Human Immunodeficiency Virus Type 1 Matrix Protein Assembles on Membranes as a Hexamer

Alfadhli, Ayna; Huseby, Douglas L.; Kapit, Eliot; Colman, Dalbinder; Barklis, Eric

doi:10.1128/jvi.02122-06

Cited by 50 publications

(65 citation statements)

References 62 publications

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“…Sendai virus and influenza virus matrix proteins have also been shown to form helical polymers and a lattice structure at the cell membrane (2,4,14,15,26). Last, the human immunodeficiency virus type 1 matrix protein has been shown recently to form hexamer rings when bound to a lipid membrane (1).…”

Section: Discussionmentioning

confidence: 99%

Role for Amino Acids ₂₁₂ KLR ₂₁₄ of Ebola Virus VP40 in Assembly and Budding

McCarthy

Johnson²,

Zhang

et al. 2007

J Virol

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Ebola virus VP40 is able to produce virus-like particles (VLPs) in the absence of other viral proteins. At least three domains within VP40 are thought to be required for efficient VLP release: the late domain (L-domain), membrane association domain (M-domain), and self-interaction domain (I-domain). While the L-domain of Ebola VP40 has been well characterized, the exact mechanism by which VP40 mediates budding through the M-and I-domains remains unclear. To identify additional domains important for VP40 assembly/budding, amino acids 212 KLR 214 were targeted for mutagenesis based on the published crystal structure of VP40. These residues are part of a loop connecting two beta sheets in the C-terminal region and thus are potentially important for overall structure and/or oligomerization of VP40. A series of alanine substitutions were generated in the KLR region of VP40, and these mutants were examined for VLP budding, intracellular localization, and oligomerization. Our results indicated that (i) 212 KLR 214 residues of VP40 are important for efficient release of VP40 VLPs, with Leu213 being the most critical; (ii) VP40 KLR mutants displayed altered patterns of cellular localization compared to that of wild-type VP40 (VP40-WT); and (iii) self-assembly of VP40 KLR mutants into oligomers was altered compared to that of VP40-WT. These results suggest that 12 KLR 214 residues of VP40 are important for proper assembly/oligomerization of VP40 which subsequently leads to efficient budding of VLPs.Ebola virus (EBOV) and Marburg virus are members of the negative-stranded RNA virus family Filoviridae (7). Filoviruses are associated with severe hemorrhagic fevers and high mortality and morbidity with fatality rates reaching 90% for EBOV Zaire strain. There are currently no approved vaccines or therapeutics for treatment of filovirus infections (8,22).The EBOV VP40 matrix protein is the most abundant protein in virions (6) and is able to produce virus-like particles (VLPs) in the absence of other viral proteins (13,17,24,30). Ebola VP40 VLPs are virtually identical in size and morphology to infectious Ebola virions (3,17,18). VP40 contains overlapping late domains (L-domains) at the N terminus that interact with host proteins to mediate separation of newly made virions from the plasma membrane (13,17,21,29). Previous work has shown that, in addition to the L-domain, VP40 possesses a membrane association domain(s) [M-domain(s)] and a self-interaction domain(s) [I-domain(s)] which have yet to be completely characterized. The putative M-domain and I-domain of Ebola VP40 are thought to be located in the Cterminal and N-terminal halves of the protein, respectively (5,17,25,27,29,30). Structural studies by Dessen et al. elucidated the crystal structure of VP40 and demonstrated that the N-and C-terminal domains were structurally similar beta-sandwiches connected by a flexible linker consisting of residues 188 to 202 (5). Trypsin treatment of bacterially purified VP40 (residues 31 to 326) resulted in cleavage after Lys212 and dissoc...

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Section: Discussionmentioning

confidence: 99%

Role for Amino Acids ₂₁₂ KLR ₂₁₄ of Ebola Virus VP40 in Assembly and Budding

McCarthy

Johnson²,

Zhang

et al. 2007

J Virol

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“…In addition, exposure of the myr group is coupled with protein trimerization (57). Despite the evidence that MA can form trimers and assemble on membrane as hexamers (87,88), the role of MA oligomerization in stabilizing Gag-Gag interactions is still controversial (57, 88 -93). Our data presented here show that exposure of the myr group upon binding to CaM does not induce the formation of MA trimer.…”

Section: Discussionmentioning

confidence: 99%

Binding of Calmodulin to the HIV-1 Matrix Protein Triggers Myristate Exposure

Ghanam

Fernandez

Fledderman

et al. 2010

Journal of Biological Chemistry

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Steady progress has been made in defining both the viral and cellular determinants of retroviral assembly and release. Although it is widely accepted that targeting of the Gag polypeptide to the plasma membrane is critical for proper assembly of HIV-1, the intracellular interactions and trafficking of Gag to its assembly sites in the infected cell are poorly understood. HIV-1 Gag was shown to interact and co-localize with calmodulin (CaM), a ubiquitous and highly conserved Ca 2؉ -binding protein expressed in all eukaryotic cells, and is implicated in a variety of cellular functions. Binding of HIV-1 Gag to CaM is dependent on calcium and is mediated by the N-terminally myristoylated matrix (myr(؉)MA) domain. Herein, we demonstrate that CaM binds to myr(؉)MA with a dissociation constant (K d ) of ϳ2 M and 1:1 stoichiometry. Strikingly, our data revealed that CaM binding to MA induces the extrusion of the myr group. However, in contrast to all known examples of CaM-binding myristoylated proteins, our data show that the myr group is exposed to solvent and not involved in CaM binding. The interactions between CaM and myr(؉)MA are endothermic and entropically driven, suggesting that hydrophobic contacts are critical for binding. As revealed by NMR data, both CaM and MA appear to engage substantial regions and/or undergo significant conformational changes upon binding. We believe that our findings will provide new insights on how Gag may interact with CaM during the HIV replication cycle.Gag is the major structural protein encoded by HIV-1 and contains all of the viral elements required to drive virus assembly (1-3). HIV-1 Gag targeting to the plasma membrane (PM) 2 is critical for proper and efficient assembly to produce progeny virions (1, 3-9). During virus maturation, Gag is cleaved into myristoylated matrix (myr(ϩ)MA), capsid, and nucleocapsid proteins, inducing major morphological reorganization of the virus (1, 2, 4, 5, 10). In many cell types, HIV-1 Gag budding and assembly has been shown to occur predominantly on the PM (4 -9, 11-18). Gag binding to the PM is mediated by the MA domain and enhanced by multimerization. Proper assembly and efficient binding of Gag to the PM requires a myristyl (myr) group as a membrane anchor and a cluster of basic residues localized within the N-terminal domain to facilitate interactions with acidic phospholipids (1,2,19,20).Steady progress has been made in defining both the viral and cellular determinants of HIV-1 assembly and release (6). However, the trafficking pathway used by Gag to reach assembly sites in the infected cell is poorly understood. Studies by Freed, Ono, and co-workers (21-23) demonstrated that the ultimate localization of HIV-1 Gag at virus assembly sites is dependent on phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P 2 ), a cellular factor localized at the inner leaflet of the PM (24 -26). Our structural studies revealed that PI(4,5)P 2 binds directly to HIV-1 MA, inducing a conformational change that triggers myr exposure (27). In addition to PI(4,5)P 2 ...

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“…The MA domain does not change structure when tethered to CA NTD , consistent with the idea that the membrane-binding "heads" of MA are connected via flexible linkers to the CA NTD hexamers below. The oligomeric state of these membrane-bound MA proteins has not yet been established, however, since the matrix layer does not form a continuous lattice [22], and MA proteins can form both trimers [25,40,43,55] and hexamers [56] in vitro. Interestingly, the MA and CA domains of RSV Gag are separated by an extended linker, called p10, which contributes to Gag assembly by forming an α-helical bundle with the neighboring subunit in the Gag hexamer ( [57], V. Vogt, pers.…”

Section: Gag-gag Lattice Interactions In the Immature Virion Are Medimentioning

confidence: 99%

The structural biology of HIV assembly

Ganser‐Pornillos

Yeager

Sundquist

2008

Current Opinion in Structural Biology

413

422

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HIV assembly and replication proceed through formation of morphologically distinct immature and mature viral capsids that are organized by the Gag polyprotein (immature) and by the fully processed CA protein (mature). The Gag polyprotein is composed of three folded polypeptides (MA, CA, and NC) and three smaller peptides (SP1, SP2, and p6) that function together to coordinate membrane binding and Gag-Gag lattice interactions in immature virions. Following budding, HIV maturation is initiated by proteolytic processing of Gag, which induces conformational changes in the CA domain and results in assembly of the distinctive conical capsid. Retroviral capsids are organized following the principles of fullerene cones, and the hexagonal CA lattice is stabilized by three distinct interfaces. Recently identified inhibitors of viral maturation act by disrupting the final stage of Gag processing, or by inhibiting formation of a critical intermolecular CA-CA interface in the mature capsid. Following release into a new host cell, the capsid disassembles and host cell factors can potently restrict this stage of retroviral replication. Here, we review the structures of immature and mature HIV virions, focusing on recent studies that have defined the global organization of the immature Gag lattice, identified sites likely to undergo conformational changes during maturation, revealed the molecular structure of the mature capsid lattice, demonstrated that capsid architectures are conserved, identified the first capsid assembly inhibitors, and begun to uncover the remarkable biology of the mature capsid.

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Human Immunodeficiency Virus Type 1 Matrix Protein Assembles on Membranes as a Hexamer

Cited by 50 publications

References 62 publications

Role for Amino Acids ₂₁₂ KLR ₂₁₄ of Ebola Virus VP40 in Assembly and Budding

Role for Amino Acids ₂₁₂ KLR ₂₁₄ of Ebola Virus VP40 in Assembly and Budding

Binding of Calmodulin to the HIV-1 Matrix Protein Triggers Myristate Exposure

The structural biology of HIV assembly

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Human Immunodeficiency Virus Type 1 Matrix Protein Assembles on Membranes as a Hexamer

Cited by 50 publications

References 62 publications

Role for Amino Acids 212 KLR 214 of Ebola Virus VP40 in Assembly and Budding

Role for Amino Acids 212 KLR 214 of Ebola Virus VP40 in Assembly and Budding

Binding of Calmodulin to the HIV-1 Matrix Protein Triggers Myristate Exposure

The structural biology of HIV assembly

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Role for Amino Acids ₂₁₂ KLR ₂₁₄ of Ebola Virus VP40 in Assembly and Budding

Role for Amino Acids ₂₁₂ KLR ₂₁₄ of Ebola Virus VP40 in Assembly and Budding