ß-D-N4-hydroxycytidine (NHC, EIDD-1931) inhibits chikungunya virus replication in mosquito cells and ex vivo Aedes aegypti guts, but not when ingested during blood-feeding.

Chikungunya virus (CHIKV) is a mosquito-borne virus transmitted by Aedes mosquitoes. While there are no antiviral therapies currently available to treat CHIKV infections, several licensed oral drugs have shown significant anti-CHIKV activity in cells and in mouse models. However, the efficacy in mosquitoes has not yet been assessed. Such cross-species antiviral activity could be favorable, since virus inhibition in the mosquito vector might prevent further transmission to vertebrate hosts. Here, we explored the antiviral effect of ß-d-N4-hydroxycytidine (NHC, EIDD-1931), the active metabolite of molnupiravir, on CHIKV replication in Aedes aegypti mosquitoes. Antiviral assays in mosquito cells and in ex vivo cultured mosquito guts showed that NHC had significant antiviral activity against CHIKV. Exposure to a clinically relevant concentration of NHC did not affect Ae. aegypti lifespan when delivered via a bloodmeal, but it slightly reduced the number of eggs developed in the ovaries. When mosquitoes were exposed to a blood meal containing both CHIKV and NHC, the compound did not significantly reduce virus infection and dissemination in the mosquitoes. This was confirmed by modelling and could be explained by pharmacokinetic analysis, which revealed that by 6 h post-blood-feeding, 90% of NHC had been cleared from the mosquito bodies. Our data show that NHC inhibited CHIKV replication in mosquito cells and gut tissue, but not in vivo when mosquitoes were provided with a CHIKV-infectious bloodmeal spiked with NHC. The pipeline presented in this study offers a suitable approach to identify anti-arboviral drugs that may impede replication in mosquitoes.

The impact of arthritogenic viruses in oral tissues.

Arthritis and periodontitis are inflammatory diseases that share several immunopathogenic features. The expansion in the study of virus-induced arthritis has shed light on how this condition could impact other parts of the human body, including the mouth. Viral arthritis is an inflammatory joint disease caused by several viruses, most notably the alphaviruses Chikungunya virus (CHIKV), Sindbis virus (SINV), Ross River virus (RRV), Mayaro virus (MAYV), and O'nyong'nyong virus (ONNV). These viruses can induce an upsurge of matrix metalloproteinases and immune-inflammatory mediators such as Interleukin-6 (IL6), IL-1ß, tumor necrosis factor, chemokine ligand 2, and receptor activator of nuclear factor kappa-B ligand in the joint and serum of infected individuals. This can lead to the influx of inflammatory cells to the joints and associated muscles as well as osteoclast activation and differentiation, culminating in clinical signs of swelling, pain, and bone resorption. Moreover, several data indicate that these viral infections can affect other sites of the body, including the mouth. The human oral cavity is a rich and diverse microbial ecosystem, and viral infection can disrupt the balance of microbial species, causing local dysbiosis. Such events can result in oral mucosal damage and gingival bleeding, which are indicative of periodontitis. Additionally, infection by RRV, CHIKV, SINV, MAYV, or ONNV can trigger the formation of osteoclasts and upregulate pro-osteoclastogenic inflammatory mediators, interfering with osteoclast activation. As a result, these viruses may be linked to systemic conditions, including oral manifestations. Therefore, this review focuses on the involvement of alphavirus infections in joint and oral health, acting as potential agents associated with oral mucosal inflammation and alveolar bone loss. The findings of this review demonstrate how alphavirus infections could be linked to the comorbidity between arthritis and periodontitis and may provide a better understanding of potential therapeutic management for both conditions.

Live-attenuated chikungunya virus vaccine.

Although Chikungunya fever does not a have a high fatality rate (<10%), it has a huge morbidity toll due to lingering chronic arthralgia. The recent FDA approval of Ixchiq, a vaccine designed to prevent infection caused by the chikungunya virus (CHIKV), provides hope that its use can prevent future CHIKV outbreaks. To view this Bench to Bedside, open or download the PDF.

Transcriptome analysis of Huh7 cells upon Chikungunya virus infection and capsid transfection reveals regulation of distinct cellular and metabolic pathways.

Chikungunya virus (CHIKV) causes persistent arthritis and neurological problems imposing a huge burden globally. The present study aims to understand the interaction mechanism of Chikungunya virus and CHIKV-capsid in Huh7 cells. The RNA-sequencing and qRT-PCR method was used for the transcript and gene profiles of CHIKV virus and CHIKV capsid alone. Transcriptional analysis showed capsid induced 1114 and 956 differentially expressed genes (DEGs) to be upregulated and downregulated respectively, while in virus, 933 genes were upregulated and 956 were downregulated. Total 202 DEGs were common in both capsid and virus; and nine were validated using qRT-PCR. Identified DEGs were found to be associated with metabolic pathways such as Diabetes, cardiac disease, and visual impairment. Further, knock-down study on one of the DEGs (MafA) responsible for insulin regulation showed low viral proteins expression suggesting a reduction in virus-infection. Thus, the study provides insight into the interplay of the virus-host factors assisting virus replication.

Identification of 2,4-Diaminoquinazoline Derivative as a Potential Small-Molecule Inhibitor against Chikungunya and Ross River Viruses.

Alphaviruses are serious zoonotic threats responsible for significant morbidity, causing arthritis or encephalitis. So far, no licensed drugs or vaccines are available to combat alphaviral infections. About 300,000 chikungunya virus (CHIKV) infections have been reported in 2023, with more than 300 deaths, including reports of a few cases in the USA as well. The discovery and development of small-molecule drugs have been revolutionized over the last decade. Here, we employed a cell-based screening approach using a series of in-house small-molecule libraries to test for their ability to inhibit CHIKV replication. DCR 137, a quinazoline derivative, was found to be the most potent inhibitor of CHIKV replication in our screening assay. Both, the cytopathic effect, and immunofluorescence of infected cells were reduced in a dose-dependent manner with DCR 137 post-treatment. Most importantly, DCR 137 was more protective than the traditional ribavirin drug and reduced CHIKV plaque-forming units by several log units. CHIKV-E2 protein levels were also reduced in a dose-dependent manner. Further, DCR 137 was probed for its antiviral activity against another alphavirus, the Ross River virus, which revealed effective inhibition of viral replication. These results led to the identification of a potential quinazoline candidate for future optimization that might act as a pan-alphavirus inhibitor.

Mitochondrial protein BNIP3 regulates Chikungunya virus replication in the early stages of infection.

Chikungunya virus (CHIKV) is a human pathogen causing outbreaks of febrile illness for which vaccines and specific treatments remain unavailable. Autophagy-related (ATG) proteins and autophagy receptors are a set of host factors that participate in autophagy, but have also shown to function in other unrelated cellular pathways. Although autophagy is reported to both inhibit and enhance CHIKV replication, the specific role of individual ATG proteins remains largely unknown. Here, a siRNA screen was performed to evaluate the importance of the ATG proteome and autophagy receptors in controlling CHIKV infection. We observed that 7 out of 50 ATG proteins impact the replication of CHIKV. Among those, depletion of the mitochondrial protein and autophagy receptor BCL2 Interacting Protein 3 (BNIP3) increased CHIKV infection. Interestingly, BNIP3 controls CHIKV independently of autophagy and cell death. Detailed analysis of the CHIKV viral cycle revealed that BNIP3 interferes with the early stages of infection. Moreover, the antiviral role of BNIP3 was found conserved across two distinct CHIKV genotypes and the closely related Semliki Forest virus. Altogether, this study describes a novel and previously unknown function of the mitochondrial protein BNIP3 in the control of the early stages of the alphavirus viral cycle.

Chikungunya virus perturbs the Wnt/ß-catenin signaling pathway for efficient viral infection.

IMPORTANCE: Being obligate parasites, viruses use various host cell machineries in effectively replicating their genome, along with virus-encoded enzymes. In order to carry out infection and pathogenesis, viruses are known to manipulate fundamental cellular processes in cells and interfere with host gene expression. Several viruses interact with the cellular proteins involved in the Wnt/ß-catenin pathway; however, reports regarding the involvement of protein components of the Wnt/ß-catenin pathway in Chikungunya virus (CHIKV) infection are scarce. Additionally, there are currently no remedies or vaccines available for CHIKV. This is the first study to report that modulation of the Wnt/ß-catenin pathway is crucial for effective CHIKV infection. These investigations deepen the understanding of the underlying mechanisms of CHIKV infection and offer new avenue for developing effective countermeasures to efficiently manage CHIKV infection.

The 8-bromobaicalein alleviated chikungunya-induced musculoskeletal inflammation and reduced the viral load in healthy adult mice.

Chikungunya virus is a re-emerging arbovirus that has caused epidemic outbreaks in recent decades. Patients in older age groups with high viral load and severe immunologic response during acute infection are likely to develop chronic arthritis and severe joint pain. Currently, no antiviral drug is available. Previous studies suggested that a flavone derivative, 8-bromobaicalein, was a potential dengue and Zika replication inhibitor in a cell-based system targeting flaviviral polymerase. Here we characterized that 8-bromobaicalein inhibited chikungunya virus replication with EC50 of 0.49 ± 0.11 µM in Vero cells. The molecular target predicted at viral nsP1 methyltransferase using molecular binding and fragment molecular orbital calculation. Additionally, oral administration of 250 mg/kg twice daily treatment alleviated chikungunya-induced musculoskeletal inflammation and reduced viral load in healthy adult mice. Pharmacokinetic analysis indicated that the 250 mg/kg administration maintained the compound level above EC99.9 for 12 h. Therefore, 8-bromobaicalein should be a potential candidate for further development as a pan-arboviral drug.

Equine Polyclonal Antibodies Prevent Acute Chikungunya Virus Infection in Mice.

Chikungunya virus (CHIKV) is a mosquito-transmitted pathogen that causes chikungunya disease (CHIK); the disease is characterized by fever, muscle ache, rash, and arthralgia. This arthralgia can be debilitating and long-lasting, seriously impacting quality of life for years. Currently, there is no specific therapy available for CHIKV infection. We have developed a despeciated equine polyclonal antibody (CHIKV-EIG) treatment against CHIKV and evaluated its protective efficacy in mouse models of CHIKV infection. In immunocompromised (IFNAR-/-) mice infected with CHIKV, daily treatment for five consecutive days with CHIKV-EIG administered at 100 mg/kg starting on the day of infection prevented mortality, reduced viremia, and improved clinical condition as measured by body weight loss. These beneficial effects were seen even when treatment was delayed to 1 day after infection. In immunocompetent mice, CHIKV-EIG treatment reduced virus induced arthritis (including footpad swelling), arthralgia-associated cytokines, viremia, and tissue virus loads in a dose-dependent fashion. Collectively, these results suggest that CHIKV-EIG is effective at preventing CHIK and could be a viable candidate for further development as a treatment for human disease.

Dual pattern recognition receptor ligands CL401, CL413, and CL429 as adjuvants for inactivated chikungunya virus.

Chikungunya virus (CHIKV) is responsible for incapacitating joint pains and is a significant health hazard in many countries. Though a definite need for a CHIKV vaccine is felt, long disappearance of CHIKV from circulation in humans has been a concern for vaccine development. Use of two separate pattern recognition receptor ligands has been shown to enhance immune response to the administered antigen. In addition, intradermal delivery of vaccine tends to mimic the natural mode of CHIKV infection. Therefore, in this study, we explored whether intradermal and intramuscular immunization with inactivated CHIKV (I-CHIKV) supplemented with dual pattern-recognition receptor ligands, CL401, CL413, and CL429, is an effective approach to enhancing antibody response to CHIKV. Our in vivo data show that I-CHIKV supplemented with these chimeric PRR ligands induces enhanced neutralizing antibody response after intradermal delivery, but is less efficient after intramuscular immunization. These results suggest that intradermal delivery of I-CHIKV with chimeric adjuvants is a possible way to elicited a better antibody response.

Colocalization of Chikungunya Virus with Its Receptor MXRA8 during Cell Attachment, Internalization, and Membrane Fusion.

Arthritogenic alphaviruses, including chikungunya virus (CHIKV), preferentially target joint tissues and cause chronic rheumatic disease that adversely impacts the quality of life of patients. Viruses enter target cells via interaction with cell surface receptor(s), which determine the viral tissue tropism and pathogenesis. Although MXRA8 is a recently identified receptor for several clinically relevant arthritogenic alphaviruses, its detailed role in the cell entry process has not been fully explored. We found that in addition to its localization on the plasma membrane, MXRA8 is present in acidic organelles, endosomes, and lysosomes. Moreover, MXRA8 is internalized into cells without a requirement for its transmembrane and cytoplasmic domains. Confocal microscopy and live cell imaging revealed that MXRA8 interacts with CHIKV at the cell surface and then enters cells along with CHIKV particles. At the moment of membrane fusion in the endosomes, many viral particles are still colocalized with MXRA8. These findings provide insight as to how MXRA8 functions in alphavirus internalization and suggest possible targets for antiviral development. IMPORTANCE The globally distributed arthritogenic alphaviruses have infected millions of humans and induce rheumatic disease, such as severe polyarthralgia/polyarthritis, for weeks to years. Alphaviruses infect target cells through receptor(s) followed by clathrin-mediated endocytosis. MXRA8 was recently identified as an entry receptor that shapes the tropism and pathogenesis for multiple arthritogenic alphaviruses, including chikungunya virus (CHIKV). Nonetheless, the exact functions of MXRA8 during the process of viral cell entry remain undetermined. Here, we have provided compelling evidence for MXRA8 as a bona fide entry receptor that mediates the uptake of alphavirus virions. Small molecules that disrupt MXRA8-dependent binding of alphaviruses or internalization steps could serve as a platform for unique classes of antiviral drugs.

A chikungunya virus-like particle vaccine induces broadly neutralizing and protective antibodies against alphaviruses in humans.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes epidemics of acute and chronic musculoskeletal disease. Here, we analyzed the human B cell response to a CHIKV-like particle-adjuvanted vaccine (PXVX0317) from samples obtained from a phase 2 clinical trial in humans (NCT03483961). Immunization with PXVX0317 induced high levels of neutralizing antibody in serum against CHIKV and circulating antigen-specific B cells up to 6 months after immunization. Monoclonal antibodies (mAbs) generated from peripheral blood B cells of three PXVX0317-vaccinated individuals on day 57 after immunization potently neutralized CHIKV infection, and a subset of these inhibited multiple related arthritogenic alphaviruses. Epitope mapping and cryo-electron microscopy defined two broadly neutralizing mAbs that uniquely bind to the apex of the B domain of the E2 glycoprotein. These results demonstrate the inhibitory breadth and activity of the human B cell response induced by the PXVX0317 vaccine against CHIKV and potentially other related alphaviruses.

Chikungunya Virus Infection and Gonotrophic Cycle Shape Aedes aegypti Oviposition Behavior and Preferences.

Targeting gravid females through chemical lures is a promising strategy in vector control; however, it requires the understanding of the factors susceptible to alter female oviposition behavior. Here, we evaluated the effect of infection with chikungunya virus (CHIKV) and the number of gonotrophic cycles (GCs) on oviposition activity in A. aegypti. Dual choice oviposition assays were performed, where dodecanoic acid, pentadecanoic acid, n-heneicosane and a Sargasssum fluitans (Børgesen) Børgesen extract were tested in uninfected females and females infected with CHIKV, at the 1st and 2nd GC. Infected females displayed a lower percentage of oviposition and a higher number of eggs laid at the 1st GC. Then, the combined effects of GC and CHIKV were observed on oviposition preferences, with a chemical-dependent effect. For instance, the deterrent effect of n-heneicosane and pentadecanoic acid increased at the 2nd GC in infected females. These results allow for a deeper understanding of the mechanisms involved in oviposition site selection and highlight the need for taking into account physiological stage changes to increase the control programs' efficacy.

Effect of Viral Strain and Host Age on Clinical Disease and Viral Replication in Immunocompetent Mouse Models of Chikungunya Encephalomyelitis.

The alphavirus chikungunya virus (CHIKV) represents a reemerging public health threat as mosquito vectors spread and viruses acquire advantageous mutations. Although primarily arthritogenic in nature, CHIKV can produce neurological disease with long-lasting sequelae that are difficult to study in humans. We therefore evaluated immunocompetent mouse strains/stocks for their susceptibility to intracranial infection with three different CHIKV strains, the East/Central/South African (ECSA) lineage strain SL15649 and Asian lineage strains AF15561 and SM2013. In CD-1 mice, neurovirulence was age- and CHIKV strain-specific, with SM2013 inducing less severe disease than SL15649 and AF15561. In 4-6-week-old C57BL/6J mice, SL15649 induced more severe disease and increased viral brain and spinal cord titers compared to Asian lineage strains, further indicating that neurological disease severity is CHIKV-strain-dependent. Proinflammatory cytokine gene expression and CD4+ T cell infiltration in the brain were also increased with SL15649 infection, suggesting that like other encephalitic alphaviruses and with CHIKV-induced arthritis, the immune response contributes to CHIKV-induced neurological disease. Finally, this study helps overcome a current barrier in the alphavirus field by identifying both 4-6-week-old CD-1 and C57BL/6J mice as immunocompetent, neurodevelopmentally appropriate mouse models that can be used to examine CHIKV neuropathogenesis and immunopathogenesis following direct brain infection.

Innate immune pathway modulator screen identifies STING pathway activation as a strategy to inhibit multiple families of arbo and respiratory viruses.

RNA viruses continue to remain a threat for potential pandemics due to their rapid evolution. Potentiating host antiviral pathways to prevent or limit viral infections is a promising strategy. Thus, by testing a library of innate immune agonists targeting pathogen recognition receptors, we observe that Toll-like receptor 3 (TLR3), stimulator of interferon genes (STING), TLR8, and Dectin-1 ligands inhibit arboviruses, Chikungunya virus (CHIKV), West Nile virus, and Zika virus to varying degrees. STING agonists (cAIMP, diABZI, and 2',3'-cGAMP) and Dectin-1 agonist scleroglucan demonstrate the most potent, broad-spectrum antiviral function. Furthermore, STING agonists inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and enterovirus-D68 (EV-D68) infection in cardiomyocytes. Transcriptome analysis reveals that cAIMP treatment rescue cells from CHIKV-induced dysregulation of cell repair, immune, and metabolic pathways. In addition, cAIMP provides protection against CHIKV in a chronic CHIKV-arthritis mouse model. Our study describes innate immune signaling circuits crucial for RNA virus replication and identifies broad-spectrum antivirals effective against multiple families of pandemic potential RNA viruses.

Dengue and chikungunya virus loads in the mosquito Aedes aegypti are determined by distinct genetic architectures.

Aedes aegypti is the primary vector of the arboviruses dengue (DENV) and chikungunya (CHIKV). These viruses exhibit key differences in their vector interactions, the latter moving more quicky through the mosquito and triggering fewer standard antiviral pathways. As the global footprint of CHIKV continues to expand, we seek to better understand the mosquito's natural response to CHIKV-both to compare it to DENV:vector coevolutionary history and to identify potential targets in the mosquito for genetic modification. We used a modified full-sibling design to estimate the contribution of mosquito genetic variation to viral loads of both DENV and CHIKV. Heritabilities were significant, but higher for DENV (40%) than CHIKV (18%). Interestingly, there was no genetic correlation between DENV and CHIKV loads between siblings. These data suggest Ae. aegypti mosquitoes respond to the two viruses using distinct genetic mechanisms. We also examined genome-wide patterns of gene expression between High and Low CHIKV families representing the phenotypic extremes of viral load. Using RNAseq, we identified only two loci that consistently differentiated High and Low families: a long non-coding RNA that has been identified in mosquito screens post-infection and a distant member of a family of Salivary Gland Specific (SGS) genes. Interestingly, the latter gene is also associated with horizontal gene transfer between mosquitoes and the endosymbiotic bacterium Wolbachia. This work is the first to link the SGS gene to a mosquito phenotype. Understanding the molecular details of how this gene contributes to viral control in mosquitoes may, therefore, also shed light on its role in Wolbachia.

Ethyl palmitate, an anti-chikungunya virus principle from Sauropus androgynus, a medicinal plant used to alleviate fever in ethnomedicine.

ETHNOPHARMACOLOGICAL RELEVANCE: Sauropus androgynus is a medicinal shrub used for the treatment of fever in ethnomedical traditions in various Southeast Asian countries. AIM OF THE STUDY: This study was aimed to identify antiviral principles from S. androgynus against Chikungunya virus (CHIKV), a major mosquito-borne pathogen that re-emerged in the last decade, and to unravel their mechanism of action. MATERIALS AND METHODS: Hydroalcoholic extract of S. androgynus leaves was screened for anti-CHIKV activity using cytopathic effect (CPE) reduction assay. The extract was subjected to activity guided isolation and the resultant pure molecule was characterized by GC-MS, Co-GC and Co-HPTLC. The isolated molecule was further evaluated for its effect by plaque reduction assay, Western blot and immunofluorescence assays. In silico docking with CHIKV envelope proteins and molecular dynamics simulation (MD) analyses were used to elucidate its possible mechanism of action. RESULTS: S. androgynus hydroalcoholic extract showed promising anti-CHIKV activity and its active component, obtained by activity guided isolation, was identified as ethyl palmitate (EP), a fatty acid ester. At 1 µg/mL, EP led to 100% inhibition of CPE and a significant 3 log10 reduction in CHIKV replication in Vero cells at 48 h post-infection. EP was highly potent with an EC50 of 0.0019 µg/mL (0.0068 µM) and a very high selectivity index. EP treatment significantly reduced viral protein expression, and time of addition studies revealed that it acts at the stage of viral entry. A strong binding to the viral envelope protein E1 homotrimer during entry, thus preventing viral fusion, was identified as a possible mechanism by which EP imparts its antiviral effect. CONCLUSIONS: S. androgynus contains EP as a potent antiviral principle against CHIKV. This justifies the use of the plant against febrile infections, possibly caused by viruses, in various ethnomedical systems. Our results also prompt more studies on fatty acids and their derivatives against viral diseases.

The protein disulfide isomerase inhibitor 3-methyltoxoflavin inhibits Chikungunya virus.

Chikungunya virus (CHIKV) is the etiological agent of chikungunya fever, a (re)emerging arbovirus infection, that causes severe and often persistent arthritis, as well as representing a serious health concern worldwide for which no antivirals are currently available. Despite efforts over the last decade to identify and optimize new inhibitors or to reposition existing drugs, no compound has progressed to clinical trials for CHIKV and current prophylaxis is based on vector control, which has shown limited success in containing the virus. Our efforts to rectify this situation were initiated by screening 36 compounds using a replicon system and ultimately identified the natural product derivative 3-methyltoxoflavin with activity against CHIKV using a cell-based assay (EC50 200 nM, SI = 17 in Huh-7 cells). We have additionally screened 3-methyltoxoflavin against a panel of 17 viruses and showed that it only additionally demonstrated inhibition of the yellow fever virus (EC50 370 nM, SI = 3.2 in Huh-7 cells). We have also showed that 3-methyltoxoflavin has excellent in vitro human and mouse microsomal metabolic stability, good solubility and high Caco-2 permeability and it is not likely to be a P-glycoprotein substrate. In summary, we demonstrate that 3-methyltoxoflavin has activity against CHIKV, good in vitro absorption, distribution, metabolism and excretion (ADME) properties as well as good calculated physicochemical properties and may represent a valuable starting point for future optimization to develop inhibitors for this and other related viruses.

The alphavirus nonstructural protein 2 NTPase induces a host translational shut-off through phosphorylation of eEF2 via cAMP-PKA-eEF2K signaling.

Chikungunya virus (CHIKV) is a reemerging alphavirus. Since 2005, it has infected millions of people during outbreaks in Africa, Asia, and South/Central America. CHIKV replication depends on host cell factors at many levels and is expected to have a profound effect on cellular physiology. To obtain more insight into host responses to infection, stable isotope labeling with amino acids in cell culture and liquid chromatography-tandem mass spectrometry were used to assess temporal changes in the cellular phosphoproteome during CHIKV infection. Among the ~3,000 unique phosphorylation sites analyzed, the largest change in phosphorylation status was measured on residue T56 of eukaryotic elongation factor 2 (eEF2), which showed a >50-fold increase at 8 and 12 h p.i. Infection with other alphaviruses (Semliki Forest, Sindbis and Venezuelan equine encephalitis virus (VEEV)) triggered a similarly strong eEF2 phosphorylation. Expression of a truncated form of CHIKV or VEEV nsP2, containing only the N-terminal and NTPase/helicase domains (nsP2-NTD-Hel), sufficed to induce eEF2 phosphorylation, which could be prevented by mutating key residues in the Walker A and B motifs of the NTPase domain. Alphavirus infection or expression of nsP2-NTD-Hel resulted in decreased cellular ATP levels and increased cAMP levels. This did not occur when catalytically inactive NTPase mutants were expressed. The wild-type nsP2-NTD-Hel inhibited cellular translation independent of the C-terminal nsP2 domain, which was previously implicated in directing the virus-induced host shut-off for Old World alphaviruses. We hypothesize that the alphavirus NTPase activates a cellular adenylyl cyclase resulting in increased cAMP levels, thus activating PKA and subsequently eukaryotic elongation factor 2 kinase. This in turn triggers eEF2 phosphorylation and translational inhibition. We conclude that the nsP2-driven increase of cAMP levels contributes to the alphavirus-induced shut-off of cellular protein synthesis that is shared between Old and New World alphaviruses. MS Data are available via ProteomeXchange with identifier PXD009381.

DNA Damage Response Signaling Is Crucial for Effective Chikungunya Virus Replication.

Viruses utilize a plethora of strategies to manipulate the host pathways and hijack host machineries for efficient replication. Several DNA and few RNA viruses are reported to interact with proteins involved in DNA damage responses (DDRs). As the DDR pathways have never been explored in alphaviruses, this investigation intended to understand the importance of the DDR pathways in chikungunya virus (CHIKV) infection in vitro, in vivo, and ex vivo models. The study revealed that CHIKV infection activated the Chk2 and Chk1 proteins associated with the DDR signaling pathways in Vero, RAW264.7, and C2C12 cells. The comet assay revealed an increase in DNA damage by 95%. Inhibition of both ATM-ATR kinases by the ATM/ATR kinase inhibitor (AAKi) showed a drastic reduction in the viral particle formation in vitro. Next, the treatment of CHIKV-infected C57BL/6 mice with this drug reduced the disease score substantially with a 93% decrease in the viral load. The same was observed in human peripheral blood mononuclear cell (hPBMC)-derived monocyte-macrophage populations. Additionally, silencing of Chk2 and Chk1 reduced viral progeny formation by 91.2% and 85.5%, respectively. Moreover, CHIKV-nsP2 was found to interact with Chk2 and Chk1 during CHIKV infection. Furthermore, CHIKV infection induced cell cycle arrest in G1 and G2 phases. In conclusion, this work demonstrated for the first time the mechanistic insights regarding the induction of the DDR pathways by CHIKV that might contribute to the designing of effective therapeutics for the control of this virus infection in the future. IMPORTANCE Being intracellular parasites, viruses require several host cell machineries for effectively replicating their genome, along with virus-encoded enzymes. One of the strategies involves hijacking of the DDR pathways. Several DNA and few RNA viruses interact with the cellular proteins involved in the DDR pathways; however, reports regarding the involvement of Chk2 and Chk1 in alphavirus infection are limited. This is the first study to report that modulation of DDR pathways is crucial for effective CHIKV infection. It also reveals an interaction of CHIKV-nsP2 with two crucial host factors, namely, Chk2 and Chk1, for efficient viral infection. Interestingly, CHIKV infection was found to cause DNA damage and arrest the cell cycle in G1 and G2 phases for efficient viral infection. This information might facilitate the development of effective therapeutics for controlling CHIKV infection in the future.