Mapping and Characterization of the N-Terminal I Domain of Human Immunodeficiency Virus Type 1 Pr55Gag

ABSTRACT Human immunodeficiency virus (HIV) type 1 particles assemble at the plasma membrane of cells in a manner similar to that of the type C oncoretroviruses. The Pr55Gag molecule directs the assembly process and is sufficient for particle assembly in the absence of all other viral gene products. The I domain is an assembly domain that has been previously localized to the nucleocapsid (NC) region of Gag. In this study we utilized a series of Gag-green fluorescent protein (GFP) fusion proteins to precisely identify sequences that constitute the N-terminal I domain of Pr55Gag. The minimal sequence required for the I domain was localized to the extreme N terminus of NC. Two basic residues (arginine 380 and arginine 384) within the initial seven residues of NC were found to be critical for the function of the N-terminal I domain. The presence of positive charge alone in these two positions, however, was not sufficient to mediate the formation of dense Gag particles. The I domain was required for the formation of detergent-resistant complexes of Gag protein, and confocal microscopy demonstrated that the I domain was also required for the formation of punctate foci of Gag proteins at the plasma membrane. Electron microscopic analysis of cells expressing Gag-GFP fusion constructs with an intact I domain revealed numerous retrovirus-like particles (RVLPs) budding from the plasma membrane, while I domain-deficient constructs failed to generate visible RVLPs. These results provide evidence that Gag-Gag interactions mediated by the I domain play a central role in the assembly of HIV particles.

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.00819-08 ◽

2008 ◽

Vol 82 (20) ◽

pp. 9937-9950 ◽

Cited By ~ 54

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.

Download Full-text

Sequence of Human Immunodeficiency Virus Type 1 (HIV-1) Gag Localization and Oligomerization Monitored with Live Confocal Imaging of a Replication-Competent, Fluorescently Tagged HIV-1

Journal of Virology ◽

10.1128/jvi.01088-07 ◽

2007 ◽

Vol 81 (22) ◽

pp. 12596-12607 ◽

Cited By ~ 126

Author(s):

Wolfgang Hübner ◽

Ping Chen ◽

Armando Del Portillo ◽

Yuxin Liu ◽

Ronald E. Gordon ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Plasma Membrane ◽

Fluorescent Protein ◽

Resonance Energy ◽

Confocal Imaging ◽

Hiv Protease ◽

Viral Gene ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The assembly of infectious human immunodeficiency virus (HIV) requires that Gag transport and oligomerization be coordinated with its association with other viral proteins, viral RNAs, and cellular membranes. We have developed a replication-competent HIV type 1 molecular clone that carries a Gag-internal or interdomain green fluorescent protein (iGFP) fusion to reveal a physiologically accurate temporal sequence of Gag localization and oligomerization during the formation of infectious HIV. This recombinant HIV is as infectious as native HIV in single-round infectivity assays, validating its use for trafficking studies. It replicates robustly in permissive MT4 cells and is infectious, yet it spreads poorly in other T-cell lines. Immunofluorescence of Gag-iGFP showed a pattern very similar to that of native Gag. However, the intense plasma membrane Gag-iGFP fluorescence contrasts markedly with its immunofluorescence at this site, indicating that many Gag epitopes can be masked by oligomerization. Consistent with this, fluorescence resonance energy transfer studies visualized intense Gag oligomerization at the plasma membrane and weaker oligomerization at cytoplasmic sites. Four-dimensional, time-lapse confocal imaging reveals a temporal progression of Gag distribution over hours in which Gag is initially diffusely localized within the cytoplasm. Plasma membrane signals then accumulate as Gag levels increase and vesicular association appears late, only after plasma membrane site signals have reached high intensity. Lastly, the cell rounds up and HIV protease activation induces diffuse fluorescence throughout the cell. These distinct phases reveal a natural progression of Gag trafficking during the viral gene expression program. HIV Gag-iGFP is a useful tool for dissecting mechanisms of viral assembly and transmission.

Download Full-text

Mobility of Human Immunodeficiency Virus Type 1 Pr55Gag in Living Cells

Journal of Virology ◽

10.1128/jvi.02159-05 ◽

2006 ◽

Vol 80 (17) ◽

pp. 8796-8806 ◽

Cited By ~ 28

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.

Download Full-text

Roles of Matrix, p2, and N-Terminal Myristoylation in Human Immunodeficiency Virus Type 1 Gag Assembly

Journal of Virology ◽

10.1128/jvi.74.1.16-23.2000 ◽

2000 ◽

Vol 74 (1) ◽

pp. 16-23 ◽

Cited By ~ 60

Author(s):

Yuko Morikawa ◽

David J. Hockley ◽

Milan V. Nermut ◽

Ian M. Jones

Keyword(s):

Human Immunodeficiency Virus ◽

Plasma Membrane ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Particle Assembly ◽

Gag Protein ◽

Gag Proteins ◽

Immunodeficiency Virus ◽

Gag Assembly

ABSTRACT Human immunodeficiency virus type 1 Gag protein is cotranslationally myristoylated at the N terminus and targeted to the plasma membrane, where virus particle assembly occurs. Particle assembly requires the ordered multimerization of Gag proteins, yet there is little direct evidence of intermediates of the reaction or of the domains that lead to each stage of the oligomerization process. In this study, following the expression in insect cells of C-terminally truncated Gag proteins and their purification, both the multimeric nature of each Gag protein and the ability to form Gag virus-like particles (VLP) were analyzed. Our results show that (i) the matrix (MA) domain forms a trimer and contributes to a similar level of oligomerization of the assembly-competent Gag; (ii) the p2 domain, located at the capsid/nucleocapsid junction, is essential for a higher order of multimerization (>1,000 kDa); (iii) the latter multimerization is accompanied by a change in Gag assembly morphology from tubes to spheres and results in VLP production; and (iv) N-terminal myristoylation is not required for either of the multimerization stages but plays a key role in conversion of these multimers to Gag VLP. We suggest that the Gag trimer and the >1,000-kDa multimer are intermediates in the assembly reaction and form before Gag targeting to the plasma membrane. Our data identify a minimum of three stages for VLP development and suggest that each stage involves a separate domain, MA, p2, or N-terminal myristoylation, each of which contributes to HIV particle assembly.

Download Full-text

Analysis of Human Immunodeficiency Virus Type 1 Gag Dimerization-Induced Assembly

Journal of Virology ◽

10.1128/jvi.79.23.14498-14506.2005 ◽

2005 ◽

Vol 79 (23) ◽

pp. 14498-14506 ◽

Cited By ~ 32

Author(s):

Ayna Alfadhli ◽

Tenzin Choesang Dhenub ◽

Amelia Still ◽

Eric Barklis

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Protein Assembly ◽

Particle Assembly ◽

Gag Protein ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Terminal Domains

ABSTRACT The nucleocapsid (NC) domains of retrovirus precursor Gag (PrGag) proteins play an essential role in virus assembly. Evidence suggests that NC binding to viral RNA promotes dimerization of PrGag capsid (CA) domains, which triggers assembly of CA N-terminal domains (NTDs) into hexamer rings that are interconnected by CA C-terminal domains. To examine the influence of dimerization on human immunodeficiency virus type 1 (HIV-1) Gag protein assembly in vitro, we analyzed the assembly properties of Gag proteins in which NC domains were replaced with cysteine residues that could be linked via chemical treatment. In accordance with the model that Gag protein pairing triggers assembly, we found that cysteine cross-linking or oxidation reagents induced the assembly of virus-like particles. However, efficient assembly also was observed to be temperature dependent or required the tethering of NTDs. Our results suggest a multistep pathway for HIV-1 Gag protein assembly. In the first step, Gag protein pairing through NC-RNA interactions or C-terminal cysteine linkage fosters dimerization. Next, a conformational change converts assembly-restricted dimers or small oligomers into assembly-competent ones. At the final stage, final particle assembly occurs, possibly through a set of larger intermediates.

Download Full-text

Kinetic Analysis of Human Immunodeficiency Virus Type 1 Assembly Reveals the Presence of Sequential Intermediates

Journal of Virology ◽

10.1128/jvi.74.13.5845-5855.2000 ◽

2000 ◽

Vol 74 (13) ◽

pp. 5845-5855 ◽

Cited By ~ 90

Author(s):

Marc Tritel ◽

Marilyn D. Resh

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Particle Production ◽

Particle Assembly ◽

Gag Protein ◽

Immunodeficiency Virus ◽

Membrane Bound ◽

Hiv 1

ABSTRACT The assembly and budding of lentiviruses, such as human immunodeficiency virus type 1 (HIV-1), are mediated by the Gag protein precursor, but the molecular details of these processes remain poorly defined. In this study, we have combined pulse-chase techniques with density gradient centrifugation to identify, isolate, and characterize sequential kinetic intermediates in the lentivirus assembly process. We show that newly synthesized HIV-1 Gag rapidly forms cytoplasmic protein complexes that are resistant to detergent treatment, sensitive to protease digestion, and degraded intracellularly. A subpopulation of newly synthesized Gag binds membranes within 5 to 10 min and over several hours assembles into membrane-bound complexes of increasing size and/or density that can be resolved on Optiprep density gradients. These complexes likely represent assembly intermediates because they are not observed with assembly-defective Gag mutants and can be chased into extracellular viruslike particles. At steady state, nearly all of the Gag is present as membrane-bound complexes in various stages of assembly. The identification of sequential assembly intermediates provides the first demonstration that HIV-1 particle assembly proceeds via an ordered process. Assembly intermediates should serve as attractive targets for the design of antiviral agents that interfere with the process of particle production.

Download Full-text

Analysis of the Assembly Function of the Human Immunodeficiency Virus Type 1 Gag Protein Nucleocapsid Domain

Journal of Virology ◽

10.1128/jvi.72.3.1782-1789.1998 ◽

1998 ◽

Vol 72 (3) ◽

pp. 1782-1789 ◽

Cited By ~ 161

Author(s):

Yaqiang Zhang ◽

Haoyu Qian ◽

Zachary Love ◽

Eric Barklis

Keyword(s):

Human Immunodeficiency Virus ◽

Virus Particle ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Assembly Process ◽

Particle Assembly ◽

Gag Protein ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Previous studies have shown that in addition to its function in specific RNA encapsidation, the human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) is required for efficient virus particle assembly. However, the mechanism by which NC facilitates the assembly process is not clearly established. Formally, NC could act by constraining the Pr55 gag polyprotein into an assembly-competent conformation or by masking residues which block the assembly process. Alternatively, the capacity of NC to bind RNA or make interprotein contacts might affect particle assembly. To examine its role in the assembly process, we replaced the NC domain in Pr55 gag with polypeptide domains of known function, and the chimeric proteins were analyzed for their abilities to direct the release of virus-like particles. Our results indicate that NC does not mask inhibitory domains and does not act passively, by simply providing a stable folded monomeric structure. However, replacement of NC by polypeptides which form interprotein contacts permitted efficient virus particle assembly and release, even when RNA was not detected in the particles. These results suggest that formation of interprotein contacts by NC is essential to the normal HIV-1 assembly process.

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.00308-07 ◽

2007 ◽

Vol 81 (14) ◽

pp. 7476-7490 ◽

Cited By ~ 75

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.

Download Full-text