Characterization of Cytoplasmic Gag-Gag Interactions by Dual-Color Z-Scan Fluorescence Fluctuation Spectroscopy

Fogarty, Keir; Chen, Yan; Grigsby, Iwen F.; Macdonald, Patrick J.; Smith, Elizabeth M.; Johnson, Jolene; Rawson, Jonathan M. O.; Mansky, Louis M.; Mueller, Joachim D.

doi:10.1016/j.bpj.2011.02.008

Cited by 34 publications

(53 citation statements)

References 29 publications

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“…Based on in vivo cross-linking, Kutluay and Bieniasz (89) concluded that the most abundant HIV-1 species in the cytoplasm is monomeric. Fogarty et al (90) came to a similar conclusion using multiphoton microscopy coupled with quantitative fluorescence fluctuation analysis of Gag-GFP (91,92). With this technology, it is possible to estimate the actual molar concentration of Gag-GFP in the cytoplasm.…”

Section: Amentioning

confidence: 84%

Effect of Multimerization on Membrane Association of Rous Sarcoma Virus and HIV-1 Matrix Domain Proteins

Dick

Kamynina

Vogt

2013

J Virol

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In most retroviruses, plasma membrane (PM) association of the Gag structural protein is a critical step in viral assembly, relying in part on interaction between the highly basic Gag MA domain and the negatively charged inner leaflet of the PM. Assembly is thought to begin with Gag dimerization followed by multimerization, resulting in a hexameric lattice. To directly address the role of multimerization in membrane binding, we fused the MA domains of Rous sarcoma virus (RSV) and HIV-1 to the chemically inducible dimerization domain FK506-binding protein (FKBP) or to the hexameric protein CcmK4 from cyanobacteria. The cellular localization of the resulting green fluorescent protein (GFP)-tagged chimeric proteins was examined by fluorescence imaging, and the association of the proteins with liposomes was quantified by flotation in sucrose gradients, following synthesis in a reticulocyte extract or as purified proteins. Four lipid compositions were tested, representative of liposomes commonly reported in flotation experiments. By themselves, GFP-tagged RSV and HIV-1 MA proteins were largely cytoplasmic, but both hexamerized proteins were highly concentrated at the PM. Dimerization led to partial PM localization for HIV-1 MA. These in vivo effects of multimerization were reproduced in vitro. In flotation analyses, the intact RSV and HIV-1 Gag proteins were more similar to multimerized MA than to monomeric MA. RNA is reported to compete with acidic liposomes for HIV-1 Gag binding, and thus we also examined the effects of RNase treatment or tRNA addition on flotation. tRNA competed with liposomes in the case of some but not all lipid compositions and ionic strengths. Taken together, our results further underpin the model that multimerization is critical for PM association of retroviral Gag proteins. In addition, they suggest that the modulation of membrane binding by RNA, as previously reported for HIV-1, may not hold for RSV.

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Section: Amentioning

confidence: 84%

Effect of Multimerization on Membrane Association of Rous Sarcoma Virus and HIV-1 Matrix Domain Proteins

Dick

Kamynina

Vogt

2013

J Virol

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“…The fluorescently labeled Gag excited during the scan gives rise to a z-scan intensity profile (Fig. 2B and D), which provides information about the axial distribution of the Gag-eYFP at the scan location (61). Fitting of the z-scan intensity profile separates the fluorescence contributions from the cytoplasmic and the PM-bound protein populations (35,36).…”

Section: Methodsmentioning

confidence: 99%

“…First, the intensity profile of the red channel (mCherry) was corrected for spectral cross talk from the eYFP signal and fit to determine the location and thickness of the cytoplasmic volume occupied by mCherry (61). Second, the intensity trace of eYFP was fit to a model that allows Gag-eYFP to be located at the ventral and dorsal PM and in the cytoplasm with the cytoplasmic location and thickness determined by the fit of the mCherry z-scan intensity profile.…”

Section: Methodsmentioning

confidence: 99%

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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“…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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Characterization of Cytoplasmic Gag-Gag Interactions by Dual-Color Z-Scan Fluorescence Fluctuation Spectroscopy

Cited by 34 publications

References 29 publications

Effect of Multimerization on Membrane Association of Rous Sarcoma Virus and HIV-1 Matrix Domain Proteins

Effect of Multimerization on Membrane Association of Rous Sarcoma Virus and HIV-1 Matrix Domain Proteins

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

In Situ Quantification of Protein Binding to the Plasma Membrane

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