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Acidic pH-Induced Conformational Changes in Chikungunya Virus Fusion Protein E1: a Spring-Twisted Region in the Domain I-III Linker Acts as a Hinge Point for Swiveling Motion of Domains.

ABSTRACT: Chikungunya virus (CHIKV), a mosquito-transmitted alphavirus, enters a cell through endocytosis, followed by viral and cell membrane fusion. The fusion protein, E1, undergoes an acid pH-induced pre- to postfusion conformation change during membrane fusion. As part of the conformation change, E1 dissociates from the receptor-binding protein, E2, and swivels its domains I and II over domain III to form an extended intermediate and then eventually to form a postfusion hairpin homotrimer. In this study, we tested if the domain I-III linker acts as a "hinge" for the swiveling motion of E1 domains. We found a conserved spring-twisted structure in the linker, stabilized by a salt bridge between a conserved arginine-aspartic acid pair, as a "hinge point" for domain swiveling. Molecular dynamics (MD) simulation of the CHIKV E1 or E2-E1 structure predicted that the spring-twisted region untwists at pH 5.5. Corroborating the prediction, introduction of a "cystine staple" at the hinge point, replacing the conserved arginine-aspartic acid pair with cysteine residues, resulted in loss of fusion activity of E1. MD simulation also predicted domain I-III swiveling at acidic pH. We tested if breaking the His 331-Lys 16 H bond between domains I and III, seen only in the prefusion conformation, is important for domain swiveling. When domains I and III are "stapled" by introducing a disulfide bond in between, E1 showed loss of fusion activity, implying that domain I and III dissociation is a critical acid pH-induced step in membrane fusion. However, replacement of His 331 with an acidic residue did not affect the pH threshold for fusion, suggesting His 331 is not an acid-sensing residue.IMPORTANCE Aedes mosquito-transmitted viruses such as the Zika, dengue, and chikungunya viruses have spread globally. CHIKV, similar to many other enveloped viruses, enters cells in sequential steps: step 1 involves receptor binding followed by endocytosis, and step 2 involves viral-cell membrane fusion in the endocytic vesicle. The viral envelope surface protein, E1, performs membrane fusion. E1 is triggered to undergo conformational changes by acidic pH of the maturing endosome. Different domains of E1 rearrange during the pre- to postfusion conformation change. Using in silico analysis of the E1 structure and different biochemical experiments, we explained a structural mechanism of key conformational changes in E1 triggered by acidic pH. We noted two important structural changes in E1 at acidic pH. In the first, a spring-twisted region in a loop connecting two domains (I and III) untwists, bringing a swiveling motion of domains on each other. In the second, breaking of interactions between domains I and III and domain separation are required for membrane fusion. This knowledge will help devise new therapeutic strategies to block conformation changes in E1 and thus viral entry.

SUBMITTER: Sahoo B

PROVIDER: S-EPMC7654277 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Chikungunya virus (CHIKV) is a reemerging arthropod-borne alphavirus and a serious threat to human health. Therefore, efforts toward elucidating how this virus causes disease and the molecular mechanisms underlying steps of the viral replication cycle are crucial. Using an in vivo transmission system that allows intrahost evolution, we identified an emerging CHIKV variant carrying a mutation in the E1 glycoprotein (V156A) in the serum of mice and saliva of mosquitoes. E1 V156A has since emerged in humans during an outbreak in Brazil, cooccurring with a second mutation, E1 K211T, suggesting an important role for these residues in CHIKV biology. Given the emergence of these variants, we hypothesized that they function to promote CHIKV infectivity and subsequent disease. Here, we show that E1 V156A and E1 K211T modulate virus attachment and fusion and impact binding to heparin, a homolog of heparan sulfate, a key entry factor on host cells. These variants also exhibit differential neutralization by antiglycoprotein monoclonal antibodies, suggesting structural impacts on the particle that may be responsible for altered interactions at the host membrane. Finally, E1 V156A and E1 K211T exhibit increased titers in an adult arthritic mouse model and induce increased foot-swelling at the site of injection. Taken together, this work has revealed new roles for E1 where discrete regions of the glycoprotein are able to modulate cell attachment and swelling within the host. IMPORTANCE Alphaviruses represent a growing threat to human health worldwide. The reemerging alphavirus chikungunya virus (CHIKV) has rapidly spread to new geographic regions in the last several decades, causing overwhelming outbreaks of disease, yet there are no approved vaccines or therapeutics. The CHIKV glycoproteins are key determinants of CHIKV adaptation and virulence. In this study, we identify and characterize the emerging E1 glycoprotein variants, V156A and K211T, that have since emerged in nature. We demonstrate that E1 V156A and K211T function in virus attachment to cells, a role that until now has only been attributed to specific residues of the CHIKV E2 glycoprotein. We also demonstrate E1 V156A and K211T increase foot-swelling of the ipsilateral foot in mice infected with these variants. Observing that these variants and other pathogenic variants occur at the E1-E1 interspike interface, we highlight this structurally important region as critical for multiple steps during CHIKV infection. Together, these studies further define the function of E1 in CHIKV infection and can inform the development of therapeutic or preventative strategies.

Project description:BackgroundChikungunya fever is a pandemic disease caused by the mosquito-borne Chikungunya virus (CHIKV). E1 glycoprotein mediation of viral membrane fusion during CHIKV infection is a crucial step in the release of viral genome into the host cytoplasm for replication. How the E1 structure determines membrane fusion and whether other CHIKV structural proteins participate in E1 fusion activity remain largely unexplored.MethodsA bicistronic baculovirus expression system to produce recombinant baculoviruses for cell-based assay was used. Sf21 insect cells infected by recombinant baculoviruses bearing wild type or single-amino-acid substitution of CHIKV E1 and EGFP (enhanced green fluorescence protein) were employed to investigate the roles of four E1 amino acid residues (G91, V178, A226, and H230) in membrane fusion activity.ResultsWestern blot analysis revealed that the E1 expression level and surface features in wild type and mutant substituted cells were similar. However, cell fusion assay found that those cells infected by CHIKV E1-H230A mutant baculovirus showed little fusion activity, and those bearing CHIKV E1-G91D mutant completely lost the ability to induce cell-cell fusion. Cells infected by recombinant baculoviruses of CHIKV E1-A226V and E1-V178A mutants exhibited the same membrane fusion capability as wild type. Although the E1 expression level of cells bearing monomeric-E1-based constructs (expressing E1 only) was greater than that of cells bearing 26S-based constructs (expressing all structural proteins), the sizes of syncytial cells induced by infection of baculoviruses containing 26S-based constructs were larger than those from infections having monomeric-E1 constructs, suggesting that other viral structure proteins participate or regulate E1 fusion activity. Furthermore, membrane fusion in cells infected by baculovirus bearing the A226V mutation constructs exhibited increased cholesterol-dependences and lower pH thresholds. Cells bearing the V178A mutation exhibited a slight decrease in cholesterol-dependence and a higher-pH threshold for fusion.ConclusionsCells expressing amino acid substitutions of conserved protein E1 residues of E1-G91 and E1-H230 lost most of the CHIKV E1-mediated membrane fusion activity. Cells expressing mutations of less-conserved amino acids, E1-V178A and E1-A226V, retained membrane fusion activity to levels similar to those expressing wild type E1, but their fusion properties of pH threshold and cholesterol dependence were slightly altered.

Project description:BACKGROUND: CHIKV is a mosquito-borne emerging pathogen that has a major health impact in humans in tropical zones around the globe. A new variant of the virus, E1-A226V caused a large outbreak in the Indian Ocean islands and India from 2004-2007. CHIKV outbreak was initially reported in Dongguan region of Guangdong in 2010 in China, another smaller CHIKV outbreak was found in Yangjiang region of Guangdong two weeks later. The viral agent causing the two outbreaks was inferred to be the new E1-A226V variant and Yangjiang CHIKV might be introduced from Dongguan. To confirm the hypothesis and determine the origin of CHIKV causing the outbreaks, we described Yangjiang outbreak in this study, and the molecular characterization of CHIKV from Yangjiang and Dongguang outbreaks were analyzed. RESULTS: 27 clinical cases of CHIK fever were reported in outbreak in Yangjiang region. Sera sample from 12 clinical cases were collected from the outbreak, and nucleic acid and antibody tests for CHIKV were performed using Real-time RT-PCR and indirect immunofluorescence. Positive samples of Real-time RT-PCR were subjected to viral isolation. The results showed 3/12 samples positive for Real-time RT-PCR. 7/12 and 4/12 samples were positive for IgM and IgG against CHIKV respectively, two virus strains were isolated. Four viral genomes from Dongguan and Yangjiang were sequenced, characterized and phylogeneticly analyzed. Phylogenetic analysis revealed that the four seqeunced viruses had the closest relationship (99.4~99.6% identify) with the Singapore 2008 isolate belonging to the Indian ocean clade. A common mutation at the site of the E1-A226V was observed among four viruses. Four and three aa substitutions were detected in the CHIKV sequence from the Dongguan and Yangjiang outbreak strains respectively. CONCLUSION: CHIKV with an E1-A226V mutation that originated from Southeast Asia isolates caused two outbreaks in China in 2010, and originated from two different infectious sources.

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Acidic pH-Induced Conformational Changes in Chikungunya Virus Fusion Protein E1: a Spring-Twisted Region in the Domain I-III Linker Acts as a Hinge Point for Swiveling Motion of Domains.

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