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Conformational changes in Chikungunya virus E2 protein upon heparan sulfate receptor binding explain mechanism of E2-E1 dissociation during viral entry.

ABSTRACT: Receptor binding is the first step in viral cell entry. In enveloped virus cell entry, viral and host membrane fusion follows receptor binding. Viral surface receptor-binding protein associates with membrane fusion protein and masks its structure, to prevent pre-mature fusion activity. Dissociation of receptor-binding protein from fusion protein is an essential step before membrane fusion. Mechanism of receptor binding leading to dissociation of receptor binding and fusion protein is poorly understood in alphaviruses. Chikungunya virus (CHIKV), an alphavirus, re-emerged as a global pathogen in recent past. CHIKV surface envelope proteins, E2 and E1, function as receptor binding and fusion protein, respectively. Site of heparan sulfate (HS) receptor binding on E2-E1 heterodimer and its effect on E2-E1 heterodimer conformation is not known. Using molecular docking, we mapped HS binding to a positively charged pocket on E2 that is structurally conserved in alphaviruses. Based on our results from docking and sequence analysis, we identified a novel HS-binding sequence motif in E2. Purified E2 binds to heparin and HS specifically through charge interactions. Binding affinity of E2 to HS is comparable with other known HS-protein interactions (K d ? 1.8 ?M). Mutation of charged residues in the predicted HS-binding motif of E2 to alanine resulted in reduction of HS binding. Molecular dynamics (MD) simulations on E2, after docking HS, predicted allosteric domain movements. Fluorescence spectroscopy, far-UV circular dichroism spectroscopy, fluorescence resonance energy transfer experiments on HS-bound E2 corroborate our findings from MD simulations. We propose a mechanism where receptor-binding results in allosteric domain movements in E2, explaining E2-E1 dissociation.

SUBMITTER: Sahoo B

PROVIDER: S-EPMC6597851 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Conformational changes in Chikungunya virus E2 protein upon heparan sulfate receptor binding explain mechanism of E2-E1 dissociation during viral entry.

Sahoo Bibekananda B Chowdary Tirumala Kumar TK

Bioscience reports 20190628 6

Receptor binding is the first step in viral cell entry. In enveloped virus cell entry, viral and host membrane fusion follows receptor binding. Viral surface receptor-binding protein associates with membrane fusion protein and masks its structure, to prevent pre-mature fusion activity. Dissociation of receptor-binding protein from fusion protein is an essential step before membrane fusion. Mechanism of receptor binding leading to dissociation of receptor binding and fusion protein is poorly unde ...[more]

PMID: 31167876

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Project description:By targeting cells that provide protection against infection, HIV-1 causes acquired immunodeficiency syndrome. Infection starts when gp120, the viral envelope glycoprotein, binds to CD4 and to a chemokine receptor usually CCR5 or CXCR4. As many microorganisms, HIV-1 also interacts with heparan sulfate (HS), a complex group of cell surface associated anionic polysaccharides. It has been thought that this binding, occurring at a step prior to CD4 recognition, increases infectivity by pre-concentrating the virion particles at the cell surface. Early work, dating from before the identification of CCR5 and CXCR4, showed that a variety of HS mimetics bind to the gp120 V3 loop through electrostatic interactions, compete with cell surface associated HS to bind the virus and consequently, neutralize the infectivity of a number of T-cell line-adapted HIV-1 strains. However, progress made to better understand HIV-1 attachment and entry, coupled with the recent identification of additional gp120 regions mediating HS recognition, have considerably modified this view. Firstly, the V3 loop from CXCR4-using viruses is much more positively charged compared to those using CCR5. HS inhibition of cell attachment is thus restricted to CXCR4-using viruses (such as T-cell line-adapted HIV-1). Secondly, studies aiming at characterizing the gp120/HS complex revealed that HS binding was far more complex than previously thought: in addition to the V3 loop of CXCR4 tropic gp120, HS interacts with several other cryptic areas of the protein, which can be induced upon CD4 binding, and are conserved amongst CCR5 and CXCR4 viruses. In view of these data, this review will detail the present knowledge on HS binding to HIV-1, with regards to attachment and entry processes. It will discuss the perspective of targeting the gp120 co-receptor binding site with HS mimetic compounds, a strategy that recently gave rise to entry inhibitors that work in the low nanomolar range, independently of co-receptor usage.

Project description:Cell culture-adapted strains of Sindbis virus (SINV) initially attach to cells by the ability to interact with heparan sulfate (HS) through selective mutation for positively charged amino acid (aa) scattered in E2 glycoprotein (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72: 7357-7366, 1998). Here we have further confirmed that interaction of E2 protein with HS is crucial for cellular infection of SINV based on the reverse genetic system of XJ-160 virus, a Sindbis-like virus (SINLV). Both SINV YN87448 and SINLV XJ-160 displayed similar infectivity on BHK-21, Vero, or C6/36 cells, but XJ-160 failed to infect mouse embryonic fibroblast (MEF) cells. The molecular mechanisms underlying the selective infectivity of XJ-160 were approached by substituting the E1, E2, or both genes of XJ-160 with that of YN87448, and the chimeric virus was denominated as XJ-160/E1, XJ-160/E2, or XJ-160/E1E2, respectively. In contrast to the parental XJ-160, all chimeric viruses became infectious to wild-type MEF cells (MEF-wt). While MEF-Ext(-/-) cells, producing shortened HS chains, were resistant not only to XJ-160, but also to YN87448 as well as the chimeric viruses, indicating that the inability of XJ-160 to infect MEF-wt cells likely due to its incompetent discrimination of cellular HS. Treatment with heparin or HS-degrading enzyme resulted in a substantial decrease in plaque formation by YN87448, XJ-160/E2, and XJ-160/E1E2, but had marginal effect on XJ-160 and XJ-160/E1, suggesting that E2 glycoprotein from YN87448 plays a more important role than does E1 in mediating cellular HS-related cell infection. In addition, the peptide containing 145-150 aa from E2 gene of YN87448 specifically bound to heparin, while the corresponding peptide from the E2 gene of XJ-160 essentially showed no binding to heparin. As a new dataset, these results clearly confirm an essential role of E2 glycoprotein, especially the domain of 145-150 aa, in SINV cellular infection through the interaction with HS.

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Conformational changes in Chikungunya virus E2 protein upon heparan sulfate receptor binding explain mechanism of E2-E1 dissociation during viral entry.

Publications

Conformational changes in Chikungunya virus E2 protein upon heparan sulfate receptor binding explain mechanism of E2-E1 dissociation during viral entry.

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