ALIX Is a Lys63-Specific Polyubiquitin Binding Protein that Functions in Retrovirus Budding

Dowlatshahi, Dara P.; Sandrin, Virginie; Vivona, Sandro; Shaler, Thomas A.; Kaiser, Stephen E.; Melandri, Francesco; Sundquist, Wesley I.; Kopito, Ron R.

doi:10.1016/j.devcel.2012.10.023

Cited by 74 publications

(76 citation statements)

References 48 publications

(71 reference statements)

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“…8). This finding is consistent with several studies that suggested that ubiquitin is involved in viral budding: (i) proteasome inhibitor treatments block the budding of paramyxoviruses, including PIV5, NiV, and SeV (26,31,58); (ii) potential ubiquitination of the MeV M protein has been observed in cells expressing the M protein together with HA-Ub (38); and (iii) ESCRT factors such as ALIX can bind to ubiquitin and enhance viral budding (59,60).…”

Section: Discussionsupporting

confidence: 79%

A Leucine Residue in the C Terminus of Human Parainfluenza Virus Type 3 Matrix Protein Is Essential for Efficient Virus-Like Particle and Virion Release

Zhang

Ding

et al. 2014

J Virol

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Paramyxovirus particles, like other enveloped virus particles, are formed by budding from membranes of infected cells, and matrix (M) proteins are critical for this process. To identify the M protein important for this process, we have characterized the budding of the human parainfluenza virus type 3 (HPIV3) M protein. Our results showed that expression of the HPIV3 M protein alone is sufficient to initiate the release of virus-like particles (VLPs). Electron microscopy analysis confirmed that VLPs are morphologically similar to HPIV3 virions. We identified a leucine (L302) residue within the C terminus of the HPIV3 M protein that is critical for M protein-mediated VLP production by regulating the ubiquitination of the M protein. When L302 was mutated into A302, ubiquitination of M protein was defective, the release of VLPs was abolished, and the membrane binding and budding abilities of M protein were greatly weakened, but the M L302A mutant retained oligomerization activity and had a dominant negative effect on M protein-mediated VLP production. Furthermore, treatment with a proteasome inhibitor also inhibited M protein-mediated VLP production and viral budding. Finally, recombinant HPIV3 containing the M L302A mutant could not be rescued. These results suggest that L302 acts as a critical regulating signal for the ubiquitination of the HPIV3 M protein and virion release. IMPORTANCEHuman parainfluenza virus type 3 (HPIV3) is an enveloped virus with a nonsegmented negative-strand RNA genome. It can cause severe respiratory tract diseases, such as bronchiolitis, pneumonia, and croup in infants and young children. However, no valid antiviral therapy or vaccine is currently available. Thus, further elucidation of its assembly and budding will be helpful in the development of novel therapeutic approaches. Here, we show that a leucine residue (L302) located at the C terminus of the HPIV3 M protein is essential for efficient production of virus-like particles (VLPs). Furthermore, we found L302 regulated M protein-mediated VLP production via regulation of M protein ubiquitination. Recombinant HPIV3 containing the M L302A mutant is growth defective. These findings provide new insight into the critical role of M protein-mediated VLP production and virion release of a residue that does not belong to L domain and may advance our understanding of HPIV3 viral assembly and budding.

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Section: Discussionsupporting

confidence: 79%

A Leucine Residue in the C Terminus of Human Parainfluenza Virus Type 3 Matrix Protein Is Essential for Efficient Virus-Like Particle and Virion Release

Zhang

Ding

et al. 2014

J Virol

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“…ALIX is a conserved protein in eukaryotes that was implicated in cytokinesis, ILV, and exosome biogenesis and endosomal sorting (28). Human ALIX was also suggested to play a role during viral infection and budding (29)(30)(31). Mammalian ALIX, yeast Bro1p, and their Arabidopsis homolog were all shown to interact with ESCRT-III via the charged multivesicular body protein 4/sucrose nonfermenting 7p (CHMP4/Snf7p) subunit (32)(33)(34)(35).…”

Section: Significancementioning

confidence: 99%

“…The V-domain of both Bro1p and human ALIX was shown to mediate the interaction with ubiquitin in intracellular trafficking (29,31,44). Because the amino acid sequence homology in the Arabidopsis ALIX V-domain is relatively moderate (25% amino acid identity with human ALIX and 17% with Bro1p), we tested whether it could bind ubiquitin.…”

Section: Alix Binds Ubiquitin and Is Involved In Ubiquitin-dependent mentioning

confidence: 99%

Arabidopsis ALIX is required for the endosomal localization of the deubiquitinating enzyme AMSH3

Kalinowska

Nagel

Goodman

et al. 2015

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

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Ubiquitination is a signal for various cellular processes, including for endocytic degradation of plasma membrane cargos. Ubiquitinating as well as deubiquitinating enzymes (DUBs) can regulate these processes by modifying the ubiquitination status of target protein. Although accumulating evidence points to the important regulatory role of DUBs, the molecular basis of their regulation is still not well understood. Associated molecule with the SH3 domain of signal transduction adaptor molecule (STAM) (AMSH) is a conserved metalloprotease DUB in eukaryotes. AMSH proteins interact with components of the endosomal sorting complex required for transport (ESCRT) and are implicated in intracellular trafficking. To investigate how the function of AMSH is regulated at the cellular level, we carried out an interaction screen for the Arabidopsis AMSH proteins and identified the Arabidopsis homolog of apoptosis-linked gene-2 interacting protein X (ALIX) as a protein interacting with AMSH3 in vitro and in vivo. Analysis of alix knockout mutants in Arabidopsis showed that ALIX is essential for plant growth and development and that ALIX is important for the biogenesis of the vacuole and multivesicular bodies (MVBs). Cell biological analysis revealed that ALIX and AMSH3 colocalize on late endosomes. Although ALIX did not stimulate AMSH3 activity in vitro, in the absence of ALIX, AMSH3 localization on endosomes was abolished. Taken together, our data indicate that ALIX could function as an important regulator for AMSH3 function at the late endosomes.Arabidopsis | intracellular trafficking | ubiquitin |

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“…(1) it lacks the ability to recognize ubiquitin directly, although recent reports indicate ES-CRT-III-interacting protein Bro1/Alix (ALG2-interacting protein X) can directly bind ubiquitin (Dowlatshahi et al 2012); (2) it displays no lipid specificity for PtdIns(3)P, implying why it can directly function in membrane remodeling at both endosomes as well as the plasma membrane; and (3) ESCRT-III does not form a stable complex, but rather assembles transiently during membrane sculpting and fission events. ESCRT-III is additionally unique because this membrane-remodeling machine induces invaginations that protrude away from the cell cytoplasm.…”

mentioning

confidence: 99%

Molecular Mechanisms of the Membrane Sculpting ESCRT Pathway

Henne

Stenmark

Emr

2013

Cold Spring Harbor Perspectives in Biology

388

313

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The endosomal sorting complexes required for transport (ESCRT) drive multivesicular body (MVB) biogenesis and cytokinetic abscission. Originally identified through genetics and cell biology, more recent work has begun to elucidate the molecular mechanisms of ESCRTmediated membrane remodeling, with special focus on the ESCRT-III complex. In particular, several light and electron microscopic studies provide high-resolution imaging of ESCRT-III rings and spirals that purportedly drive MVB morphogenesis and abscission. These studies highlight unifying principles to ESCRT-III function, in particular: (1) the ordered assembly of the ESCRT-III monomers into a heteropolymer, (2) ESCRT-III as a dynamic complex, and (3) the role of the AAA ATPase Vps4 as a contributing factor in membrane scission. Mechanistic comparisons of ESCRT-III function in MVB morphogenesis and cytokinesis suggest common mechanisms in membrane remodeling. THE ESCRT PATHWAY IN MVB BIOGENESIS AND CYTOKINESIS

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ALIX Is a Lys63-Specific Polyubiquitin Binding Protein that Functions in Retrovirus Budding

Cited by 74 publications

References 48 publications

A Leucine Residue in the C Terminus of Human Parainfluenza Virus Type 3 Matrix Protein Is Essential for Efficient Virus-Like Particle and Virion Release

A Leucine Residue in the C Terminus of Human Parainfluenza Virus Type 3 Matrix Protein Is Essential for Efficient Virus-Like Particle and Virion Release

Arabidopsis ALIX is required for the endosomal localization of the deubiquitinating enzyme AMSH3

Molecular Mechanisms of the Membrane Sculpting ESCRT Pathway

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