FRET Analysis of the Promiscuous yet Specific Interactions of the HIV-1 Vpu Transmembrane Domain

Cole, Gregory B.; Reichheld, Sean E.; Sharpe, Simon

doi:10.1016/j.bpj.2017.09.010

Cited by 6 publications

(8 citation statements)

References 54 publications

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“…Data from this study show that peptides of Vpu’s TMD have a lower affinity for TMD peptides of NTB-A, PVR, and BST-2 than for known TM helix dimers (43). The interaction between Vpu and dimeric BST-2, however, remained more energetically favorable than Vpu–NTB-A and Vpu-PVR interactions, potentially due to the additive effects of multiple TMD-TMD interactions (43). BST-2 forms disulfide-linked dimers, and BST-2 dimerization is required for its antiviral function (7, 44–46).…”

Section: Discussionmentioning

confidence: 87%

“…One can speculate that Vpu’s TMD could have a higher affinity for BST-2’s TMD than for NTB-A’s or PVR’s TMD. A recent study by Cole et al characterized the heterooligomer formation between the TMD of Vpu and its substrates in a lipid environment using Förster resonance energy transfer (FRET) (43). Data from this study show that peptides of Vpu’s TMD have a lower affinity for TMD peptides of NTB-A, PVR, and BST-2 than for known TM helix dimers (43).…”

Section: Discussionmentioning

confidence: 99%

“…A recent study by Cole et al characterized the heterooligomer formation between the TMD of Vpu and its substrates in a lipid environment using Förster resonance energy transfer (FRET) (43). Data from this study show that peptides of Vpu’s TMD have a lower affinity for TMD peptides of NTB-A, PVR, and BST-2 than for known TM helix dimers (43). The interaction between Vpu and dimeric BST-2, however, remained more energetically favorable than Vpu–NTB-A and Vpu-PVR interactions, potentially due to the additive effects of multiple TMD-TMD interactions (43).…”

Section: Discussionmentioning

confidence: 99%

“…The preference of Vpu for BST-2 could therefore result from its higher affinity for BST-2 dimers than for other monomeric substrates. In the study of Cole et al, PVR and NTB-A TMDs were found to compete with BST-2 TMD for the binding of the Vpu TMD peptide in a lipid environment (43). It is possible that this reduced Vpu–BST-2 interaction detected in the presence of NTB-A’s or PVR’s TMD is not sufficient to have a significant impact on Vpu function.…”

Section: Discussionmentioning

confidence: 99%

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Upregulation of BST-2 by Type I Interferons Reduces the Capacity of Vpu To Protect HIV-1-Infected Cells from NK Cell Responses

Prévost

Pickering

Mumby

et al. 2019

mBio

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The HIV-1 accessory protein Vpu enhances viral release by counteracting the restriction factor BST-2. Furthermore, Vpu promotes NK cell evasion by downmodulating cell surface NTB-A and PVR, known ligands of the NK cell receptors NTB-A and DNAM-1, respectively. While it has been established that Vpu’s transmembrane domain (TMD) is required for the interaction and intracellular sequestration of BST-2, NTB-A, and PVR, it remains unclear how Vpu manages to target these proteins simultaneously. In this study, we show that upon upregulation, BST-2 is preferentially downregulated by Vpu over its other TMD substrates. We found that type I interferon (IFN)-mediated BST-2 upregulation greatly impairs the ability of Vpu to downregulate NTB-A and PVR. Our results suggest that occupation of Vpu by BST-2 affects its ability to downregulate other TMD substrates. Accordingly, knockdown of BST-2 increases Vpu’s potency to downmodulate NTB-A and PVR in the presence of type I IFN treatment. Moreover, we show that expression of human BST-2, but not that of the macaque orthologue, decreases Vpu’s capacity to downregulate NTB-A. Importantly, we show that type I IFNs efficiently sensitize HIV-1-infected cells to NTB-A- and DNAM-1-mediated direct and antibody-dependent NK cell responses. Altogether, our results reveal that type I IFNs decrease Vpu’s polyfunctionality, thus reducing its capacity to protect HIV-1-infected cells from NK cell responses. IMPORTANCE The restriction factor BST-2 and the NK cell ligands NTB-A and PVR are among a growing list of membrane proteins found to be downregulated by HIV-1 Vpu. BST-2 antagonism enhances viral release, while NTB-A and PVR downmodulation contributes to NK cell evasion. However, it remains unclear how Vpu can target multiple cellular factors simultaneously. Here we provide evidence that under physiological conditions, BST-2 is preferentially targeted by Vpu over NTB-A and PVR. Specifically, we show that type I IFNs decrease Vpu’s polyfunctionality by upregulating BST-2, thus reducing its capacity to protect HIV-1-infected cells from NK cell responses. This indicates that there is a hierarchy of Vpu substrates upon IFN treatment, revealing that for the virus, targeting BST-2 as part of its resistance to IFN takes precedence over evading NK cell responses. This reveals a potential weakness in HIV-1’s immunoevasion mechanisms that may be exploited therapeutically to harness NK cell responses against HIV-1.

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Section: Discussionmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Upregulation of BST-2 by Type I Interferons Reduces the Capacity of Vpu To Protect HIV-1-Infected Cells from NK Cell Responses

Prévost

Pickering

Mumby

et al. 2019

mBio

View full text Add to dashboard Cite

show abstract

“…This is best characterized in the case of BST-2 and Vpu: an anti-parallel interaction occurs between an alanine-face of Vpu’s TMD helix and a face of BST-2’s TMD helix that displays bulky hydrophobic side chains (Figure 4) [27]. How this seemingly bland face of the Vpu TMD specifically selects cellular targets such as NTB-A [10], PVR [95], and CCR7 [96] in addition to BST-2 is an open question [97], but modulation of these proteins requires the primary sequence of the Vpu TMD. In contrast, the modulation of CD1d by Vpu does not [98,99], and the modulation of HLA-C by Vpu requires bulky hydrophobic residues in the TMD rather than the alanines [9,100].…”

Section: Vpu Nef and The Cell Biologic Mechanisms Behind Their Mmentioning

confidence: 99%

Plasma Membrane-Associated Restriction Factors and Their Counteraction by HIV-1 Accessory Proteins

Ramirez

Sharma

Singh

et al. 2019

Cells

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The plasma membrane is a site of conflict between host defenses and many viruses. One aspect of this conflict is the host’s attempt to eliminate infected cells using innate and adaptive cell-mediated immune mechanisms that recognize features of the plasma membrane characteristic of viral infection. Another is the expression of plasma membrane-associated proteins, so-called restriction factors, which inhibit enveloped virions directly. HIV-1 encodes two countermeasures to these host defenses: The membrane-associated accessory proteins Vpu and Nef. In addition to inhibiting cell-mediated immune-surveillance, Vpu and Nef counteract membrane-associated restriction factors. These include BST-2, which traps newly formed virions at the plasma membrane unless counteracted by Vpu, and SERINC5, which decreases the infectivity of virions unless counteracted by Nef. Here we review key features of these two antiviral proteins, and we review Vpu and Nef, which deplete them from the plasma membrane by co-opting specific cellular proteins and pathways of membrane trafficking and protein-degradation. We also discuss other plasma membrane proteins modulated by HIV-1, particularly CD4, which, if not opposed in infected cells by Vpu and Nef, inhibits viral infectivity and increases the sensitivity of the viral envelope glycoprotein to host immunity.

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Toward a broad‐spectrum peptide‐based inhibitor of small multidrug resistance efflux pumps

2023

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As an alternative approach to conventional antibiotics, here we explore a novel, rational drug design to target a resistance mechanism in antibiotic‐resistant bacteria—the small multidrug resistance (SMR) efflux pump. SMRs are a membrane protein that consist of four transmembrane (TM) helices, which homodimerize via TM4 in an anti‐parallel fashion through a GG7 motif. To target this interaction, we synthesized a series of peptides, typified by Ac‐A‐(Sar)3‐IIGMMLISAGVLI‐KKK‐NH2 (Sar = N‐methyl‐glycine, GG7 underlined), with a sequence derived from Escherichia coli TM4, designed to bind to—and competitively disrupt—the TM4–TM4 interaction site. The peptides also contain an uncharged N‐terminal sarcosine (N‐methyl‐glycine) tag to promote membrane insertion, and a C‐terminal tri‐lysine tag to direct the peptides toward the negatively‐charged bacterial membrane. As we found that the GG7 dimerization motif was highly conserved across bacterial species, including a range of priority pathogens, the peptides were tested for interspecies inhibitory activity where we varied the SMR overexpressed in E. coli bacterium and the peptide. The peptides included the TM4 sequence of E. coli (EmrE), Pseudomonas aeruginosa (PAsmr), and Mycobacterium tuberculosis (Mmr). We performed a series of assays measuring the efflux rates of the fluorescent toxin ethidium bromide, finding that the PAsmr peptide was broadly active against all SMRs expressed in E. coli. Interestingly, a peptide derived from E. coli TM4, but where the GG7 motif was scrambled, showed high activity against several SMRs. Overall, the efflux inhibitors designed herein show promise not only for improved treatment of bacterial infections, but more generally, may provide a successful approach to targeting and disrupting membrane‐embedded protein–protein interactions.

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FRET Analysis of the Promiscuous yet Specific Interactions of the HIV-1 Vpu Transmembrane Domain

Cited by 6 publications

References 54 publications

Upregulation of BST-2 by Type I Interferons Reduces the Capacity of Vpu To Protect HIV-1-Infected Cells from NK Cell Responses

Upregulation of BST-2 by Type I Interferons Reduces the Capacity of Vpu To Protect HIV-1-Infected Cells from NK Cell Responses

Plasma Membrane-Associated Restriction Factors and Their Counteraction by HIV-1 Accessory Proteins

Toward a broad‐spectrum peptide‐based inhibitor of small multidrug resistance efflux pumps

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