Role of the C terminus Gag protein in human immunodeficiency virus type 1 virion assembly and maturation

ABSTRACT The retroviral Gag precursor plays an important role in the assembly of virion particles. The capsid (CA) protein of the Gag molecule makes a major contribution to this process. In the crystal structure of the free CA protein of the human immunodeficiency virus type 1 (HIV-1), 11 residues of the C terminus were found to be unstructured, and to date no information exists on the structure of these residues in the context of the Gag precursor molecule. We performed phylogenetic analysis and demonstrated a high degree of conservation of these 11 amino acids. Deletion of this cluster or introduction of various point mutations into these residues resulted in significant impairment of particle infectivity. In this cluster, two putative structural regions were identified, residues that form a hinge region (353-VGGP-356) and those that contribute to an α-helix (357-GHKARVL-363). Overall, mutations in these regions resulted in inhibition of virion production, but mutations in the hinge region demonstrated the most significant reduction. Although all the Gag mutants appeared to have normal Gag-Gag and Gag-RNA interactions, the hinge mutants were characterized by abnormal formation of cytoplasmic Gag complexes. Gag proteins with mutations in the hinge region demonstrated normal membrane association but aberrant rod-like membrane structures. More detailed analysis of these structures in one of the mutants demonstrated abnormal trapped Gag assemblies. These data suggest that the conserved CA C terminus is important for HIV-1 virion assembly and release and define a putative target for drug design geared to inhibit the HIV-1 assembly process.

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Particle Size Determinants in the Human Immunodeficiency Virus Type 1 Gag Protein

Journal of Virology ◽

10.1128/jvi.72.6.4667-4677.1998 ◽

1998 ◽

Vol 72 (6) ◽

pp. 4667-4677 ◽

Cited By ~ 38

Author(s):

Laurence Garnier ◽

Lee Ratner ◽

Benjamin Rovinski ◽

Shi-Xian Cao ◽

John W. Wills

Keyword(s):

Human Immunodeficiency Virus ◽

Particle Size ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Gag Protein ◽

Important Region ◽

C Terminus ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The retroviral Gag protein plays the central role in the assembly process and can form membrane-enclosed, virus-like particles in the absence of any other viral products. These particles are similar to authentic virions in density and size. Three small domains of the human immunodeficiency virus type 1 (HIV-1) Gag protein have been previously identified as being important for budding. Regions that lie outside these domains can be deleted without any effect on particle release or density. However, the regions of Gag that control the size of HIV-1 particles are less well understood. In the case of Rous sarcoma virus (RSV), the size determinant maps to the CA (capsid) and adjacent spacer sequences within Gag, but systematic mapping of the HIV Gag protein has not been reported. To locate the size determinants of HIV-1, we analyzed a large collection of Gag mutants. To our surprise, all mutants with defects in the MA (matrix), CA, and the N-terminal part of NC (nucleocapsid) sequences produced dense particles of normal size, suggesting that oncoviruses (RSV) and lentiviruses (HIV-1) have different size-controlling elements. The most important region found to be critical for determining HIV-1 particle size is the p6 sequence. Particles lacking all or small parts of p6 were uniform in size distribution but very large as measured by rate zonal gradients. Further evidence for this novel function of p6 was obtained by placing this sequence at the C terminus of RSV CA mutants that produce heterogeneously sized particles. We found that the RSV-p6 chimeras produced normally sized particles. Thus, we present evidence that the entire p6 sequence plays a role in determining the size of a retroviral particle.

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Critical Role of Enhanced CD4 Affinity in Laboratory Adaptation of Human Immunodeficiency Virus Type 1

AIDS Research and Human Retroviruses ◽

10.1089/08892220050042819 ◽

2000 ◽

Vol 16 (9) ◽

pp. 871-882 ◽

Cited By ~ 27

Author(s):

Emily J. Platt ◽

Susan L. Kozak ◽

David Kabat

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Critical Role ◽

Laboratory Adaptation ◽

Immunodeficiency Virus

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Cellular Membrane-Binding Ability of the C-Terminal Cytoplasmic Domain of Human Immunodeficiency Virus Type 1 Envelope Transmembrane Protein gp41

Journal of Virology ◽

10.1128/jvi.75.20.9925-9938.2001 ◽

2001 ◽

Vol 75 (20) ◽

pp. 9925-9938 ◽

Cited By ~ 39

Author(s):

Steve S.-L. Chen ◽

Sheau-Fen Lee ◽

Chin-Tien Wang

Keyword(s):

Amino Acids ◽

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Membrane Binding ◽

Cytoplasmic Tail ◽

Cellular Membrane ◽

C Terminus ◽

Immunodeficiency Virus

ABSTRACT The amphipathic α-helices located in the cytoplasmic tail of the envelope (Env) transmembrane glycoprotein gp41 of human immunodeficiency virus type 1 have been implicated in membrane association and cytopathicity. Deletion of the last 12 amino acids in the C terminus of this domain severely impairs infectivity. However, the nature of the involvement of the cytoplasmic tail in Env-membrane interactions in cells and the molecular basis for the defect in infectivity of this mutant virus are still poorly understood. In this study we examined the interaction of the cytoplasmic tail with membranes in living mammalian cells by expressing a recombinant cytoplasmic tail fragment and an Escherichia coli β-galactosidase/cytoplasmic tail fusion protein, both of them lacking gp120, the gp41 ectodomain, and the transmembrane region. We found through cell fractionation, in vivo membrane flotation, and confocal immunofluorescence studies that the cytoplasmic tail contained determinants to be routed to a perinuclear membrane region in cells. Further mapping showed that each of the three lentivirus lytic peptide (LLP-1, LLP-2, and LLP-3) sequences conferred this cellular membrane-targeting ability. Deletion of the last 12 amino acids from the C terminus abolished the ability of the LLP-1 motif to bind to membranes. High salt extraction, in vitro transcription and translation, and posttranslational membrane binding analyses indicated that the β-galactosidase/LLP fusion proteins were inserted into membranes via the LLP sequences. Subcellular fractionation and confocal microscopy studies revealed that each of the LLP motifs, acting in a position-independent manner, targeted non-endoplasmic reticulum (ER)-associated β-galactosidase and enhanced green fluorescence protein to the ER. Our study provides a basis for the involvement of the gp41 cytoplasmic tail during Env maturation and also supports the notion that the membrane apposition of the C-terminal cytoplasmic tail plays a crucial role in virus-host interaction.

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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.

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