Analysis of HIV-1 Matrix-Envelope Cytoplasmic Tail Interactions

Alfadhli, Ayna; Staubus, August O.; Tedbury, Philip R.; Novikova, Mariia; Freed, Eric O.; Barklis, Eric

doi:10.1128/jvi.01079-19

Cited by 42 publications

(51 citation statements)

References 63 publications

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“…We have also demonstrated rescue of the 74LG MA trimerization defect in the 34VI/43FL/74LG triple mutant in recently published work, using recombinant proteins for an in vitro MA UV cross-linking assay and to demonstrate MA-CT interaction (43). That study demonstrates that 34VI/43FL/74LG recovers MA trimerization and CT interaction, which had been lost in the 74LG mutant; MA trimerization and CT interaction had previously been demonstrated for the WT MA protein (44).…”

Section: Discussionsupporting

confidence: 69%

HIV-1 Matrix Trimerization-Impaired Mutants Are Rescued by Matrix Substitutions That Enhance Envelope Glycoprotein Incorporation

Tedbury

Novikova

Alfadhli

et al. 2019

J Virol

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The matrix (MA) domain of HIV-1 Gag plays key roles in virus assembly by targeting the Gag precursor to the plasma membrane and directing the incorporation of the viral envelope (Env) glycoprotein into virions. The latter function appears to be in part dependent on trimerization of the MA domain of Gag during assembly, as disruption of the MA trimer interface impairs Env incorporation. Conversely, many MA mutations that impair Env incorporation can be rescued by compensatory mutations in the trimer interface. In this study, we sought to investigate further the biological significance of MA trimerization by isolating and characterizing compensatory mutations that rescue MA trimer interface mutants with severely impaired Env incorporation. By serially propagating MA trimerization-defective mutants in T cell lines, we identified a number of changes in MA, both within and distant from the trimer interface. The compensatory mutations located within or near the trimer interface restored Env incorporation and particle infectivity and permitted replication in culture. The structure of the MA lattice was interrogated by measuring the cleavage of the murine leukemia virus (MLV) transmembrane Env protein by the viral protease in MLV Env-pseudotyped HIV-1 particles bearing the MA mutations and by performing crystallographic studies of in vitro-assembled MA lattices. These results demonstrate that rescue is associated with structural alterations in MA organization and rescue of MA domain trimer formation. Our data highlight the significance of the trimer interface of the MA domain of Gag as a critical site of protein-protein interaction during HIV-1 assembly and establish the functional importance of trimeric MA for Env incorporation. IMPORTANCE The immature Gag lattice is a critical structural feature of assembling HIV-1 particles, which is primarily important for virion formation and release. While Gag forms a hexameric lattice, driven primarily by the capsid domain, the MA domain additionally trimerizes where three Gag hexamers meet. MA mutants that are defective for trimerization are deficient for Env incorporation and replication, suggesting a requirement for trimerization of the MA domain of Gag in Env incorporation. This study used a gain-of-function, forced viral evolution approach to rescue HIV-1 mutants that are defective for MA trimerization. Compensatory mutations that rescue virus replication do so by restoring Env incorporation and MA trimer formation. This study supports the importance of MA domain trimerization in HIV-1 replication and the potential of the trimer interface as a therapeutic target.

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Section: Discussionsupporting

confidence: 69%

HIV-1 Matrix Trimerization-Impaired Mutants Are Rescued by Matrix Substitutions That Enhance Envelope Glycoprotein Incorporation

Tedbury

Novikova

Alfadhli

et al. 2019

J Virol

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“…This hypothesis was further supported by the finding that Q63R mutation promoted interaction with gp41CT without altering the organization of MA on a membrane layer (41). A correlation between MA trimerization and gp41CT binding was also suggested in a biochemical study involving MA mutants and MACA proteins, supplemented with inositol polyphosphates (64).…”

Section: Discussionmentioning

confidence: 73%

Structural characterization of HIV-1 matrix mutants implicated in envelope incorporation

Eastep

Ghanam

Green

et al. 2020

Preprint

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During the late phase of HIV-1 infection, viral Gag polyproteins are targeted to the plasma membrane (PM) for assembly. Gag localization at the PM is a prerequisite for the incorporation of the envelope protein (Env) into budding particles. Gag assembly and Env incorporation are mediated by the N-terminal myristoylated matrix (MA) domain of Gag. Nonconservative mutations in the trimer interface of MA (A45E, T70R, and L75G) were found to impair Env incorporation and infectivity, leading to the hypothesis that MA trimerization is an obligatory step for Env incorporation. Conversely, Env incorporation can be rescued by a compensatory mutation in the MA trimer interface (Q63R). The impact of these MA mutations on the structure and trimerization properties of MA is not known. In this study, we employed NMR spectroscopy, x-ray crystallography, and sedimentation techniques to characterize the structure and trimerization properties of HIV-1 MA A45E, Q63R, T70R, and L75G mutant proteins. NMR data revealed that these point mutations did not alter the overall structure and folding of MA but caused minor structural perturbations in the trimer interface. Analytical ultracentrifugation data indicated that mutations had a minimal effect on the MA monomertrimer equilibrium. The high-resolution x-ray structure of the unmyristoylated MA Q63R protein revealed hydrogen bonding between the side chains of Arg-63 and Ser-67 located in the center of the trimer interface, providing the first structural evidence for a stabilization of the trimer form. These findings advance our knowledge of the interplay of MA trimerization and Env incorporation into HIV-1 particles.

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“…WT HIV-1 MA has been reported to trimerize variously as a 3D crystal (34), at high concentration in solution (35), when bound to biomimetic membranes (36) and in the mature viral particle (37). Trimerization of MA is thought to be important to accommodate the large cytoplasmic tail of the viral envelope protein for its proper packaging into infectious virus (37–39). Although the concentrations at which MA has been reported to trimerize in solution are significantly higher (~70 μM half point) than those probed by DC z-scan, PM binding likely favors oligomerization through increased MA-MA proximity due to partitioning into the reduced (effectively 2-dimensional) volume at the PM.…”

Section: Discussionmentioning

confidence: 99%

Sensitive Detection of Protein Binding to the Plasma Membrane with Dual-Color Z-Scan Fluorescence

Angert

Karuka

Hennen

et al. 2019

Preprint

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Delicate and transitory protein engagement at the plasma membrane (PM) is crucial to a broad range of cellular functions including cell motility, signal transduction, and virus replication. Here we describe a dual color (DC) extension of the fluorescence z-scan technique which has proven successful for quantification of peripheral membrane protein binding to the PM in living cells. We demonstrate that the co-expression of a second distinctly colored fluorescent protein provides a soluble reference species, which delineates the extent of the cell cytoplasm and lowers the detection threshold of z-scan PM binding measurements by an order of magnitude. DC z-scan generates an intensity profile for each detection channel that contains information on the axial distribution of the peripheral membrane and reference protein. Fit models for DC z-scan are developed and verified using simple model systems. Next, we apply the quantitative DC z-scan technique to investigate the binding of two peripheral membrane protein systems for which previous z-scan studies failed to detect binding: human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein and lipidation-deficient mutants of the fibroblast growth factor receptor substrate 2α. Our findings show that these mutations severely disrupt PM association of fibroblast growth factor receptor substrate 2α but do not eliminate it. We further detected binding of HIV-1 MA to the PM using DC z-scan. Interestingly, our data indicate that HIV-1 MA binds cooperatively to the PM with a dissociation coefficient of Kd ~16 μM and Hill coefficient of n ~2. SIGNIFICANCE Protein binding to the plasma membrane of cells plays an important role in a multitude of cell functions and disease processes. Quantitative binding studies of protein/membrane interactions are almost exclusively limited to in vitro systems and may produce results that poorly mimic the authentic interactions in living cells. We report quantitative measurements of plasma membrane binding directly in living cells by using dual color z-scan fluorescence, which improves the detection threshold by an order of magnitude compared to our previous single color technique. This advance allowed us to examine the role of mutations on binding affinity and identify the presence of cooperative binding in protein systems with relevance to HIV/AIDS and cancer biology.

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Analysis of HIV-1 Matrix-Envelope Cytoplasmic Tail Interactions

Cited by 42 publications

References 63 publications

HIV-1 Matrix Trimerization-Impaired Mutants Are Rescued by Matrix Substitutions That Enhance Envelope Glycoprotein Incorporation

HIV-1 Matrix Trimerization-Impaired Mutants Are Rescued by Matrix Substitutions That Enhance Envelope Glycoprotein Incorporation

Structural characterization of HIV-1 matrix mutants implicated in envelope incorporation

Sensitive Detection of Protein Binding to the Plasma Membrane with Dual-Color Z-Scan Fluorescence

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