Interrelationship between Cytoplasmic Retroviral Gag Concentration and Gag–Membrane Association

Fogarty, Keir; Berk, Serkan; Grigsby, Iwen F.; Chen, Yan; Mansky, Louis M.; Mueller, Joachim D.

doi:10.1016/j.jmb.2013.11.025

Cited by 38 publications

(46 citation statements)

References 72 publications

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“…5B), indicating that the majority of HIV/HIV-N Gag was unable to translocate to the PM. This agrees well with previous findings that indicate HIV-1 WT Gag-Gag interactions must occur before efficient Gag translocation to the PM (34).…”

supporting

confidence: 93%

“…HTLV-1 and HIV-1 Gag proteins have markedly different Gag-Gag interactions and PM trafficking phenotypes based on previous studies (33,34). While HIV-1 Gag-Gag interactions occur in the cytoplasm, HTLV-1 Gag-Gag interactions initiate at the PM.…”

mentioning

confidence: 91%

“…5A), indicative of a concentration-dependent change from Gag monomer to oligomer (34). Unlike HIV-1 WT Gag, HTLV-1 WT Gag was found at the PM (Fig.…”

mentioning

confidence: 95%

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Disparate Contributions of Human Retrovirus Capsid Subdomains to Gag-Gag Oligomerization, Virus Morphology, and Particle Biogenesis

Martin

Mendonça

Angert

et al. 2017

J Virol

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The capsid domain (CA) of the retroviral Gag protein is a primary determinant of Gag oligomerization, which is a critical step for immature Gag lattice formation and virus particle budding. Although the human immunodeficiency virus type 1 (HIV-1) CA carboxy-terminal domain (CTD) is essential for CA-CA interactions, the CA CTD has been suggested to be largely dispensable for human T-cell leukemia virus type 1 (HTLV-1) particle biogenesis. To more clearly define the roles of the HTLV-1 CA amino-terminal domain (NTD) and CA CTD in particle biogenesis, we generated and analyzed a panel of Gag proteins with chimeric HIV-1/HTLV-1 CA domains. Subcellular distribution and protein expression levels indicated that Gag proteins with a chimeric HIV-1 CA NTD/HTLV-1 CA CTD did not result in Gag oligomerization regardless of the parent Gag background. Furthermore, chimeric Gag proteins with the HTLV-1 CA NTD produced particles phenotypically similar to HTLV-1 immature particles, highlighting the importance of the HTLV-1 CA NTD in HTLV-1 immature particle morphology. Taken together, these observations support the conclusion that the HTLV-1 CA NTD can functionally replace the HIV-1 CA CTD, but the HIV-1 CA NTD cannot replace the HTLV-1 CA CTD, indicating that the HTLV-1 CA subdomains provide distinct contributions to Gag-Gag oligomerization, particle morphology, and biogenesis. Furthermore, we have shown for the first time that HIV-1 and HTLV-1 Gag domains outside the CA (e.g., matrix and nucleocapsid) impact Gag oligomerization as well as immature particle size and morphology. IMPORTANCE A key aspect in virus replication is virus particle assembly, which is a poorly understood process for most viruses. For retroviruses, the Gag structural protein is the primary driver of virus particle biogenesis, and the CA CTD is the primary determinant of Gag-Gag interactions for HIV-1. In this study, the HTLV-1 capsid amino-terminal domain was found to provide distinct contributions to Gag-Gag oligomerization, particle morphology, and biogenesis. This study provides information that will aid efforts for discovery of therapeutic targets for intervention.

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supporting

confidence: 93%

mentioning

confidence: 91%

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Disparate Contributions of Human Retrovirus Capsid Subdomains to Gag-Gag Oligomerization, Virus Morphology, and Particle Biogenesis

Martin

Mendonça

Angert

et al. 2017

J Virol

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“…Gag multimerization is a critical component of the ability of Gag to bind stably to the PM. RSV MA and HIV MA with and without GFP are not localized to the PM (18,19,(58)(59)(60)(61)(62) The plateaus correspond to equilibrium responses at given protein concentrations. Gag remained bound to the membrane after the buffer rinse, while MA was almost completely removed.…”

Section: Resultsmentioning

confidence: 99%

Membrane Binding of the Rous Sarcoma Virus Gag Protein Is Cooperative and Dependent on the Spacer Peptide Assembly Domain

Dick

Barros

Jin

et al. 2016

J Virol

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The principles underlying membrane binding and assembly of retroviral Gag proteins into a lattice are understood. However, little is known about how these processes are related. Using purified Rous sarcoma virus Gag and Gag truncations, we studied the interrelation of Gag-Gag interaction and Gag-membrane interaction. Both by liposome binding and by surface plasmon resonance on a supported bilayer, Gag bound to membranes much more tightly than did matrix (MA), the isolated membrane binding domain. In principle, this difference could be explained either by protein-protein interactions leading to cooperativity in membrane binding or by the simultaneous interaction of the N-terminal MA and the C-terminal nucleocapsid (NC) of Gag with the bilayer, since both are highly basic. However, we found that NC was not required for strong membrane binding. Instead, the spacer peptide assembly domain (SPA), a putative 24-residue helical sequence comprising the 12-residue SP segment of Gag and overlapping the capsid (CA) C terminus and the NC N terminus, was required. SPA is known to be critical for proper assembly of the immature Gag lattice. A single amino acid mutation in SPA that abrogates assembly in vitro dramatically reduced binding of Gag to liposomes. In vivo, plasma membrane localization was dependent on SPA. Disulfide cross-linking based on ectopic Cys residues showed that the contacts between Gag proteins on the membrane are similar to the known contacts in virus-like particles. Taken together, we interpret these results to mean that Gag membrane interaction is cooperative in that it depends on the ability of Gag to multimerize. IMPORTANCEThe retroviral structural protein Gag has three major domains. The N-terminal MA domain interacts directly with the plasma membrane (PM) of cells. The central CA domain, together with immediately adjoining sequences, facilitates the assembly of thousands of Gag molecules into a lattice. The C-terminal NC domain interacts with the genome, resulting in packaging of viral RNA. For assembly in vitro with purified Gag, in the absence of membranes, binding of NC to nucleic acid somehow facilitates further Gag-Gag interactions that lead to formation of the Gag lattice. The contributions of MA-mediated membrane binding to virus particle assembly are not well understood. Here, we report that in the absence of nucleic acid, membranes provide a platform that facilitates Gag-Gag interactions. This study demonstrates that the binding of Gag, but not of MA, to membranes is cooperative and identifies SPA as a major factor that controls this cooperativity. L ate in the retroviral life cycle, the structural protein Gag localizes to the inner leaflet of the plasma membrane (PM), where it assembles around the viral genomic RNA, leading to budding from the cell. Gag proteins include three major domains, the Nterminal matrix (MA) domain, which mediates membrane binding; the central capsid (CA) domain, which mediates Gag-Gag interactions during virion assembly; and the C-terminal nucleocapsid (NC...

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“…Finally we applied our technique to study the membrane interactions of the matrix domain of human T-cell leukemia virus type 1 (HTLV-1) Gag. We previously observed that the Gag polyprotein and matrix (MA), its membrane-binding domain, of HTLV-1 interact with the plasma membrane at low cytoplasmic concentrations, which is in stark contrast with the corresponding proteins of the human immunodeficiency virus type 1 (HIV-1) (11)(12)(13). Here we expand our investigation of HTLV-1 virus assembly by characterizing the membrane binding curve for the MA domain of HTLV-1 Gag and the lipid-specificity of the interaction.…”

Section: Introductionmentioning

confidence: 99%

In Situ Quantification of Protein Binding to the Plasma Membrane

Smith

Hennen

Chen

et al. 2015

Biophysical Journal

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This study presents a fluorescence-based assay that allows for direct measurement of protein binding to the plasma membrane inside living cells. An axial scan through the cell generates a fluorescence intensity profile that is analyzed to determine the membrane-bound and cytoplasmic concentrations of a peripheral membrane protein labeled by the enhanced green fluorescent protein (EGFP). The membrane binding curve is constructed by mapping those concentrations for a population of cells with a wide range of protein expression levels, and a fit of the binding curve determines the number of binding sites and the dissociation coefficient. We experimentally verified the technique, using myosin-1C-EGFP as a model system and fit its binding curve. Furthermore, we studied the protein-lipid interactions of the membrane binding domains from lactadherin and phospholipase C-δ1 to evaluate the feasibility of using competition binding experiments to identify specific lipid-protein interactions in living cells. Finally, we applied the technique to determine the lipid specificity, the number of binding sites, and the dissociation coefficient of membrane binding for the Gag matrix domain of human T-lymphotropic virus type 1, which provides insight into early assembly steps of the retrovirus.

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Interrelationship between Cytoplasmic Retroviral Gag Concentration and Gag–Membrane Association

Cited by 38 publications

References 72 publications

Disparate Contributions of Human Retrovirus Capsid Subdomains to Gag-Gag Oligomerization, Virus Morphology, and Particle Biogenesis

Disparate Contributions of Human Retrovirus Capsid Subdomains to Gag-Gag Oligomerization, Virus Morphology, and Particle Biogenesis

Membrane Binding of the Rous Sarcoma Virus Gag Protein Is Cooperative and Dependent on the Spacer Peptide Assembly Domain

In Situ Quantification of Protein Binding to the Plasma Membrane

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