Kinetically coupled folding of a single HIV-1 glycoprotein 41 complex in viral membrane fusion and inhibition

HIV-1 glycoprotein 41 (gp41) mediates viral entry into host cells by coupling its folding energy to membrane fusion. Gp41 folding is blocked by fusion inhibitors, including the commercial drug T20, to treat HIV/AIDS. However, gp41 folding intermediates, energy, and kinetics are poorly understood. Here, we identified the folding intermediates of a single gp41 trimer-of-hairpins and measured their associated energy and kinetics using high-resolution optical tweezers. We found that folding of gp41 hairpins was energetically independent but kinetically coupled: Each hairpin contributed a folding energy of ∼−23 kBT, but folding of one hairpin successively accelerated the folding rate of the next one by ∼20-fold. Membrane-mimicking micelles slowed down gp41 folding and reduced the stability of the six-helix bundle. However, the stability was restored by cooperative folding of the membrane-proximal external region. Surprisingly, T20 strongly inhibited gp41 folding by actively displacing the C-terminal hairpin strand in a force-dependent manner. The inhibition was abolished by a T20-resistant gp41 mutation. The energetics and kinetics of gp41 folding established by us provides a basis to understand viral membrane fusion, infection, and therapeutic intervention.

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P2X1 Selective Antagonists Block HIV-1 Infection through Inhibition of Envelope Conformation-Dependent Fusion

Journal of Virology ◽

10.1128/jvi.01622-19 ◽

2019 ◽

Vol 94 (6) ◽

Cited By ~ 1

Author(s):

Alexandra Y. Soare ◽

Hagerah S. Malik ◽

Natasha D. Durham ◽

Tracey L. Freeman ◽

Raymond Alvarez ◽

...

Keyword(s):

Membrane Fusion ◽

Chronic Inflammation ◽

Neutralizing Antibody ◽

Extracellular Nucleotides ◽

Productive Infection ◽

Variable Regions ◽

Viral Membrane ◽

P2x1 Receptor ◽

Viral Membrane Fusion ◽

Hiv 1

ABSTRACT Purinergic receptors are well-established modulators of inflammatory processes, primarily through detection of extracellular nucleotides that are released by dying or infected cells. Emerging literature has demonstrated that inhibition of these inflammatory receptors can block HIV-1 productive infection and HIV-1-associated inflammation. The specificity of receptor type and mechanism of interaction has not yet been determined. Here, we characterize the inhibitory activity of P2X1 receptor antagonists, NF279 and NF449, in cell lines, primary cells, and a variety of HIV-1 envelope (Env) clades. NF279 and NF449 blocked productive infection at the level of viral membrane fusion, with a range of inhibitory activities against different HIV-1 Env isolates. A mutant virus carrying a truncation deletion of the C-terminal tail of HIV-1 Env glycoprotein 41 (gp41) showed reduced sensitivity to P2X1 antagonists, indicating that the sensitivity of inhibition by these molecules may be modulated by Env conformation. In contrast, a P2X7 antagonist, A438079, had a limited effect on productive infection and fusion. NF279 and NF449 interfered with the ability of the gp120 variable regions 1 and 2 (V1V2)-targeted broadly neutralizing antibody PG9 to block productive infection, suggesting that these drugs may antagonize HIV-1 Env at gp120 V1V2 to block viral membrane fusion. Our observations indicate that P2X1 antagonism can inhibit HIV-1 replication at the level of viral membrane fusion through interaction with Env. Future studies will probe the nature of these compounds in inhibiting HIV-1 fusion and the development of small molecules to block HIV-1 entry via this mechanism. IMPORTANCE While effective treatment can lower the severe morbidity and mortality associated with HIV-1 infection, patients infected with HIV-1 suffer from significantly higher rates of noncommunicable comorbidities associated with chronic inflammation. Emerging literature suggests a key role for P2X1 receptors in mediating this chronic inflammation, but the mechanism is still unknown. Here, we demonstrate that HIV-1 infection is reduced by P2X1 receptor antagonism. This inhibition is mediated by interference with HIV-1 Env and can impact a variety of viral clades. These observations highlight the importance of P2X1 antagonists as potential novel therapeutics that could serve to block a variety of different viral clades with additional benefits for their anti-inflammatory properties.

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Interferon-Induced Transmembrane Protein 3 Is a Virus-Associated Protein Which Suppresses Porcine Reproductive and Respiratory Syndrome Virus Replication by Blocking Viral Membrane Fusion

Journal of Virology ◽

10.1128/jvi.01350-20 ◽

2020 ◽

Vol 94 (24) ◽

Author(s):

Angke Zhang ◽

Hong Duan ◽

Huijun Zhao ◽

Huancheng Liao ◽

Yongkun Du ◽

...

Keyword(s):

Membrane Fusion ◽

Transmembrane Protein ◽

Fluorescent Labeling ◽

Host Cells ◽

Respiratory Syndrome Virus ◽

Type I ◽

Antiviral Immune Response ◽

Viral Membrane ◽

Viral Membrane Fusion

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) infection eliminates production of type I interferons (IFNs) in host cells, which triggers an antiviral immune response through the induction of downstream IFN-stimulated genes (ISGs), thus escaping the fate of host-mediated clearance. The IFN-induced transmembrane 3 (IFITM3) has recently been identified as an ISG and plays a pivotal role against enveloped RNA viruses by restricting cell entry. However, the role of IFITM3 in PRRSV replication is unknown. The present study demonstrated that overexpression of IFITM3 suppresses PRRSV replication, while silencing of endogenous IFITM3 prominently promoted PRRSV replication. Additionally, it was shown that IFITM3 undergoes S-palmitoylation and ubiquitination modification, and both posttranslational modifications contribute to the anti-PRRSV activity of IFITM3. Further study showed that PRRSV particles are transported into endosomes and then into lysosomes during the early stages of infection, and confocal microscopy results revealed that PRRSV particles are transported to IFITM3-positive cellular vesicles. By using a single virus particle fluorescent labeling technique, we confirmed that IFITM3 can restrict PRRSV membrane fusion by inducing accumulation of cholesterol in cellular vesicles. Additionally, we found that both endogenous and exogenous IFITM3 are incorporated into newly producing PRRS virions and diminish viral intrinsic infectivity. By using cell coculture systems, we found that IFITM3 effectively restricted PRRSV intercellular transmission, which may have been caused by disrupted membrane fusion and reduced viral infectivity. In conclusion, our results demonstrate, for the first time, that swine IFITM3 interferes with the life cycle of PRRSV, and possibly other enveloped arteritis viruses, at multiple steps. IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS), which is caused by PRRS virus (PRRSV), is of great economic significance to the swine industry. Due to the complicated immune escape mechanisms of PRRSV, there are no effective vaccines or therapeutic drugs currently available against PRRS. Identification of cellular factors and underlying mechanisms that establish an effective antiviral state against PRRSV can provide unique strategies for developing antiviral vaccines or drugs. As an interferon (IFN)-stimulated gene, the role of IFN-induced transmembrane 3 (IFITM3) in PRRSV infection has not been reported as of yet. In the present study, it was shown that IFITM3 can exert a potent anti-PRRSV effect, and PRRS virions are trafficked to IFITM3-containing cell vesicles, where viral membrane fusion is impaired by cholesterol accumulation that is induced by IFITM3. Additionally, both endogenous and exogenous IFITM3 are incorporated into newly assembled progeny virions, and this decreased their intrinsic infectivity.

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Cell-to-Cell Transfer of HIV-1 via Virological Synapses Leads to Endosomal Virion Maturation that Activates Viral Membrane Fusion

Cell Host & Microbe ◽

10.1016/j.chom.2011.10.015 ◽

2011 ◽

Vol 10 (6) ◽

pp. 551-562 ◽

Cited By ~ 96

Author(s):

Benjamin M. Dale ◽

Gregory P. McNerney ◽

Deanna L. Thompson ◽

Wolfgang Hubner ◽

Kevin de los Reyes ◽

...

Keyword(s):

Membrane Fusion ◽

Cell Transfer ◽

Viral Membrane ◽

Virological Synapses ◽

Cell To Cell Transfer ◽

Viral Membrane Fusion ◽

Hiv 1

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Purinergic Receptors: Elucidating the Role of These Immune Mediators in HIV-1 Fusion

10.20944/preprints202001.0299.v1 ◽

2020 ◽

Author(s):

Tracey L. Freeman ◽

Talia H. Swartz

Keyword(s):

Membrane Fusion ◽

Purinergic Receptors ◽

Purinergic Receptor ◽

Extracellular Nucleotides ◽

Viral Membrane ◽

Immune Mediators ◽

Receptor Interactions ◽

Viral Membrane Fusion ◽

Hiv 1

Purinergic receptors are inflammatory mediators activated by extracellular nucleotides released by dying or injured cells. Several studies have described an important role for these receptors in HIV-1 entry, particularly regarding their activity on HIV-1 viral membrane fusion. Several reports identify purinergic receptor antagonists that inhibit HIV-1 membrane fusion; these drugs are suspected to act through antagonizing Env-chemokine receptor interactions. They also appear to abrogate activity of downstream mediators that potentiate activation of the NLRP3 inflammasome pathway. Here we review the literature on purinergic receptors, the drugs that inhibit their function, and the evidence implicating these receptors in HIV-1 entry.

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Purinergic Receptors: Elucidating the Role of these Immune Mediators in HIV-1 Fusion

Viruses ◽

10.3390/v12030290 ◽

2020 ◽

Vol 12 (3) ◽

pp. 290

Author(s):

Tracey L. Freeman ◽

Talia H. Swartz

Keyword(s):

Membrane Fusion ◽

Purinergic Receptors ◽

Purinergic Receptor ◽

Extracellular Nucleotides ◽

Viral Membrane ◽

Immune Mediators ◽

Receptor Interactions ◽

Viral Membrane Fusion ◽

Hiv 1

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P2X1 selective antagonists block HIV-1 infection through inhibition of envelope conformation-dependent fusion

10.1101/783464 ◽

2019 ◽

Cited By ~ 1

Author(s):

Alexandra Y. Soare ◽

Hagerah S. Malik ◽

Natasha D. Durham ◽

Tracey L. Freeman ◽

Raymond Alvarez ◽

...

Keyword(s):

Membrane Fusion ◽

Chronic Inflammation ◽

Neutralizing Antibody ◽

Productive Infection ◽

Viral Membrane ◽

P2x1 Receptor ◽

Fusion Inhibition ◽

Inflammatory Processes ◽

Viral Membrane Fusion ◽

Hiv 1

AbstractPurinergic receptors detect extracellular ATP and promote inflammatory processes. Emerging literature has demonstrated that inhibition of these proinflammatory receptors can block HIV-1 productive infection. The specificity of receptor type and mechanism of interaction has not yet been determined. Here we characterize the inhibitory activity of P2X1 receptor antagonists, NF279 and NF449 in cell lines, primary cells, and in a variety of envelope clades. NF279 and NF449 blocked productive infection at the level of viral membrane fusion with a range of inhibitory activities against different HIV-1 envelopes. A mutant virus carrying a truncation deletion of the C-terminal tail of HIV-1 envelope (Env) glycoprotein 41 (gp41) showed reduced sensitivity to P2X1 antagonists, indicating that the sensitivity of inhibition by these molecules is modulated by Env conformation. By contrast, a P2X7 antagonist, A438079, had limited effect on productive infection and fusion. Inhibition with NF449 interfered with the ability of the V1V2 targeted broadly neutralizing antibody PG9 to block productive infection, suggesting that these drugs may antagonize HIV-1 Env at gp120 V1V2 to block viral membrane fusion. Our observations indicate that P2X1 antagonism can inhibit HIV-1 replication at the level of viral membrane fusion through interaction with Env. Future studies will probe the nature of these compounds in inhibiting HIV-1 fusion and in development of a different class of small molecules to block HIV-1 entry.IMPORTANCEWhile effective treatment can lower the severe morbidity and mortality associated with HIV-1 infection, patients infected with HIV-1 suffer from significantly higher rates of non-communicable comorbidities associated with chronic inflammation. Emerging literature suggests a key role for P2X1 receptors in mediating this chronic inflammation but the mechanism is still unknown. Here, we demonstrate that HIV-1 infection is reduced by P2X1 receptor antagonism. This inhibition is mediated by interference with HIV-1 Env and can impact a variety of viral clades. These observations highlight the importance of P2X1 antagonists as potential novel therapeutics that could serve to block a variety of different viral clades with additional benefits for their anti-inflammatory properties.

Download Full-text