Envelope Protein-Targeting Zika Virus Entry Inhibitors.

Zika virus (ZIKV; family, Flaviviridae), which causes congenital Zika syndrome, Guillain-Barré Syndrome, and other severe diseases, is transmitted mainly by mosquitoes; however, the virus can be transmitted through other routes. Among the three structural and seven nonstructural proteins, the surface envelope (E) protein of ZIKV plays a critical role in viral entry and pathogenesis, making it a key target for the development of effective entry inhibitors. This review article describes the life cycle, genome, and encoded proteins of ZIKV, illustrates the structure and function of the ZIKV E protein, summarizes E protein-targeting entry inhibitors (with a focus on those based on natural products and small molecules), and highlights challenges that may potentially hinder the development of effective inhibitors of ZIKV infection. Overall, the article will provide useful guidance for further development of safe and potent ZIKV entry inhibitors targeting the viral E protein.

Polypyrimidine Tract-Binding Protein Enhances Zika Virus Translation by Binding to the 5' UTR of Internal Ribosomal Entry Site.

Objectives To identify the 5' untranslated region of Zika virus (ZIKV5'UTR) RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site (IRES) located in ZIKV5'UTR and virus production. Methods Interacting proteins in U251 cells were captured using tRSA-tagged ZIKV 5'UTR RNA and tRSA-ZIKV 5'UTR RNA-binding proteins were visualized by SDS-PAGE silver staining. Subsequently, liquid chromatography-tandem mass spectrometry (LC-MS/MS), bioinformatics analysis, and western blot were used to identify the candidate proteins binding to ZIKV5'UTR. Dicistronic expression assay and plaque forming assay were performed to analyze the effect of the binding protein on ZIKV IRES activity and ZIKV production. Results tRSA RNA pull-down assay, LC-MS/MS, and western blot analysis showed that polypyrimidine tract-binding protein (PTB) bound to the ZIKV 5'UTR Furthermore, dual luciferase reporter assay revealed that overexpression of PTB significantly enhanced the IRES activity of ZIKV (t = 10.220, P < 0.001), while PTB knockdown had the opposite effect (t = 4.897, P < 0.01). Additionally, virus plaque forming assay demonstrated that up-regulation of PTB expression significantly enhanced viral titer (t = 6.400, P < 0.01), whereas reducing PTB expression level weakened virus infectivity (t = 5.055, P < 0.01). Conclusion PTB positively interacts with the ZIKV 5'UTR and enhances IRES activity and virus production.

Orthoflaviviral Inhibitors in Clinical Trials, Preclinical In Vivo Efficacy Targeting NS2B-NS3 and Cellular Antiviral Activity via Competitive Protease Inhibition.

Orthoflaviviruses, including zika (ZIKV), West Nile (WNV), and dengue (DENV) virus, induce severely debilitating infections and contribute significantly to the global disease burden, yet no clinically approved antiviral treatments exist. This review offers a comprehensive analysis of small-molecule drug development targeting orthoflaviviral infections, with a focus on NS2B-NS3 inhibition. We systematically examined clinical trials, preclinical efficacy studies, and modes of action for various viral replication inhibitors, emphasizing allosteric and orthosteric drugs inhibiting NS2B-NS3 protease with in vivo efficacy and in vitro-tested competitive NS2B-NS3 inhibitors with cellular efficacy. Our findings revealed that several compounds with in vivo preclinical efficacy failed to show clinical antiviral efficacy. NS3-NS4B inhibitors, such as JNJ-64281802 and EYU688, show promise, recently entering clinical trials, underscoring the importance of developing novel viral replication inhibitors targeting viral machinery. To date, the only NS2B-NS3 inhibitor that has undergone clinical trials is doxycycline, however, its mechanism of action and clinical efficacy as viral growth inhibitor require additional investigation. SYC-1307, an allosteric inhibitor, exhibits high in vivo efficacy, while temoporfin and methylene blue represent promising orthosteric non-competitive inhibitors. Compound 71, a competitive NS2B-NS3 inhibitor, emerges as a leading preclinical candidate due to its high cellular antiviral efficacy, minimal cytotoxicity, and favorable in vitro pharmacokinetic parameters. Challenges remain in developing competitive NS2B-NS3 inhibitors, including appropriate biochemical inhibition assays as well as the selectivity and conformational flexibility of the protease, complicating effective antiviral treatment design.

Geographic Distribution and Temporal Trends of Zika Virus Outbreaks in India (2016-2023): Insights From the Integrated Disease Surveillance Programme Data.

Introduction This study examines the geographic distribution and temporal trends of Zika virus (ZIKV) outbreaks in India from 2016 to 2023 using data from the Integrated Disease Surveillance Programme (IDSP). The burden of ZIKV in India has risen due to its rapid spread and significant health impacts. Existing literature highlights seasonal and geographic patterns but lacks a comprehensive, long-term analysis specific to India. This study addresses this gap by analyzing trends over seven years to inform better public health responses. Methods A secondary data analysis was conducted using publicly available data from the IDSP on reported Zika cases from January 2016 to December 2023. Descriptive statistical methods and geographic information system (GIS) mapping techniques were employed to analyze the geographic distribution and temporal trends of ZIKV outbreaks in India. The data were analyzed and visualized using R software version 4.3.2 (R Foundation for Statistical Computing, Vienna, Austria), with heat maps and choropleth maps to identify hotspots, and line diagrams to identify temporal trends. Results Zika outbreaks predominantly occurred during the post-monsoon season, accounting for 47.62% (n = 10) of the total 21 outbreaks, followed by the monsoon season with 33.33% (n = 7), and summer with 19.05% (n = 4). Two deaths were reported during a significant outbreak in Madhya Pradesh in 2018. Temporal trends indicated notable spikes in cases in 2018 (131 cases) and 2021 (234 cases), with no cases reported in 2019 and 2020. The geographic distribution maps highlighted significant concentrations of ZIKV outbreaks in specific districts within Uttar Pradesh, Madhya Pradesh, and Kerala. Discussion The study identified seasonal patterns, with most cases occurring in the post-monsoon season. The geographic spread of the ZIKV was observed in eight states from 2016 to 2023. GIS identified three hotspots in Uttar Pradesh, Madhya Pradesh, and Kerala. Conclusion The study highlights the need for heightened surveillance and targeted intervention preparedness during high-risk seasons. Enhancing testing facilities and data reporting systems could improve outbreak identification, management, and response.

Raman laser intensity and sample clarification on biochemical monitoring over Zika-VLP upstream stages.

In the current biopharmaceutical scenario, constant bioprocess monitoring is crucial for the quality and integrity of final products. Thus, process analytical techniques, such as those based on Raman spectroscopy, have been used as multiparameter tracking methods in pharma bioprocesses, which can be combined with chemometric tools, like Partial Least Squares (PLS) and Artificial Neural Networks (ANN). In some cases, applying spectra pre-processing techniques before modeling can improve the accuracy of chemometric model fittings to observed values. One of the biological applications of these techniques could have as a target the virus-like particles (VLP), a vaccine production platform for viral diseases. A disease that has drawn attention in recent years is Zika, with large-scale production sometimes challenging without an appropriate monitoring approach. This work aimed to define global models for Zika VLP upstream production monitoring with Raman considering different laser intensities (200 mW and 495 mW), sample clarification (with or without cells), spectra pre-processing approaches, and PLS and ANN modeling techniques. Six experiments were performed in a benchtop bioreactor to collect the Raman spectral and biochemical datasets for modeling calibration. The best models generated presented a mean absolute error and mean relative error respectively of 3.46 × 105 cell/mL and 35 % for viable cell density (Xv); 4.1 % and 5 % for cell viability (CV); 0.245 g/L and 3 % for glucose (Glc); 0.006 g/L and 18 % for lactate (Lac); 0.115 g/L and 26 % for glutamine (Gln); 0.132 g/L and 18 % for glutamate (Glu); 0.0029 g/L and 3 % for ammonium (NH4+); and 0.0103 g/L and 2 % for potassium (K+). Sample without conditioning (with cells) improved the models' adequacy, except for Glutamine. ANN better predicted CV, Gln, Glu, and K+, while Xv, Glc, Lac, and NH4+ presented no statistical difference between the chemometric tools. For most of the assessed experimental parameters, there was no statistical need for spectra pre-filtering, for which the models based on the raw spectra were selected as the best ones. Laser intensity impacts quality model predictions in some parameters, Xv, Gln, and K+ had a better performance with 200 mW of intensity (for PLS, ANN, and ANN, respectively), for CV the 495 mW laser intensity was better (for PLS), and for the other biochemical variables, the use of 200 or 495 mW did not impact model fitting adequacy.

Bis-benzylisoquinoline alkaloids inhibit flavivirus entry and replication by compromising endolysosomal trafficking and autophagy.

Flaviviruses, such as dengue virus (DENV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV), represent a substantial public health challenge as there are currently no approved treatments available. Here, we investigated the antiviral effects of bis-benzylisoquinoline alkaloids (BBAs) on flavivirus infections. We evaluated five specific BBAs-berbamine, tetrandrine, iso-tetrandrine, fangchinoline, and cepharanthine-and found that they effectively inhibited infections by ZIKV, DENV, or JEV by blocking virus entry and genome replication stages in the flavivirus life cycle. Furthermore, we synthesized a fluorophore-conjugated BBA and showed that BBAs targeted endolysosomes, causing lysosomal pH alkalization. Mechanistic studies on inhibiting ZIKV infection by BBAs revealed that these compounds blocked TRPML channels, leading to lysosomal dysfunction and reducing the expression of NCAM1, a key receptor for the entry of ZIKV into cells, thereby decreasing cells susceptibility to ZIKV infection. Additionally, BBAs inhibited the fusion of autophagosomes and lysosomes, significantly reducing viral RNA replication. Collectively, our results suggest that BBAs inhibit flavivirus entry and replication by compromising endolysosomal trafficking and autophagy, respectively, underscoring the potential of BBAs as therapeutic agents against flavivirus infections.

Fetal Zika virus inoculation in macaques revealed control of the fetal viral load during pregnancy.

BACKGROUND: Early pregnancy Zika virus (ZIKV) infection is associated with major brain damage in fetuses, leading to microcephaly in 0.6-5.0% of cases, but the underlying mechanisms remain largely unknown. METHODS: To understand the kinetics of ZIKV infection during fetal development in a nonhuman primate model, four cynomolgus macaque fetuses were exposed in utero through echo-guided intramuscular inoculation with 103 PFU of ZIKV at 70-80 days of gestation, 2 controls were mock inoculated. Clinical, immuno-virological and ultrasound imaging follow-ups of the mother/fetus pairs were performed until autopsy after cesarean section 1 or 2 months after exposure (n = 3 per group). RESULTS: ZIKV was transmitted from the fetus to the mother and then replicate in the peripheral blood of the mother from week 1 to 4 postexposure. Infected fetal brains tended to be smaller than those of controls, but not the femur lengths. High level of viral RNA ws found after the first month in brain tissues and placenta. Thereafter, there was partial control of the virus in the fetus, resulting in a decreased number of infected tissue sections and a decreased viral load. Immune cellular and humoral responses were effectively induced. CONCLUSIONS: ZIKV infection during the second trimester of gestation induces short-term brain injury, and although viral genomes persist in tissues, most of the virus is cleared before delivery.

Zika Virus NS1 Protein Detection Using Gold Nanoparticle-Assisted Dynamic Light Scattering.

The Zika virus (ZIKV) is a global health threat due to its rapid spread and severe health implications, including congenital abnormalities and neurological complications. Differentiating ZIKV from other arboviruses such as dengue virus (DENV) is crucial for effective diagnosis and treatment. This study presents the development of a biosensor for detecting the ZIKV non-structural protein 1 (NS1) using gold nanoparticles (AuNPs) functionalized with monoclonal antibodies employing dynamic light scattering (DLS). The biosensor named ZINS1-mAb-AuNP exhibited specific binding to the ZIKV NS1 protein, demonstrating high colloidal stability indicated by a hydrodynamic diameter (DH) of 140 nm, detectable via DLS. In the absence of the protein, the high ionic strength medium caused particle aggregation. This detection method showed good sensitivity and specificity, with a limit of detection (LOD) of 0.96 µg mL-1, and avoided cross-reactivity with DENV2 NS1 and SARS-CoV-2 spike proteins. The ZINS1-mAb-AuNP biosensor represents a promising tool for the early and accurate detection of ZIKV, facilitating diagnostic and treatment capabilities for arboviral infections.

Prevalence of dengue, Zika, and chikungunya virus infections among mosquitoes in Asia: A systematic review and meta-analysis.

BACKGROUND: Dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) continue to pose significant public health risks. This study aims to assess the prevalence of these arbovirus infections in field-caught mosquitoes across Asia. METHODS: Studies published after the year 2000 on DENV, ZIKV, and/or CHIKV infections in Asian mosquitoes were identified from Embase, Scopus, PubMed, and Ovid. A random-effects model estimated the pooled prevalence, defined as the overall prevalence from included studies, adjusted for variability among the studies. Meta-regression models were used to evaluate the association between predictors and their prevalence. RESULTS: A total of 2529 articles were retrieved; 57 met the inclusion criteria. Pooled prevalence of DENV, ZIKV, and CHIKV infections in Asian mosquitoes were 5.85%, 2.15%, and 1.26%, respectively. Subgroup analysis revealed varying DENV prevalence across regions: East Asia (3.32%), South Asia (5.26%), and Southeast Asia (6.92%). Univariate regression analysis demonstrated significant associations between mosquito capture site and DENV prevalence (P < 0.001), and between study region and ZIKV prevalence (P = 0.005). However, no significant predictors were identified for CHIKV prevalence. CONCLUSION: Our findings provide reference pooled summary estimates of arbovirus infections in mosquitoes, offering crucial insight into the regional disease burden and - guidance in the development and implementation of arbovirus surveillance in mosquitoes.

Integrative analysis of molecular pathways and morphological anomalies associated with congenital Zika syndrome.

Congenital Zika syndrome (CZS) comprises a set of clinical manifestations that can be presented by neonates born to mothers infected by the Zika virus (ZIKV). CZS-associated phenotypes include neurological, skeletal, and systemic alterations and long-term developmental sequelae. One of the most frequently reported clinical conditions is microcephaly characterized by a reduction in head circumference and cognitive complications. Nevertheless, the associations among the diverse signaling pathways underlying CZS phenotypes remain to be elucidated. To shed light on CZS, we have extensively reviewed the morphological anomalies resulting from ZIKV infection, as well as genes and proteins of interest obtained from the published literature. With this list of genes or proteins, we performed computational analyses to explore the cellular processes, molecular mechanisms, and molecular pathways related to ZIKV infection. Therefore, in this review, we comprehensively describe the morphological abnormalities caused by congenital ZIKV infection and, through the analysis noted above, propose common molecular pathways altered by ZIKV that could explain both central nervous system and craniofacial skeletal alterations.

Addition of nucleotide adjuvants enhances the immunogenicity of a recombinant subunit vaccine against the Zika virus in BALB/c mice.

Zika virus (ZIKV) infection remains a global public health problem. After the "Public Health Emergencies of International Concern" declared in February 2016, the incidence of new infections by this pathogen has been decreasing in many areas. However, there is still a likely risk that ZIKV will spread to more countries. To date, there is no vaccine or antiviral drug available to prevent or treat Zika virus infection. In the Zika vaccine development, those based on protein subunits are attractive as a non-replicable platform due to their potentially enhanced safety profile to be used in all populations. However, these vaccines frequently require multiple doses and adjuvants to achieve protective immunity. In this study we show the immunological evaluation of new formulations of the recombinant protein ZEC, which combines regions of domain III of the envelope and the capsid from ZIKV. Two nucleotide-based adjuvants were used to enhance the immunity elicited by the vaccine candidate ZEC. ODN 39M or c-di-AMP was incorporated as immunomodulator into the formulations combined with aluminum hydroxide. Following immunizations in immunocompetent BALB/c mice, the formulations stimulated high IgG antibodies. Although the IgG subtypes suggested a predominantly Th1-biased immune response by the formulation including the ODN 39M, cellular immune responses measured by IFNγ secretion from spleen cells after in vitro stimulations were induced by both immunomodulators. These results demonstrate the capacity of both immunomodulators to enhance the immunogenicity of the recombinant subunit ZEC as a vaccine candidate against ZIKV.

Multi-omics analysis of antiviral interactions of Elizabethkingia anophelis and Zika virus.

The microbial communities residing in the mosquito midgut play a key role in determining the outcome of mosquito pathogen infection. Elizabethkingia anophelis, originally isolated from the midgut of Anopheles gambiae possess a broad-spectrum antiviral phenotype, yet a gap in knowledge regarding the mechanistic basis of its interaction with viruses exists. The current study aims to identify pathways and genetic factors linked to E. anophelis antiviral activity. The understanding of E. anophelis antiviral mechanism could lead to novel transmission barrier tools to prevent arboviral outbreaks. We utilized a non-targeted multi-omics approach, analyzing extracellular lipids, proteins, metabolites of culture supernatants coinfected with ZIKV and E. anophelis. We observed a significant decrease in arginine and phenylalanine levels, metabolites that are essential for viral replication and progression of viral infection. This study provides insights into the molecular basis of E. anophelis antiviral phenotype. The findings lay a foundation for in-depth mechanistic studies.

Zika virus capsid protein closed structure modulates binding to host lipid systems.

Zika virus (ZIKV), a mosquito-borne Flavivirus of international concern, causes congenital microcephaly in newborns and Guillain-Barré syndrome in adults. ZIKV capsid (C) protein, one of three key structural proteins, is essential for viral assembly and encapsidation. In dengue virus, a closely related flavivirus, the homologous C protein interacts with host lipid systems, namely intracellular lipid droplets, for successful viral replication. Here, we investigate ZIKV C interaction with host lipid systems, showing that it binds host lipid droplets but, contrary to expected, in an unspecific manner. Contrasting with other flaviviruses, ZIKV C also does not bind very-low density-lipoproteins. Comparing with other Flavivirus, capsid proteins show that ZIKV C structure is particularly thermostable and seems to be locked into an auto-inhibitory conformation due to a disordered N-terminal, hence blocking specific interactions and supporting the experimental differences observed. Such distinct structural features must be considered when targeting capsid proteins in drug development.

The Role of TIM-1 and CD300a in Zika Virus Infection Investigated with Cell-Based Electrical Impedance.

Orthoflaviviruses cause a major threat to global public health, and no antiviral treatment is available yet. Zika virus (ZIKV) entry, together with many other viruses, is known to be enhanced by phosphatidylserine (PS) receptors such as T-cell immunoglobulin mucin domain protein 1 (TIM-1). In this study, we demonstrate for the first time, using cell-based electrical impedance (CEI) biosensing, that ZIKV entry is also enhanced by expression of CD300a, another PS receptor. Furthermore, inhibiting CD300a in immature monocyte-derived dendritic cells partially but significantly inhibits ZIKV replication. As we have previously demonstrated that CEI is a useful tool to study Orthoflavivirus infection in real time, we now use this technology to determine how these PS receptors influence the kinetics of in vitro ZIKV infection. Results show that ZIKV entry is highly sensitive to minor changes in TIM-1 expression, both after overexpression of TIM-1 in infection-resistant HEK293T cells, as well as after partial knockout of TIM-1 in susceptible A549 cells. These results are confirmed by quantification of viral copy number and viral infectivity, demonstrating that CEI is highly suited to study and compare virus-host interactions. Overall, the results presented here demonstrate the potential of targeting this universal viral entry pathway.

Endogenous ZAP affects Zika virus RNA interactome.

One of the most recent advances in the analysis of viral RNA-cellular protein interactions is the Comprehensive Identification of RNA-binding Proteins by Mass Spectrometry (ChIRP-MS). Here, we used ChIRP-MS in mock-infected and Zika-infected wild-type cells and cells knockout for the zinc finger CCCH-type antiviral protein 1 (ZAP). We characterized 'ZAP-independent' and 'ZAP-dependent' cellular protein interactomes associated with flavivirus RNA and found that ZAP affects cellular proteins associated with Zika virus RNA. The ZAP-dependent interactome identified with ChIRP-MS provides potential ZAP co-factors for antiviral activity against Zika virus and possibly other viruses. Identifying the full spectrum of ZAP co-factors and mechanisms of how they act will be critical to understanding the ZAP antiviral system and may contribute to the development of antivirals.

Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors.

Arboviruses, transmitted by medical arthropods, pose a serious health threat worldwide. During viral infection, Post Translational Modifications (PTMs) are present on both host and viral proteins, regulating multiple processes of the viral lifecycle. In this study, a mammalian E3 ubiquitin ligase WWP2 (WW domain containing E3 ubiquitin ligase 2) is identified, which interacts with the NS1 protein of Zika virus (ZIKV) and mediates K63 and K48 ubiquitination of Lys 265 and Lys 284, respectively. WWP2-mediated NS1 ubiquitination leads to NS1 degradation via the ubiquitin-proteasome pathway, thereby inhibiting ZIKV infection in mammalian hosts. Simultaneously, it is found Su(dx), a protein highly homologous to host WWP2 in mosquitoes, is capable of ubiquitinating NS1 in mosquito cells. Unexpectedly, ubiquitination of NS1 in mosquitoes does not lead to NS1 degradation; instead, it promotes viral infection in mosquitoes. Correspondingly, the NS1 K265R mutant virus is less infectious to mosquitoes than the wild-type (WT) virus. The above results suggest that the ubiquitination of the NS1 protein confers different adaptations of ZIKV to hosts and vectors, and more importantly, this explains why NS1 K265-type strains have become predominantly endemic in nature. This study highlights the potential application in antiviral drug and vaccine development by targeting viral proteins' PTMs.

Low humidity enhances Zika virus infection and dissemination in Aedes aegypti mosquitoes.

As climate change alters Earth's biomes, it is expected the transmission dynamics of mosquito-borne viruses will change. While the effects of temperature changes on mosquito-virus interactions and the spread of the pathogens have been elucidated over the last decade, the impact of relative humidity changes is still relatively unknown. To overcome this knowledge gap, we exposed Aedes aegypti females to various humidity conditions. We measured different components of vectorial capacity such as survival, blood-feeding rates, and changes in infection and dissemination of Zika virus. Survival decreased as the humidity level decreased, while infection rates increased as the humidity level decreased. Alternatively, blood feeding rates and disseminated infection rates peaked at the intermediate 50% relative humidity treatment but were the same in the 30% and 80% relative humidity treatments. These results provide empirical evidence that Ae. aegypti exposure to low humidity can enhance Zika virus infection in the mosquito, which has important implications in predicting how climate change will impact mosquito-borne viruses.IMPORTANCEViruses transmitted by mosquitoes to humans are a major public health burden and are expected to increase under climate change. While we know that temperature is an important driver of variation in arbovirus replication in the mosquito, very little is known about how other relevant climate variables such as humidity will influence the interaction between mosquitoes and the viruses they transmit. Given the variability in humidity across environments, and the predicted changes in humidity under climate change, it is imperative that we also study the impact that it has on mosquito infection and transmission of arboviruses.

Neurovirulence of Usutu virus in human fetal organotypic brain slice cultures partially resembles Zika and West Nile virus.

Usutu (USUV), West Nile (WNV), and Zika virus (ZIKV) are neurotropic arthropod-borne viruses (arboviruses) that cause severe neurological disease in humans. However, USUV-associated neurological disease is rare, suggesting a block in entry to or infection of the brain. We determined the replication, cell tropism and neurovirulence of these arboviruses in human brain tissue using a well-characterized human fetal organotypic brain slice culture model. Furthermore, we assessed the efficacy of interferon-ß and 2'C-methyl-cytidine, a synthetic nucleoside analogue, in restricting viral replication. All three arboviruses replicated within the brain slices, with WNV reaching the highest titers, and all primarily infected neuronal cells. USUV- and WNV-infected cells exhibited a shrunken morphology, not associated with detectable cell death. Pre-treatment with interferon-ß inhibited replication of all arboviruses, while 2'C-methyl-cytidine reduced only USUV and ZIKV titers. Collectively, USUV can infect human brain tissue, showing similarities in tropism and neurovirulence as WNV and ZIKV. These data suggest that a blockade to infection of the human brain may not be the explanation for the low clinical incidence of USUV-associated neurological disease. However, USUV replicated more slowly and to lower titers than WNV, which could help to explain the reduced severity of neurological disease resulting from USUV infection.

Cleavage of SQSTM1/p62 by the Zika virus protease NS2B3 prevents autophagic degradation of viral NS3 and NS5 proteins.

Macroautophagy/autophagy plays a crucial role in inhibiting viral replication and regulating the host's immune response. The autophagy receptor SQSTM1/p62 (sequestosome 1) restricts viral replication by directing specific viral proteins to phagophores for degradation. In this study, we investigate the reciprocal relationship between Zika virus (ZIKV) and selective autophagy mediated by SQSTM1/p62. We show that NS2B3 protease encoded by ZIKV cleaves human SQSTM1/p62 at arginine 265 (R265). This cleavage also occurs with endogenous SQSTM1 in ZIKV-infected cells. Furthermore, overexpression of SQSTM1 inhibits ZIKV replication in A549 cells, while its absence increases viral titer. We have also shown that SQSTM1 impedes ZIKV replication by interacting with NS3 and NS5 and directing them to autophagic degradation, and that NS2B3-mediated cleavage could potentially alter this antiviral function of SQSTM1. Taken together, our study highlights the role of SQSTM1-mediated selective autophagy in the host's antiviral defense against ZIKV and uncovers potential viral evasion strategies that exploit the host's autophagic machinery to ensure successful infection.Abbreviation: Cas9: CRISPR-associated protein 9; Co-IP: co-immunoprecipitation; CRISPR: clustered regularly interspaced short palindromic repeats; DENV: dengue virus; GFP: green fluorescent protein; IFA: indirect immunofluorescence assay; KIR: KEAP1-interacting region; KO: knockout; LIR: MAP1LC3/LC3-interacting region; mAb: monoclonal antibody; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; pAb: polyclonal antibody; PB1: Phox/BEM1 domain; R265A, a SQSTM1 construct with the arginine (R) residue at position 265 replaced with glutamic acid (A); SQSTM1: sequestosome 1; SQSTM1-C, C-terminal fragment of SQSTM1; SQSTM1-N, N-terminal fragment of SQSTM1; SVV: Seneca Valley virus; TAX1BP1: Tax1 binding protein 1; TBD: TRAF6-binding domain; TCID50: 50% tissue culture infective dose; UBA: ubiquitin-associated domain; Ub: ubiquitin; WT: wild type; ZIKV: Zika virus; ZZ: ZZ-type zinc finger domain.

The Flavivirus Non-Structural Protein 5 (NS5): Structure, Functions, and Targeting for Development of Vaccines and Therapeutics.

Flaviviruses, including dengue (DENV), Zika (ZIKV), West Nile (WNV), Japanese encephalitis (JEV), yellow fever (YFV), and tick-borne encephalitis (TBEV) viruses, pose a significant global emerging threat. With their potential to cause widespread outbreaks and severe health complications, the development of effective vaccines and antiviral therapeutics is imperative. The flaviviral non-structural protein 5 (NS5) is a highly conserved and multifunctional protein that is crucial for viral replication, and the NS5 protein of many flaviviruses has been shown to be a potent inhibitor of interferon (IFN) signalling. In this review, we discuss the functions of NS5, diverse NS5-mediated strategies adopted by flaviviruses to evade the host antiviral response, and how NS5 can be a target for the development of vaccines and antiviral therapeutics.