Retromer Regulates HIV-1 Envelope Glycoprotein Trafficking and Incorporation into Virions

Groppelli, Elisabetta; Len, Alice C. L.; Granger, Luke A.; Jolly, Clare

doi:10.1371/journal.ppat.1004518

Cited by 61 publications

(76 citation statements)

References 60 publications

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“…Using PGT151, we were able to isolate a component of pseudovirion Env that had a range of complex-type sugars similar to that isolated from PBMC-derived Env, except for a different linkage of terminal sialic acids. These highly processed complex-type glycans might be indicative of a prolonged exposure in the Golgi or retromer-mediated sorting of virion-associated Env from the cell surface back to the Golgi, as has recently been demonstrated (64). In contrast, recombinant gp120, which is not membrane bound, displayed an additional range of smaller complex-type glycans, suggesting faster transit through the Golgi apparatus.…”

Section: Figmentioning

confidence: 56%

Cell- and Protein-Directed Glycosylation of Native Cleaved HIV-1 Envelope

Pritchard

Harvey

Bonomelli

et al. 2015

J Virol

116

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The gp120/gp41 HIV-1 envelope glycoprotein (Env) is highly glycosylated, with up to 50% of its mass consisting of N-linked glycans. This dense carbohydrate coat has emerged as a promising vaccine target, with its glycans acting as epitopes for a number of potent and broadly neutralizing antibodies (bnAbs). Characterizing the glycan structures present on native HIV-1 Env is thus a critical goal for the design of Env immunogens. In this study, we used a complementary, multistep approach involving ion mobility mass spectrometry and high-performance liquid chromatography to comprehensively characterize the glycan structures present on HIV-1 gp120 produced in peripheral blood mononuclear cells (PBMCs). The capacity of different expression systems, including pseudoviral particles and recombinant cell surface trimers, to reproduce native-like glycosylation was then assessed. A population of oligomannose glycans on gp120 was reproduced across all expression systems, supporting this as an intrinsic property of Env that can be targeted for vaccine design. In contrast, Env produced in HEK 293T cells failed to accurately reproduce the highly processed complex-type glycan structures observed on PBMC-derived gp120, and in particular the precise linkage of sialic acid residues that cap these glycans. Finally, we show that unlike for gp120, the glycans decorating gp41 are mostly complex-type sugars, consistent with the glycan specificity of bnAbs that target this region. These findings provide insights into the glycosylation of native and recombinant HIV-1 Env and can be used to inform strategies for immunogen design and preparation. IMPORTANCEDevelopment of an HIV vaccine is desperately needed to control new infections, and elicitation of HIV bnAbs will likely be an important component of an effective vaccine. Increasingly, HIV bnAbs are being identified that bind to the N-linked glycans coating the HIV envelope glycoproteins gp120 and gp41, highlighting them as important targets for vaccine design. It is therefore important to characterize the glycan structures present on native, virion-associated gp120 and gp41 for development of vaccines that accurately mimic native-Env glycosylation. In this study, we used a number of analytical techniques to precisely study the structures of both the oligomannose and complex-type glycans present on native Env to provide a reference for determining the ability of potential HIV immunogens to accurately replicate the glycosylation pattern on these native structures.T he HIV-1 envelope glycoprotein (Env) consists of a noncovalently linked trimer of gp120/gp41 heterodimers. Interaction of this trimer with CD4 and its subsequent rearrangement is essential for infection of target cells and therefore is the sole target for broadly neutralizing antibodies (bnAbs). The surface protein, gp120, is extensively modified with host-derived glycans, having a median of 25 potential N-linked glycosylation sites (PNGSs) (1) and thus making it one of the most densely glycosylated proteins known. The t...

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Section: Figmentioning

confidence: 56%

Cell- and Protein-Directed Glycosylation of Native Cleaved HIV-1 Envelope

Pritchard

Harvey

Bonomelli

et al. 2015

J Virol

116

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“…Mutations in these putative protein-binding sites were found to exhibit reduced cell-free infectivity, which was cell type dependent in the case of the prohibitin binding site mutant LL_RQ. The cellular retromer complex has also recently been shown to bind to the CT (21). Disruption of retromer-CT interaction can result in the production of virus particles with enhanced Env packaging and infectivity.…”

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confidence: 99%

“…A number of studies have examined HIV-1 genomes carrying progressive gp41 CT truncations and observed differential impacts on Env fusogenicity (1,3,26), Env packaging, and/or infectivity of cell-free virus (3,(26)(27)(28)(29)(30)(31). Other studies have also characterized small deletions and point mutations within the CT LLP regions or endocytic motif(s) that affect Env incorporation and/or infectivity of viral particles (2,6,(31)(32)(33)(34)(35), the intracellular distribution of Env (21,33), syncytium formation (2,6,34), viral fusion (6,35), and polarized budding (36).…”

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confidence: 99%

HIV-1 Cell-Free and Cell-to-Cell Infections Are Differentially Regulated by Distinct Determinants in the Env gp41 Cytoplasmic Tail

Durham

Chen

2015

J Virol

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The HIV-1 envelope (Env) glycoprotein mediates viral entry during both cell-free and cell-to-cell infection of CD4 ؉ T cells. The highly conserved long cytoplasmic tail (CT) of Env is required in a cell type-dependent manner for optimal infectivity of cell-free virus. To probe the role of the CT in cell-to-cell infection, we tested a panel of mutations in the CT region that maintain or attenuate cell-free infection to investigate whether the functions of the CT are conserved during cell-free and cell-to-cell infection. The mutations tested included truncations of structural motifs in the gp41 CT and two point mutations in lentiviral lytic peptide 3 (LLP-3) previously described as disrupting the infectivity of cell-free virus. We found that small truncations of 28 to 43 amino acids (aa) or two LLP-3 point mutations, YW_SL and LL_RQ, severely impaired single-round cell-free infectivity 10-fold or more relative to wild-type full-length CT. These mutants showed a modest 2-fold reduction in cell-to-cell infection assays. Conversely, large truncations of 93 to 124 aa severely impaired cell-to-cell infectivity 20-fold or more while resulting in a 50% increase in infectivity of cell-free viral particles when produced in 293T cells. Intermediate truncations of 46 to 90 aa showed profound impairment of both modes of infection. Our results show that the abilities of Env to support cell-free and cell-to-cell infection are genetically distinct. These differences are cell type dependent for large-CT-truncation mutants. Additionally, point mutants in LLP-3 can maintain multiround propagation from cell-to-cell in primary CD4 ؉ T cells. IMPORTANCEThe functions of HIV Env gp41 CT remain poorly understood despite being widely studied in the context of cell-free infection. We have identified domains of the gp41 CT responsible for striking selective deficiencies in either cell-free or cell-to-cell infectivity. These differences may reflect a different intrinsic regulatory influence of the CT on cell-associated versus particle-associated Env or differential interaction with host or viral proteins. Our findings provide novel insight into the key regulatory potential of the gp41 CT in cell-free and cell-to-cell HIV-1 infection, particularly for short-truncation mutants of <43 amino acids or mutants with point mutations in the LLP-3 helical domain of the CT, which are able to propagate via cell-to-cell infection in the absence of infectious cell-free virus production. These mutants may also serve as tools to further define the contributions of cellfree and cell-to-cell infection in vitro and in vivo. Human immunodeficiency virus (HIV-1) is an enveloped retrovirus that can enter permissive target cells through binding of its envelope glycoprotein (Env) to host CD4 and chemokine receptors. Env is a trimer of gp120-gp41 heterodimers and mediates viral attachment, fusion, and entry into CD4 ϩ T cells during both cell-free infection and direct cell-to-cell infection via the virological synapse (VS). The gp41 cytoplasmic tail (CT) of ...

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“…Human immunodeficiency virus (HIV-1) has been shown to use retromer-based recycling in the process of virion construction (Fig. 3A), specifically involving the two component envelope (Env) proteins (gp120 and gp41) (Blot et al, 2003;Groppelli et al, 2014). Not only do retromer proteins show intracellular colocalization with HIV-1 particles in vivo, but retromer proteins Vps26 and Vps35 have been shown to physically interact directly with the Table 1 Examples of mammalian retromer cargo proteins.…”

Section: Viral Pathogensmentioning

confidence: 99%