HIV-1 Gag Associates with Specific Uropod-Directed Microdomains in a Manner Dependent on Its MA Highly Basic Region

Llewellyn, G. Nicholas; Grover, Jonathan R.; Olety, Balaji; Ono, Akira

doi:10.1128/jvi.00040-13

Cited by 45 publications

(81 citation statements)

References 109 publications

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“…pNL4-3/Gag-mEos3.2 was described previously (56). pNL4-3/ Fyn(10)/Gag-mEos3.2, pNL4-3/Fyn(10)/6A2T/Gag-mEos3.2, pNL4-3/ Fyn(10)/HBR/RKswitch/Gag-mEos3.2, and pNL4-3/Fyn(10)/⌬MA/GagmEos3.2 were constructed using MA-containing sequences from pNL4-3/Gag-Venus versions of these constructs, which were described previously (38,39). pNL4-3/Kmyr/⌬MA/Gag-mEos3.2 was constructed by replacing the MA domain of Gag with the N-terminal, Ras-derived membrane-anchoring sequence MGSSKSKDGKKKKKKSKTK (58,59).…”

Section: Methodsmentioning

confidence: 99%

“…In polarized T cells, Gag localizes to a rear-end protrusion known as the uropod (34,47). In our previous studies, we observed that when clustering was induced by specific antibodies, a subset of uropod-directed proteins, including PSGL-1, CD43, and CD44 (here referred to as class I uropoddirected proteins), colocalized or copatched with Gag, even in nonpolarized T cells (34,39). This colocalization was found to be dependent upon the charge, but not the specific amino acid sequence, of the MA HBR (39).…”

mentioning

confidence: 99%

“…Indeed, previous studies suggested that most membrane proteins are incorporated into retrovirus particles passively, i.e., with no enrichment relative to the plasma membrane (28). During assembly, Gag has been observed to associate with plasma membrane microdomains, such as lipid rafts (29,30) and tetraspanin-enriched microdomains (TEMs) (31)(32)(33)(34)(35)(36), and reorganize these domains in ways that do not occur in uninfected cells (37)(38)(39). Therefore, at least a subset of membrane proteins (e.g., glycosylphosphatidylinositol-anchored proteins and tetraspanins) may be incorporated into virions actively (as opposed to by passive incorporation, which does not involve enrichment of membrane proteins at assembly sites) without specific interactions, other than association with the plasma membrane microdomains.…”

mentioning

confidence: 99%

“…Intriguingly, we also observed that Gag failed to copatch with a second class of uropod-directed proteins (class II); CD59, ICAM-1, and ICAM-3 were observed to localize to uropods but not to copatch with Gag in unpolarized T cells. Furthermore, ICAM-3, which copatched with the class I uropod-directed protein CD44 in uninfected cells, dissociated from CD44 in the presence of Gag through an unknown mechanism (39). Most of these membrane proteins described above are cell adhesion molecules, and some of them are implicated in modulation of immunological synapses (48)(49)(50).…”

mentioning

confidence: 99%

“…In our previous studies, we observed that when clustering was induced by specific antibodies, a subset of uropod-directed proteins, including PSGL-1, CD43, and CD44 (here referred to as class I uropoddirected proteins), colocalized or copatched with Gag, even in nonpolarized T cells (34,39). This colocalization was found to be dependent upon the charge, but not the specific amino acid sequence, of the MA HBR (39). Intriguingly, we also observed that Gag failed to copatch with a second class of uropod-directed proteins (class II); CD59, ICAM-1, and ICAM-3 were observed to localize to uropods but not to copatch with Gag in unpolarized T cells.…”

mentioning

confidence: 99%

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Basic Motifs Target PSGL-1, CD43, and CD44 to Plasma Membrane Sites Where HIV-1 Assembles

Grover

Veatch

Ono

2015

J Virol

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HIV-1 incorporates various host membrane proteins during particle assembly at the plasma membrane; however, the mechanisms mediating this incorporation process remain poorly understood. We previously showed that the HIV-1 structural protein Gag localizes to the uropod, a rear-end structure of polarized T cells, and that assembling Gag copatches with a subset, but not all, of the uropod-directed proteins, i.e., PSGL-1, CD43, and CD44, in nonpolarized T cells. The latter observation suggests the presence of a mechanism promoting virion incorporation of these cellular proteins. To address this possibility and identify molecular determinants, in the present study we examined coclustering between Gag and the transmembrane proteins in T and HeLa cells using quantitative two-color superresolution localization microscopy. Consistent with the findings of the T-cell copatching study, we found that basic residues within the matrix domain of Gag are required for Gag-PSGL-1 coclustering. Notably, the presence of a polybasic sequence in the PSGL-1 cytoplasmic domain significantly enhanced this coclustering. We also found that polybasic motifs present in the cytoplasmic tails of CD43 and CD44 also promote their coclustering with Gag. ICAM-1 and ICAM-3, uropod-directed proteins that do not copatch with Gag in T cells, and CD46, a non-uropod-directed protein, showed no or little coclustering with Gag. However, replacing their cytoplasmic tails with the cytoplasmic tail of PSGL-1 significantly enhanced their coclustering with Gag. Altogether, these results identify a novel mechanism for host membrane protein association with assembling HIV-1 Gag in which polybasic sequences present in the cytoplasmic tails of the membrane proteins and in Gag are the major determinants. IMPORTANCENascent HIV-1 particles incorporate many host plasma membrane proteins during assembly. However, it is largely unknown what mechanisms promote the association of these proteins with virus assembly sites within the plasma membrane. Notably, our previous study showed that HIV-1 structural protein Gag colocalizes with a group of uropod-directed transmembrane proteins, PSGL-1, CD43, and CD44, at the plasma membrane of T cells. The results obtained in the current study using superresolution localization microscopy suggest the presence of a novel molecular mechanism promoting the association of PSGL-1, CD43, and CD44 with assembling HIV-1 which relies on polybasic sequences in HIV-1 Gag and in cytoplasmic domains of the transmembrane proteins. This information advances our understanding of virion incorporation of host plasma membrane proteins, some of which modulate virus spread positively or negatively, and suggests a possible new strategy to enrich HIV-1-based lentiviral vectors with a desired transmembrane protein. HIV-1 assembles at the plasma membrane in most cell types. The viral structural protein Gag, synthesized as a precursor polyprotein (Pr55), initiates and coordinates viral assembly at the plasma membrane. Membrane binding and targeting of ...

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