Dataset Information

Novel screening systems for HIV-1 fusion mediated by two extra-virion heptad repeats of gp41.

ABSTRACT: Entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by its envelope protein gp41 through membrane fusion. Interaction of two extra-virion heptad repeats (HRs) in the gp41 plays a pivotal role in the fusion, and its inhibitor, enfuvirtide (T-20), blocks HIV-1 entry. To identify agents that block HIV-1 fusion, two screening methods based on detection and quantification by the enzyme-linked immunosorbent assay (ELISA) principle have been established. One method uses an alkaline phosphatase (ALP)-conjugated antibody (Ab-ELISA) and the other uses an ALP-fused HR (F-ELISA) to detect and quantify the interaction of the two HRs. The F-ELISA was more simple and rapid, since no ALP-conjugated antibody reaction was required. Both ELISAs detected all the fusion inhibitors tested except for T-20. Interaction of the two HRs was observed in both ELISAs, even in the presence of 10% dimethyl sulfoxide. Ab-ELISA performed best in a pH ranging from 6 to 8, while F-ELISA performed best at a pH ranging from 7 to 8. These results indicate that both established ELISAs are suitable for the identification of HIV-1 fusion inhibitors.

SUBMITTER: Nishikawa H

PROVIDER: S-EPMC7114109 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Novel screening systems for HIV-1 fusion mediated by two extra-virion heptad repeats of gp41.

Nishikawa Hiroki H Kodama Eiichi E Sakakibara Ayako A Fukudome Ayako A Izumi Kazuki K Oishi Shinya S Fujii Nobutaka N Matsuoka Masao M

Antiviral research 20080612 1

Entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by its envelope protein gp41 through membrane fusion. Interaction of two extra-virion heptad repeats (HRs) in the gp41 plays a pivotal role in the fusion, and its inhibitor, enfuvirtide (T-20), blocks HIV-1 entry. To identify agents that block HIV-1 fusion, two screening methods based on detection and quantification by the enzyme-linked immunosorbent assay (ELISA) principle have been established. One method uses a ...[more]

PMID: 18584890

Similar Datasets

Project description:BackgroundThe continuous evolution of SARS-CoV-2 has underscored the development of broad-spectrum prophylaxis. Antivirals targeting the membrane fusion process represent promising paradigms. Kaempferol (Kae), an ubiquitous plant flavonol, has been shown efficacy against various enveloped viruses. However, its potential in anti-SARS-CoV-2 invasion remains obscure.PurposeTo evaluate capabilities and mechanisms of Kae in preventing SARS-CoV-2 invasion.MethodsTo avoid interference of viral replication, virus-like particles (VLPs) constructed with luciferase reporter were applied. To investigate the antiviral potency of Kae, human induced pluripotent stem cells (hiPSC)-derived alveolar epithelial cells type II (AECII) and human ACE2 (hACE2) transgenic mice were utilized as in vitro and in vivo models, respectively. Using dual split protein (DSP) assays, inhibitory activities of Kae in viral fusion were determined in Alpha, Delta and Omicron variants of SARS-CoV-2, as well as in SARS-CoV and MERS-CoV. To further reveal molecular determinants of Kae in restricting viral fusion, synthetic peptides corresponding to the conserved heptad repeat (HR) 1 and 2, involved in viral fusion, and the mutant form of HR2 were explored by circular dichroism and native polyacrylamide gel electrophoresis.ResultsKae inhibited SARS-CoV-2 invasion both in vitro and in vivo, which was mainly attributed to its suppressive effects on viral fusion, but not endocytosis, two pathways that mediate viral invasion. In accordance with the proposed model of anti-fusion prophylaxis, Kae functioned as a pan-inhibitor of viral fusion, including three emerged highly pathogenic coronaviruses, and the currently circulating Omicron BQ.1.1 and XBB.1 variants of SARS-CoV-2. Consistent with the typical target of viral fusion inhibitors, Kae interacted with HR regions of SARS-CoV-2 S2 subunits. Distinct from previous inhibitory fusion peptides which prevent the formation of six-helix bundle (6-HB) by competitively interacting with HRs, Kae deformed HR1 and directly reacted with lysine residues within HR2 region, the latter of which was considered critical for the preservation of stabilized S2 during SARS-CoV-2 invasion.ConclusionsKae prevents SARS-CoV-2 infection by blocking membrane fusion and possesses a broad-spectrum anti-fusion ability. These findings provide valuable insights into potential benefits of Kae-containing botanical products as a complementary prophylaxis, especially during the waves of breakthrough infections and re-infections.

Project description:Binding of the gp120 surface subunit of the envelope glycoprotein (Env) of HIV-1 to CD4 and chemokine receptors on target cells triggers refolding of the gp41 transmembrane subunit into a six-helix bundle (6HB) that promotes fusion between virus and host cell membranes. To elucidate details of Env entry and potential differences between viruses that use CXCR4 (X4) or CCR5 (R5) coreceptors, we generated viruses that are resistant to peptide fusion inhibitors corresponding to the first heptad repeat region (HR1) of gp41 that target fusion-intermediate conformations of Env. Previously we reported that an R5 virus selected two resistance pathways, each defined by an early gp41 resistance mutation in either HR1 or the second heptad repeat (HR2), to escape inhibition by an HR1 peptide, but preferentially selected the HR1 pathway to escape inhibition by a trimer-stabilized HR1 peptide. Here, we report that an X4 virus selected the same HR1 and HR2 resistance pathways as the R5 virus to escape inhibition by the HR1 peptide. However, in contrast to the R5 virus, the X4 virus selected a unique mutation in HR2 to escape inhibition by the trimer-stabilized peptide. Significantly, both of these X4 and R5 viruses acquired gp41 resistance mutations that improved the thermostability of the six-helix bundle, but they selected different gp120 adaptive mutations. These findings show that these X4 and R5 viruses use a similar resistance mechanism to escape from HR1 peptide inhibition but different gp120-gp41 interactions to regulate Env conformational changes.IMPORTANCE HIV-1 fuses with cells when the gp41 subunit of Env refolds into a 6HB after binding to cellular receptors. Peptides corresponding to HR1 or HR2 interrupt gp41 refolding and inhibit HIV infection. Previously, we found that a CCR5 coreceptor-tropic HIV-1 acquired a key HR1 or HR2 resistance mutation to escape HR1 peptide inhibitors but only the key HR1 mutation to escape a trimer-stabilized HR1 peptide inhibitor. Here, we report that a CXCR4 coreceptor-tropic HIV-1 selected the same key HR1 or HR2 mutations to escape inhibition by the HR1 peptide but different combinations of HR1 and HR2 mutations to escape the trimer-stabilized HR1 peptide. All gp41 mutations enhance 6HB stability to outcompete inhibitors, but gp120 adaptive mutations differed between these R5 and X4 viruses, providing new insights into gp120-gp41 functional interactions affecting Env refolding during HIV entry.

Dataset Information

Novel screening systems for HIV-1 fusion mediated by two extra-virion heptad repeats of gp41.

Publications

Novel screening systems for HIV-1 fusion mediated by two extra-virion heptad repeats of gp41.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets