scholarly journals Mutations Conferring a Noncytotoxic Phenotype on Chikungunya Virus Replicons Compromise Enzymatic Properties of Nonstructural Protein 2

2014 ◽  
Vol 89 (6) ◽  
pp. 3145-3162 ◽  
Author(s):  
Age Utt ◽  
Pratyush Kumar Das ◽  
Margus Varjak ◽  
Valeria Lulla ◽  
Aleksei Lulla ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Chikungunya Virus ◽  
Nonstructural Protein ◽  
Rna Helicase ◽  
Enzymatic Activities ◽  
Host Factors ◽  
Adaptive Mutations ◽  
Nonstructural Protein 2 ◽  
Experimental Systems

ABSTRACTChikungunya virus (CHIKV) (genusAlphavirus) has a positive-sense RNA genome. CHIKV nonstructural protein 2 (nsP2) proteolytically processes the viral nonstructural polyprotein, possesses nucleoside triphosphatase (NTPase), RNA triphosphatase, and RNA helicase activities, and induces cytopathic effects in vertebrate cells. Although alphaviral nsP2 mutations can result in a noncytotoxic phenotype, the effects of such mutations on nsP2 enzymatic activities are not well understood. In this study, we introduced a P718G (PG) mutation and selected for additional mutations in CHIKV nsP2 that resulted in a CHIKV replicon with a noncytotoxic phenotype in BHK-21 cells. Combinations of PG and either an E116K (EK) substitution or a GEEGS sequence insertion after residue T648 (5A) markedly reduced RNA synthesis; however, neither PG nor 5A prevented nsP2 nuclear translocation. Introducing PG into recombinant nsP2 inhibited proteolytic cleavage of nsP1/nsP2 and nsP3/nsP4 sites, reduced GTPase and RNA helicase activities, and abolished RNA stimulation of GTPase activity. 5A and EK modulated the effects of PG. However, only the RNA helicase activity of nsP2 was reduced by both of these mutations, suggesting that defects in this activity may be linked to a noncytotoxic phenotype. These results increase our understanding of the molecular basis for the cytotoxicity that accompanies alphaviral replication. Furthermore, adaptation of the CHIKV replicon containing both 5A and PG allowed the selection of a CHIKV replicon with adaptive mutations in nsP1 and nsP3 that enable persistence in human cell line. Such cell lines represent valuable experimental systems for discovering host factors and for screening inhibitors of CHIKV replication at lower biosafety levels.IMPORTANCECHIKV is a medically important pathogen that causes febrile illness and can cause chronic arthritis. No approved vaccines or antivirals are available for CHIKV. The attenuation of CHIKV is critical to the establishment of experimental systems that can be used to conduct virus replication studies at a lower biosafety level. We applied a functional selection approach to develop, for the first time, a noncytotoxic CHIKV replicon capable of persisting in human cell lines. We anticipate that this safe and efficient research tool will be valuable for screening CHIKV replication inhibitors and for identifying and analyzing host factors involved in viral replication. We also analyzed, from virological and protein biochemistry perspectives, the functional defects caused by mutations conferring noncytotoxic phenotypes; we found that all known enzymatic activities of CHIKV nsP2, as well as its RNA-binding capability, were compromised by these mutations, which led to a reduced capacity for replication.

scholarly journals Structural insights into RNA recognition by the Chikungunya virus nsP2 helicase

2019 ◽  
Vol 116 (19) ◽  
pp. 9558-9567 ◽  
Author(s):  
Yee-Song Law ◽  
Age Utt ◽  
Yaw Bia Tan ◽  
Jie Zheng ◽  
Sainan Wang ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Chikungunya Virus ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Rna Helicase ◽  
Viral Rna ◽  
Helicase Domain ◽  
Stacking Interactions ◽  
Adaptive Mutations ◽  
Rna Backbone

Chikungunya virus (CHIKV) is transmitted to humans through mosquitoes and causes Chikungunya fever. Nonstructural protein 2 (nsP2) exhibits the protease and RNA helicase activities that are required for viral RNA replication and transcription. Unlike for the C-terminal protease, the structure of the N-terminal RNA helicase (nsP2h) has not been determined. Here, we report the crystal structure of the nsP2h bound to the conserved 3′-end 14 nucleotides of the CHIKV genome and the nonhydrolyzable transition-state nucleotide analog ADP-AlF4. Overall, the structural analysis revealed that nsP2h adopts a uniquely folded N-terminal domain followed by a superfamily 1 RNA helicase fold. The conserved helicase motifs establish polar contacts with the RNA backbone. There are three hydrophobic residues (Y161, F164, and F287) which form stacking interactions with RNA bases and thereby bend the RNA backbone. An F287A substitution that disrupted these stacking interactions increased the basal ATPase activity but decreased the RNA binding affinity. Furthermore, the F287A substitution reduced viral infectivity by attenuating subgenomic RNA synthesis. Replication of the mutant virus was restored by pseudoreversion (A287V) or adaptive mutations in the RecA2 helicase domain (T358S or V410I). Y161A and/or F164A substitutions, which were designed to disrupt the interactions with the RNA molecule, did not affect the ATPase activity but completely abolished the replication and transcription of viral RNA and the infectivity of CHIKV. Our study sheds light on the roles of the RNA helicase region in viral replication and provides insights that might be applicable to alphaviruses and other RNA viruses in general.

scholarly journals Structural and Functional Characterization of Host FHL1 Protein Interaction with Hypervariable Domain of Chikungunya Virus nsP3 Protein

2020 ◽  
Vol 95 (1) ◽  
Author(s):  
Tetyana Lukash ◽  
Tatiana Agback ◽  
Francisco Dominguez ◽  
Nikita Shiliaev ◽  
Chetan Meshram ◽  
...  
Keyword(s):  
Viral Replication ◽  
Binding Site ◽  
Cell Lines ◽  
Binding Sites ◽  
Chikungunya Virus ◽  
Biological Significance ◽  
Host Factors ◽  
Published Data ◽  
Wide Range ◽  
Infection Spread

ABSTRACT Decades of insufficient control have resulted in unprecedented spread of chikungunya virus (CHIKV) around the globe, and millions have suffered from the highly debilitating disease. Nevertheless, the current understanding of CHIKV-host interactions and adaptability of the virus to replication in mosquitoes and mammalian hosts is still elusive. Our new study shows that four-and-a-half LIM domain protein (FHL1) is one of the host factors that interact with the hypervariable domain (HVD) of CHIKV nsP3. Unlike G3BPs, FHL1 is not a prerequisite of CHIKV replication, and many commonly used cell lines do not express FHL1. However, its expression has a detectable stimulatory effect(s) on CHIKV replication, and Fhl1 knockout (KO) cell lines demonstrate slower infection spread. Nuclear magnetic resonance (NMR)-based studies revealed that the binding site of FHL1 in CHIKV nsP3 HVD overlaps that of another proviral host factor, CD2AP. The structural data also demonstrated that FHL1-HVD interaction is mostly determined by the LIM1 domain of FHL1. However, it does not mirror binding of the entire protein, suggesting that other LIM domains are involved. In agreement with previously published data, our biological experiments showed that interactions of CHIKV HVD with CD2AP and FHL1 have additive effects on the efficiency of CHIKV replication. This study shows that CHIKV mutants with extensive modifications of FHL1- or both FHL1- and CD2AP-binding sites remain viable and develop spreading infection in multiple cell types. Our study also demonstrated that other members of the FHL family can bind to CHIKV HVD and thus may be involved in viral replication. IMPORTANCE Replication of chikungunya virus (CHIKV) is determined by a wide range of host factors. Previously, we have demonstrated that the hypervariable domain (HVD) of CHIKV nsP3 contains linear motifs that recruit defined families of host proteins into formation of functional viral replication complexes. Now, using NMR-based structural and biological approaches, we have characterized the binding site of the cellular FHL1 protein in CHIKV HVD and defined the biological significance of this interaction. In contrast to previously described binding of G3BP to CHIKV HVD, the FHL1-HVD interaction was found to not be a prerequisite of viral replication. However, the presence of FHL1 has a stimulatory effect on CHIKV infectivity and, subsequently, the infection spread. FHL1 and CD2AP proteins were found to have overlapping binding sites in CHIKV HVD and additive proviral functions. Elimination of the FHL1-binding site in the nsP3 HVD can be used for the development of stable, attenuated vaccine candidates.

scholarly journals Nonstructural Protein 2 (nsP2) of Chikungunya Virus (CHIKV) Enhances Protective Immunity Mediated by a CHIKV Envelope Protein Expressing DNA Vaccine

2013 ◽  
Vol 26 (1) ◽  
pp. 75-83 ◽  
Author(s):  
Huihui Bao ◽  
Aarti A. Ramanathan ◽  
Omkar Kawalakar ◽  
Senthil G. Sundaram ◽  
Colleen Tingey ◽  
...  
Keyword(s):  
Dna Vaccine ◽  
Envelope Protein ◽  
Protective Immunity ◽  
Chikungunya Virus ◽  
Nonstructural Protein ◽  
Nonstructural Protein 2

scholarly journals Structural and Functional Studies of Chikungunya Virus nsP2

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 113
Author(s):  
Yee Song Law ◽  
Yaw Bia Tan ◽  
Orion Shih ◽  
Age Utt ◽  
Jie Zheng ◽  
...  
Keyword(s):  
Cysteine Protease ◽  
Solution Structure ◽  
Chikungunya Virus ◽  
Atp Hydrolysis ◽  
Nonstructural Protein ◽  
Rna Helicase ◽  
Full Length ◽  
Viral Rna ◽  
Helicase Domain ◽  
Functional Studies

Chikungunya virus (CHIKV) is transmitted to humans through mosquitoes and causes Chikungunya fever. Nonstructural protein 2 (nsP2) contains an N-terminal RNA helicase with both nucleotide triphosphatase and RNA triphosphatase activities, and a C-terminal cysteine protease that is responsible for polyprotein processing. Both N-terminal RNA helicase and C-terminal cysteine protease are connected through a flexible linker. Although the structure of the C-terminal cysteine protease has been solved, the structure and the conformational arrangement of full-length nsP2 remains elusive. Here, we determined the crystal structure of the helicase part of the CHIKV nsP2 (nsP2h) bound to the conserved 3′-end of the genomic RNA and the nucleotide analogue ADP-AlF4. The structure of this ternary complex revealed the molecular basis for viral RNA recognition and ATP hydrolysis by the nsP2h. Unique hydrophobic protein–RNA interactions play essential roles in viral RNA replication. We also determined the solution structure of full-length nsP2 using small-angle X-ray scattering (SAXS). The solution architecture of the nsP2 was modeled using the available high-resolution structures and program CORAL (complexes with random loops). The CORAL model revealed that nsP2 is partially unfolded and the N-terminal protease domain is arranged near the N-terminal domain of the helicase domain. These findings expand our knowledge of CHIKV and related alphaviruses and might also have broad implications for antiviral and vaccine developments against pathogenic alphaviruses.

scholarly journals Novel Mutations in nsP2 Abolish Chikungunya Virus-Induced Transcriptional Shutoff and Make the Virus Less Cytopathic without Affecting Its Replication Rates

2018 ◽  
Vol 93 (4) ◽  
Author(s):  
Ivan Akhrymuk ◽  
Tetyana Lukash ◽  
Ilya Frolov ◽  
Elena I. Frolova
Keyword(s):  
Cellular Response ◽  
Sindbis Virus ◽  
Chikungunya Virus ◽  
Type I Interferon ◽  
Nonstructural Protein ◽  
Type I ◽  
Heterologous Proteins ◽  
Old World ◽  
Nonstructural Protein 2 ◽  
Cellular Transcription

ABSTRACTAlphavirus infections are characterized by global inhibition of cellular transcription and rapid induction of a cytopathic effect (CPE) in cells of vertebrate origin. Transcriptional shutoff impedes the cellular response to alphavirus replication and prevents establishment of an antiviral state. Chikungunya virus (CHIKV) is a highly pathogenic alphavirus representative, and its nonstructural protein 2 (nsP2) plays critical roles in both inhibition of transcription and CPE development. Previously, we have identified a small peptide in Sindbis virus (SINV) nsP2 (VLoop) that determined the protein’s transcriptional inhibition function. It is located in the surface-exposed loop of the carboxy-terminal domain of nsP2 and exhibits high variability between members of different alphavirus serocomplexes. In this study, we found that SINV-specific mutations could not be directly applied to CHIKV. However, by using a new selection approach, we identified a variety of new VLoop variants that made CHIKV and its replicons incapable of inhibiting cellular transcription and dramatically less cytopathic. Importantly, the mutations had no negative effect on RNA and viral replication rates. In contrast to parental CHIKV, the developed VLoop mutants were unable to block induction of type I interferon. Consequently, they were cleared from interferon (IFN)-competent cells without CPE development. Alternatively, in murine cells that have defects in type I IFN production or signaling, the VLoop mutants established persistent, noncytopathic replication. The mutations in nsP2 VLoop may be used for development of new vaccine candidates against alphavirus infections and vectors for expression of heterologous proteins.IMPORTANCEChikungunya virus is an important human pathogen which now circulates in both the Old and New Worlds. As in the case of other Old World alphaviruses, CHIKV nsP2 not only has enzymatic functions in viral RNA replication but also is a critical inhibitor of the antiviral response and one of the determinants of CHIKV pathogenesis. In this study, we have applied a new strategy to select a variety of CHIKV nsP2 mutants that no longer exhibited transcription-inhibitory functions. The designed CHIKV variants became potent type I interferon inducers and acquired a less cytopathic phenotype. Importantly, they demonstrated the same replication rates as the parental CHIKV. Mutations in the same identified peptide of nsP2 proteins derived from other Old World alphaviruses also abolished their nuclear functions. Such mutations can be further exploited for development of new attenuated alphaviruses.

scholarly journals Structural and Functional Characterization of Host FHL1 Protein Interaction with Hypervariable Domain of Chikungunya Virus nsP3 Protein

2020 ◽  
Author(s):  
Tetyana Lukash ◽  
Tatiana Agback ◽  
Francisco Dominguez ◽  
Nikita Shiliaev ◽  
Chetan Meshram ◽  
...  
Keyword(s):  
Viral Replication ◽  
Binding Site ◽  
Cell Lines ◽  
Binding Sites ◽  
Chikungunya Virus ◽  
Host Factors ◽  
Vaccine Candidates ◽  
Positive Effects ◽  
Wide Range ◽  
Infection Spread

ABSTRACTDecades of insufficient control resulted in unprecedented spread of chikungunya virus (CHIKV) around the globe and millions already suffered from the highly debilitating disease. Nevertheless, the current understanding of CHIKV-host interactions and adaptability of the virus to replication in mosquitoes and mammalian hosts is still elusive. Our new study shows that four-and-a-half LIM domain protein (FHL1) is one of the host factors that interact with hypervariable domain (HVD) of CHIKV nsP3. Unlike G3BPs, FHL1 is not a pre-requisite of CHIKV replication, and many commonly used cell lines do not express FHL1. However, its expression has detectable stimulatory effect(s) on CHIKV replication, and the Fhl1 KO cell lines demonstrate slower infection spread. The NMR-based studies revealed that the binding site of FHL1 in CHIKV nsP3 HVD overlaps with that of another pro-viral host factor, CD2AP. The structural data also demonstrated that FHL1-HVD interaction is mostly determined by LIM1 domain of FHL1. However, it does not mirror binding of the entire protein, suggesting that other LIM domains are involved. In agreement with previously published data, our biological experiments showed that interactions of CHIKV HVD with CD2AP and FHL1 have additive positive effects on the efficiency of CHIKV replication. This study shows that CHIKV mutants with extensive modifications of FHL1- or both FHL1- and CD2AP- binding sites remain viable and develop spreading infection in multiple cell types. Thus, such modifications of HVD may improve live CHIKV vaccine candidates in terms of their safety and stability of the attenuated phenotype.IMPORTANCEReplication of chikungunya virus (CHIKV) is determined by a wide range of host factors. Previously, we have demonstrated that the hypervariable domain (HVD) of CHIKV nsP3 protein contains linear motifs that recruit defined families of host proteins into formation of functional viral replication complexes. Now, using NMR-based structural and biological approaches, we have characterized the binding site of cellular FHL1 protein in CHIKV HVD and defined the biological significance of this interaction. In contrast to previously described binding of G3BP to CHIKV HVD, the FHL1-HVD interaction was found to not be a prerequisite of viral replication. However, the presence of FHL1 has a stimulatory effect on CHIKV infectivity and subsequently, the infection spread. FHL1 and CD2AP proteins were found to have overlapping binding sites in CHIKV HVD and additive pro-viral functions. Elimination of FHL1-binding site in nsP3 HVD can be used for the development of stable, live attenuated vaccine candidates.

scholarly journals Screening and testing for a suitable untransfected cell line for SARS-CoV-2 studies

2020 ◽  
Author(s):  
Claudia Pommerenke ◽  
Ulfert Rand ◽  
Cord C. Uphoff ◽  
Stefan Nagel ◽  
Margarete Zaborski ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Human Cell ◽  
Viral Entry ◽  
Cancer Cell Line ◽  
Receptor Expression ◽  
Host Factors ◽  
Human Cell Lines ◽  
Infection Models

AbstractAt present, the novel pandemic coronavirus SARS-CoV-2 is a major global threat to human health and hence demands united research activities at different levels. Finding appropriate cell systems for drug screening and testing molecular interactions of the virus with the host cell is mandatory for drug development and understanding the mechanisms of viral entry and replication. For this, we selected human cell lines represented in the Cancer Cell Line Encyclopedia (CCLE) based on RNA-seq data determined transcript levels of ACE2 and TMPRSS2, two membrane proteins that have been identified to aid SARS-CoV-2 entry into the host cell. mRNA and protein expression of these host factors were verified via RQ-PCR and western blot. We then tested permissiveness of these cell lines towards SARS-CoV-2 infection, cytopathic effect, and viral replication finding limited correlation between receptor expression and infectability. One of the candidate cancer cell lines, the human colon cancer cell line CL-14, tested positive for SARS-CoV-2 infection. Our data argue that SARS-CoV-2 in vitro infection models need careful selection and validation since ACE2/TMPRSS2 receptor expression on its own does not guarantee permissiveness to the virus.Author summaryIn the midst of the pandemic outbreak of corona-virus SARS-CoV-2 therapeutics for disease treatment are still to be tested and the virus-host-interactions are to be elucidated. Drug testing and viral studies are commonly conducted with genetically manipulated cells. In order to find a cell model system without genetic modification we screened human cell lines for two proteins known to facilitate entry of SARS-CoV-2. We confirmed and quantified permissiveness of current cell line infection models, but dismissed a number of receptor-positive cell lines that did not support viral replication. Importantly, ACE2/TMPRSS2 co-expression seems to be necessary for viral entry but is not sufficient to predict permissiveness of various cancer cell lines. Moreover, the expression of specific splice variants and the absence of missense mutations of the host factors might hint on successful infection and virus replication of the cell lines.

scholarly journals NAP1L1 and NAP1L4 binding to Hypervariable Domain of Chikungunya Virus nsP3 Protein is bivalent and requires phosphorylation

2021 ◽  
Author(s):  
Francisco Dominguez ◽  
Nikita Shiliaev ◽  
Tetyana Lukash ◽  
Peter Agback ◽  
Oksana Palchevska ◽  
...  
Keyword(s):  
Viral Replication ◽  
Chikungunya Virus ◽  
Nonstructural Protein ◽  
Family Members ◽  
Host Factors ◽  
Nonstructural Proteins ◽  
Binding Motifs ◽  
Wide Range ◽  
New Generation ◽  
Ck2 Kinase

Chikungunya virus (CHIKV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. Within the last two decades, CHIKV has expanded its presence to both hemispheres and is currently circulating in both Old and New Worlds. Despite the severity and persistence of the arthritis it causes in humans, no approved vaccines or therapeutic means have been developed for CHIKV infection. Replication of alphaviruses, including CHIKV, is determined not only by their nonstructural proteins, but also by a wide range of host factors, which are indispensable components of viral replication complexes (vRCs). Alphavirus nsP3s contain hypervariable domains (HVDs), which encode multiple motifs that drive recruitment of cell- and virus-specific host proteins into vRCs. Our previous data suggested that NAP1 family members are a group of host factors that may interact with CHIKV nsP3 HVD. In this study, we performed a detailed investigation of the NAP1 function in CHIKV replication in vertebrate cells. Our data demonstrate that i) the NAP1-HVD interactions have strong stimulatory effects on CHIKV replication; ii) both NAP1L1 and NAP1L4 interact with the CHIKV HVD; iii) NAP1 family members interact with two motifs, which are located upstream and downstream of the G3BP-binding motifs of CHIKV HVD; iv) NAP1 proteins interact only with a phosphorylated form of CHIKV HVD and HVD phosphorylation is mediated by CK2 kinase; v) NAP1 and other families of host factors redundantly promote CHIKV replication and their bindings have additive stimulatory effects on viral replication. IMPORTANCE Cellular proteins play critical roles in the assembly of alphavirus replication complexes (vRCs). Their recruitment is determined by the viral nonstructural protein 3 (nsP3). This protein contains a long, disordered hypervariable domain (HVD), which encodes virus-specific combinations of short linear motifs interacting with host factors during vRC assembly. Our study defined the binding mechanism of NAP1 family members to CHIKV HVD and demonstrated a stimulatory effect of this interaction on viral replication. We showed that interaction with NAP1L1 is mediated by two HVD motifs and requires phosphorylation of HVD by CK2 kinase. Based on the accumulated data, we present a map of the binding motifs of the critical host factors currently known to interact with CHIKV HVD. It can be used to manipulate cell specificity of viral replication and pathogenesis, and to develop a new generation of vaccine candidates.

scholarly journals Chikungunya Virus Nonstructural Protein 2 Inhibits Type I/II Interferon-Stimulated JAK-STAT Signaling

2010 ◽  
Vol 84 (20) ◽  
pp. 10877-10887 ◽  
Author(s):  
Jelke J. Fros ◽  
Wen Jun Liu ◽  
Natalie A. Prow ◽  
Corinne Geertsema ◽  
Maarten Ligtenberg ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Sindbis Virus ◽  
Chikungunya Virus ◽  
Nuclear Translocation ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Type I ◽  
Nonstructural Protein 2 ◽  
Stat Signaling ◽  
Host Shutoff

ABSTRACT Chikungunya virus (CHIKV) is an emerging human pathogen transmitted by mosquitoes. Like that of other alphaviruses, CHIKV replication causes general host shutoff, leading to severe cytopathicity in mammalian cells, and inhibits the ability of infected cells to respond to interferon (IFN). Recent research, however, suggests that alphaviruses may have additional mechanisms to circumvent the host's antiviral IFN response. Here we show that CHIKV replication is resistant to inhibition by interferon once RNA replication has been established and that CHIKV actively suppresses the antiviral IFN response by preventing IFN-induced gene expression. Both CHIKV infection and CHIKV replicon RNA replication efficiently blocked STAT1 phosphorylation and/or nuclear translocation in mammalian cells induced by either type I or type II IFN. Expression of individual CHIKV nonstructural proteins (nsPs) showed that nsP2 was a potent inhibitor of IFN-induced JAK-STAT signaling. In addition, mutations in CHIKV-nsP2 (P718S) and Sindbis virus (SINV)-nsP2 (P726S) that render alphavirus replicons noncytopathic significantly reduced JAK-STAT inhibition. This host shutoff-independent inhibition of IFN signaling by CHIKV is likely to have an important role in viral pathogenesis.

scholarly journals Radicicol Inhibits Chikungunya Virus Replication by Targeting Nonstructural Protein-2

2021 ◽  
Author(s):  
Sangwoo Nam ◽  
Yun Ji Ga ◽  
Joo-Youn Lee ◽  
Woo-Yeon Hwang ◽  
Eunhye Jung ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Large Scale ◽  
Chikungunya Virus ◽  
Nonstructural Protein ◽  
Febrile Illness ◽  
Underlying Mechanism ◽  
Chemical Library ◽  
Screening Assay ◽  
Nonstructural Protein 2 ◽  
High Throughput Screening Assay

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes a debilitating febrile illness characterized by persistent muscle and joint pain. The widespread distribution of transmission-competent vectors, Aedes species mosquitoes, indicates the potential risk of large-scale epidemics with high attack rates that can severely impact public health globally. Despite this, currently, there are no antivirals available for the treatment of CHIKV infections. Thus, we aimed to identify potential drug candidates by screening a chemical library using a cytopathic effect-based high-throughput screening assay. As a result, we identified radicicol, a heat-shock protein 90 (Hsp90) inhibitor that effectively suppressed CHIKV replication by blocking the synthesis of both positive- and negative-strand viral RNA as well as expression of viral proteins. Interestingly, selection for viral drug-resistant variants and mutational studies revealed nonstructural protein 2 (nsP2) as a putative molecular target of radicicol. Moreover, co-immunoprecipitation and in silico modeling analyses determined that G641D mutation in the methyltransferase (MT)-like domain of nsP2 is essential for its interaction with cytoplasmic Hsp90β chaperone. Our findings collectively support the potential application of radicicol as an anti-CHIKV agent. The detailed study of the underlying mechanism of action further contributes to understanding virus-host interactions for novel therapeutics against CHIKV infection.

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