immunogenicity of Gag. Based on these findings, we sought to identify a naturally occurring sequence motif within Gag that regulates its entry into the DRiP pathway. As the PTAP late assembly domain motif in the C-terminal p6 domain of Gag has been shown to negatively regulate the ubiquitination of Gag, we analyzed the correlation between ubiquitination and MHC-I presentation of PTAP-deficient Gag. Intriguingly, mutation of PTAP not only reduces the release of virus-like particles, but also increases ubiquitination of Gag and, consistently, enhances MHC-I presentation of a Gag-derived epitope. Although the half-life of the PTAP mutant was only mildly reduced, the entry into the DRiP pathway was significantly increased, as demonstrated by short-term pulse-chase analyses under proteasome inhibition. Collectively, these results indicate that, besides driving virus release, the PTAP motif regulates the entry of Gag into the DRiP pathway and, thus, into the MHC-I pathway. Although there are no naturally occurring PTAP mutants of HIV-1, mutations of PTAP might enhance the immunogenicity of Gag and, thus, be considered for the improvement of vaccine development.

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The Nucleocapsid Region of HIV-1 Gag Cooperates with the PTAP and LYPXnL Late Domains to Recruit the Cellular Machinery Necessary for Viral Budding

Cited by 133 publications

References 82 publications

In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters

In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters

Properties and functions of the nucleocapsid protein in virus assembly

The PTAP Sequence within the p6 Domain of Human Immunodeficiency Virus Type 1 Gag Regulates Its Ubiquitination and MHC Class I Antigen Presentation

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