scholarly journals Dynamic Fluorescent Imaging of Human Immunodeficiency Virus Type 1 Gag in Live Cells by Biarsenical Labeling

2005 ◽  
Vol 79 (7) ◽  
pp. 4055-4065 ◽  
Author(s):  
Lynnie Rudner ◽  
Sascha Nydegger ◽  
Lori V. Coren ◽  
Kunio Nagashima ◽  
Markus Thali ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Fluorescent Imaging ◽  
Live Cells ◽  
Color Analysis ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Gag is the primary structural protein of the virus and is sufficient for particle formation. We utilized the recently developed biarsenical-labeling method to dynamically observe HIV-1 Gag within live cells by adding a tetracysteine tag (C-C-P-G-C-C) to the C terminus of Gag in both Pr55Gag expression and full-length proviral constructs. Membrane-permeable biarsenical compounds FlAsH and ReAsH covalently bond to this tetracysteine sequence and specifically fluoresce, effectively labeling Gag in the cell. Biarsenical labeling readily and specifically detected a tetracysteine-tagged HIV-1 Gag protein (Gag-TC) in HeLa, Mel JuSo, and Jurkat T cells by deconvolution fluorescence microscopy. Gag-TC was localized primarily at or near the plasma membrane in all cell types examined. Fluorescent two-color analysis of Gag-TC in HeLa cells revealed that nascent Gag was present mostly at the plasma membrane in distinct regions. Intracellular imaging of a Gag-TC myristylation mutant observed a diffuse signal throughout the cell, consistent with the role of myristylation in Gag localization to the plasma membrane. In contrast, mutation of the L-domain core sequence did not appreciably alter the localization of Gag, suggesting that the PTAP L domain functions at the site of budding rather than as a targeting signal. Taken together, our results show that Gag concentrates in specific plasma membrane areas rapidly after translation and demonstrate the utility of biarsenical labeling for visualizing the dynamic localization of Gag.

scholarly journals Kinesin KIF4 Regulates Intracellular Trafficking and Stability of the Human Immunodeficiency Virus Type 1 Gag Polyprotein

2008 ◽  
Vol 82 (20) ◽  
pp. 9937-9950 ◽  
Author(s):  
Nathaniel W. Martinez ◽  
Xiaoxiao Xue ◽  
Reem G. Berro ◽  
Geri Kreitzer ◽  
Marilyn D. Resh
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Particle Production ◽  
Particle Assembly ◽  
Gag Protein ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Retroviral Gag proteins are synthesized as soluble, myristoylated precursors that traffic to the plasma membrane and promote viral particle production. The intracellular transport of human immunodeficiency virus type 1 (HIV-1) Gag to the plasma membrane remains poorly understood, and cellular motor proteins responsible for Gag movement are not known. Here we show that disrupting the function of KIF4, a kinesin family member, slowed temporal progression of Gag through its trafficking intermediates and inhibited virus-like particle production. Knockdown of KIF4 also led to increased Gag degradation, resulting in reduced intracellular Gag protein levels; this phenotype was rescued by reintroduction of KIF4. When KIF4 function was blocked, Gag transiently accumulated in discrete, perinuclear, nonendocytic clusters that colocalized with endogenous KIF4, with Ubc9, an E2 SUMO-1 conjugating enzyme, and with SUMO. These studies identify a novel transit station through which Gag traffics en route to particle assembly and highlight the importance of KIF4 in regulating HIV-1 Gag trafficking and stability.

scholarly journals Cell-Type-Dependent Targeting of Human Immunodeficiency Virus Type 1 Assembly to the Plasma Membrane and the Multivesicular Body

2004 ◽  
Vol 78 (3) ◽  
pp. 1552-1563 ◽  
Author(s):  
Akira Ono ◽  
Eric O. Freed
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Multivesicular Body ◽  
Cell Type ◽  
Gag Proteins ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) assembly-and-release pathway begins with the targeting of the Gag precursor to the site of virus assembly. The molecular mechanism by which Gag is targeted to the appropriate subcellular location remains poorly understood. Based on the analysis of mutant Gag proteins, we and others have previously demonstrated that a highly basic patch in the matrix (MA) domain of Gag is a major determinant of Gag transport to the plasma membrane. In this study, we determined that in HeLa and T cells, the MA mutant Gag proteins that are defective in plasma membrane targeting form virus particles in a CD63-positive compartment, defined as the late endosome or multivesicular body (MVB). Interestingly, we find that in primary human macrophages, both wild-type (WT) and MA mutant Gag proteins are targeted specifically to the MVB. Despite the fact that particle assembly in macrophages occurs at an intracellular site rather than at the plasma membrane, we observe that WT Gag expressed in this cell type is released as extracellular virions with high efficiency. These results demonstrate that Gag targeting to and assembly in the MVB are physiologically important steps in HIV-1 virus particle production in macrophages and that particle release in this cell type may follow an exosomal pathway. To determine whether Gag targeting to the MVB is the result of an interaction between the late domain in p6Gag and the MVB sorting machinery (e.g., TSG101), we examined the targeting and assembly of Gag mutants lacking p6. Significantly, the MVB localization of Gag was still observed in the absence of p6, suggesting that an interaction between Gag and TSG101 is not required for Gag targeting to the MVB. These data are consistent with a model for Gag targeting that postulates two different cellular binding partners for Gag, one on the plasma membrane and the other in the MVB.

scholarly journals Productive Human Immunodeficiency Virus Type 1 Assembly Takes Place at the Plasma Membrane

2007 ◽  
Vol 81 (14) ◽  
pp. 7476-7490 ◽  
Author(s):  
Andrés Finzi ◽  
Alexandre Orthwein ◽  
Johanne Mercier ◽  
Éric A. Cohen
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Multivesicular Body ◽  
Subcellular Fractionation ◽  
Particle Assembly ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Gag proteins are necessary and sufficient to direct human immunodeficiency virus type 1 (HIV-1) particle assembly and budding. Recent evidence suggests that Gag targeting to late endosomal/multivesicular body (LE/MVB) compartments occurs prior to viral particle budding at the plasma membrane (PM). However, the route that Gag follows before reaching its steady-state destinations still remains a subject of debate. Using a subcellular fractionation method that separates PM from LE/MVB combined with pulse-chase labeling, we analyzed Gag trafficking in HIV-1-producing HEK 293T cells. Our results reveal that the majority of newly synthesized Gag is primarily targeted to the PM. While PM-targeted Gag was efficiently released, a significant fraction of the remaining cell surface-associated Gag was found to be subsequently internalized to LE/MVB, where it accumulated, thus accounting for the majority of LE/MVB-associated Gag. Importantly, this accumulation of Gag in LE/MVB was found to be cholesterol dependent since it was sensitive to the sterol-binding drugs filipin and methyl-β-cyclodextrin. These results point towards the PM as being the primary site of productive HIV-1 assembly in cells that also support Gag accumulation in intracellular compartments.

scholarly journals Human Immunodeficiency Virus Type 1 Matrix Inhibits and Confers Cooperativity on Gag Precursor-Membrane Interactions

2004 ◽  
Vol 78 (17) ◽  
pp. 9560-9563 ◽  
Author(s):  
David Perez-Caballero ◽  
Theodora Hatziioannou ◽  
Juan Martin-Serrano ◽  
Paul D. Bieniasz
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Specific Targeting ◽  
Immunodeficiency Virus ◽  
Targeting Signal ◽  
Hiv 1 ◽  
Globular Head

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Gag multimerization and membrane binding are required for particle formation. However, it is unclear what constitutes a minimal plasma membrane-specific targeting signal and what role the matrix (MA) globular head and other Gag domains play in membrane targeting. Here, we use membrane flotation and microscopic analysis of Gag deletion mutants to demonstrate that the HIV-1 MA globular head inhibits a plasma membrane-specific targeting signal contained within the six amino-terminal MA residues. MA-mediated inhibition is relieved by concentration-dependent Gag multimerization and imparts a high degree of cooperativity on Gag-membrane association. This cooperativity may confer temporal and spatial regulation on HIV-1 assembly.

scholarly journals Vpu and Tsg101 Regulate Intracellular Targeting of the Human Immunodeficiency Virus Type 1 Core Protein Precursor Pr55gag

2006 ◽  
Vol 80 (8) ◽  
pp. 3765-3772 ◽  
Author(s):  
Kirsi Harila ◽  
Ian Prior ◽  
Mathilda Sjöberg ◽  
Antti Salminen ◽  
Jorma Hinkula ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Core Protein ◽  
Virus Assembly ◽  
Host Protein ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Assembly of human immunodeficiency virus type 1 (HIV-1) is directed by the viral core protein Pr55 gag . Depending on the cell type, Pr55 gag accumulates either at the plasma membrane or on late endosomes/multivesicular bodies. Intracellular localization of Pr55 gag determines the site of virus assembly, but molecular mechanisms that define cell surface or endosomal targeting of Pr55 gag are poorly characterized. We have analyzed targeting of newly synthesized Pr55 gag in HeLa H1 cells by pulse-chase studies and subcellular fractionations. Our results indicated that Pr55 gag was inserted into the plasma membrane and, when coexpressed with the viral accessory protein Vpu, Pr55 gag remained at the plasma membrane and virions assembled at this site. In contrast, Pr55 gag expressed in the absence of Vpu was initially inserted into the plasma membrane, but subsequently endocytosed, and virus assembly was partially shifted to internal membranes. This endocytosis of Pr55 gag required the host protein Tsg101. These results identified a previously unknown role for Vpu and Tsg101 as regulators for the endocytic uptake of Pr55 gag and suggested that the site of HIV-1 assembly is determined by factors that regulate the endocytosis of Pr55 gag .

scholarly journals Human Immunodeficiency Virus Type 1 Assembly, Budding, and Cell-Cell Spread in T Cells Take Place in Tetraspanin-Enriched Plasma Membrane Domains

2007 ◽  
Vol 81 (15) ◽  
pp. 7873-7884 ◽  
Author(s):  
Clare Jolly ◽  
Quentin J. Sattentau
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Membrane Domains ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Cell Cell

ABSTRACT Human immunodeficiency virus type-1 (HIV-1) egress from infected CD4+ T cells is thought to be via assembly and budding at the plasma membrane and may involve components of the T-cell secretory apparatus, including tetraspanins. However, many studies on HIV-1 assembly have examined the trafficking of viral proteins in isolation, and most have used immortalized epithelial, fibroblastic, or hematopoietic cell lines that may not necessarily reflect natural infection of susceptible T cells. Here we have used immunofluorescence and cryoimmunoelectron microscopy (CEM) to examine protein transport during HIV-1 assembly in productively infected Jurkat CD4+ T cells and primary CD4+ T cells. The HIV-1 envelope glycoprotein (Env) and the core protein (Gag) colocalize strongly with CD63 and CD81 and less strongly with CD9, whereas no colocalization was seen between Env or Gag and the late endosome/lysosomal marker Lamp2. CEM revealed incorporation of CD63 and CD81 but not Lamp2 into virions budding at the plasma membrane, and this was supported by immunoprecipitation studies, confirming that HIV-1 egress in T cells is trafficked via tetraspanin-enriched membrane domains (TEMs) that are distinct from lysosomal compartments. CD63, CD81, and, to a lesser extent, CD9 were recruited to the virological synapse (VS), and antibodies against these tetraspanins reduced VS formation. We propose that HIV-1 promotes virus assembly and cell-cell transfer in T cells by targeting plasma membrane TEMs.

scholarly journals Mobility of Human Immunodeficiency Virus Type 1 Pr55Gag in Living Cells

2006 ◽  
Vol 80 (17) ◽  
pp. 8796-8806 ◽  
Author(s):  
Candace Y. Gomez ◽  
Thomas J. Hope
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Fluorescent Protein ◽  
Live Cells ◽  
Particle Assembly ◽  
Immunodeficiency Virus ◽  
Green Fluorescent

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) assembly requires the converging of thousands of structural proteins on cellular membranes to form a tightly packed immature virion. The Gag polyprotein contains all of the determinants important for viral assembly and must move around in the cell in order to form particles. This work has focused on Gag mobility in order to provide more insights into the dynamics of particle assembly. Key to these studies was the use of several fluorescently labeled Gag derivatives. We used fluorescence recovery after photobleaching as well as photoactivation to determine Gag mobility. Upon expression, Gag can be localized diffusely in the cytoplasm, associated with the plasma membrane, or in virus-like particles (VLPs). Here we show that Gag VLPs are primarily localized in the plasma membrane and do not colocalize with CD63. We have shown using full-length Gag as well as truncation mutants fused to green fluorescent protein that Gag is highly mobile in live cells when it is not assembled into VLPs. Results also showed that this mobility is highly dependent upon cholesterol. When cholesterol is depleted from cells expressing Gag, mobility is significantly decreased. Once cholesterol was replenished, Gag mobility returned to wild-type levels. Taken together, results from these mobility studies suggest that Gag is highly mobile and that as the assembly process proceeds, mobility decreases. These studies also suggest that Gag assembly must occur in cholesterol-rich domains in the plasma membrane.

scholarly journals The Host Protein Staufen1 Participates in Human Immunodeficiency Virus Type 1 Assembly in Live Cells by Influencing pr55Gag Multimerization

2007 ◽  
Vol 81 (12) ◽  
pp. 6216-6230 ◽  
Author(s):  
Laurent Chatel-Chaix ◽  
Levon Abrahamyan ◽  
Céline Fréchina ◽  
Andrew J. Mouland ◽  
Luc DesGroseillers
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Host Protein ◽  
Live Cells ◽  
Cell Fractionation ◽  
Gag Protein ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) requires the sequential activities of virus-encoded proteins during replication. The activities of several host cell proteins and machineries are also critical to the completion of virus assembly and the release of infectious virus particles from cells. One of these proteins, the double-stranded RNA-binding protein Staufen1 (Stau1), selectively associates with the HIV-1 genomic RNA and the viral precursor Gag protein, pr55Gag. In this report, we tested whether Stau1 modulates pr55Gag assembly using a new and specific pr55Gag oligomerization assay based on bioluminescence resonance energy transfer (BRET) in both live cells and extracts after cell fractionation. Our results show that both the overexpression and knockdown of Stau1 increase the pr55Gag-pr55Gag BRET levels, suggesting a role for Stau1 in regulating pr55Gag oligomerization during assembly. This effect of Stau1 on pr55Gag oligomerization was observed only in membranes, a cellular compartment in which pr55Gag assembly primarily occurs. Consistently, expression of Stau1 harboring a vSrc myristylation signal led to a 6.5-fold enrichment of Stau1 in membranes and a corresponding enhancement in the Stau1-mediated effect on pr55Gag-pr55Gag BRET, demonstrating that Stau1 acts on assembly when targeted to membranes. A role for Stau1 in the formation of particles is further supported by the detection of membrane-associated detergent-resistant pr55Gag complexes and the increase of virus-like particle release when Stau1 expression levels are modulated. Our results indicate that Stau1 influences HIV-1 assembly by modulating pr55Gag-pr55Gag interactions, as shown in a live cell interaction assay. This likely occurs when Stau1 interacts with membrane-associated assembly intermediates.

scholarly journals Human Immunodeficiency Virus Type 1 Protease Triggers a Myristoyl Switch That Modulates Membrane Binding of Pr55gag and p17MA

1999 ◽  
Vol 73 (3) ◽  
pp. 1902-1908 ◽  
Author(s):  
Luz Hermida-Matsumoto ◽  
Marilyn D. Resh
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Membrane Binding ◽  
Immunodeficiency Virus ◽  
Membrane Bound ◽  
Myristoyl Switch ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Pr55 gag gene product directs the assembly of virions at the inner surface of the cell plasma membrane. The specificity of plasma membrane binding by Pr55 gag is conferred by a combination of an N-terminal myristoyl moiety and a basic residue-rich domain. Although the myristate plus basic domain is also present in the p17MA proteolytic product formed upon Pr55 gag maturation, the ability of p17MA to bind to membranes is significantly reduced. It was previously reported that the reduced membrane binding of p17MA was due to sequestration of the myristate moiety by a myristoyl switch (W. Zhou and M. D. Resh, J. Virol. 70:8540–8548, 1996). Here we demonstrate directly that treatment of membrane-bound Pr55 gag in situ with HIV-1 protease generates p17MA, which is then released from the membrane. Pr55 gag was synthesized in reticulocyte lysates, bound to membranes, and incubated with purified HIV-1 protease. The p17MA product in the membrane-bound and soluble fractions was analyzed following proteolysis. Newly generated p17MA initially was membrane bound but then displayed a slow, time-dependent dissociation resulting in 65% solubilization. Residual p17MA could be extracted from the membranes with either high pH or high salt. Treatment of membranes from transfected COS-1 cells with protease revealed that Pr55 gag was present within sealed membrane vesicles and that the release of p17MA occurred only when detergent and salt were added. We present a model proposing that the HIV-1 protease is the “trigger” for a myristoyl switch mechanism that modulates the membrane associations of Pr55 gag and p17MA in virions and membranes.

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