Biology subjects

Tan, Y. B.

Publications and source records attributed to Tan, Y. B..

5 recordsLinked to original sources

bioRxiv2026

Distinct RNA engagements define genome import and replication elongation in alphaviruses

Membrane-associated replication complexes (RCs) are a hallmark of alphaviruses, yet the mechanisms by which viral RNA is delivered into these compartments and coordinated with RNA synthesis remain incompletely understood. Here, we present a cryo-electron microscopy (cryo-EM) structure of the Chikungunya virus (CHIKV) RC core, consisting of nsP1, nsP2, and nsP4, in complex with the replicative RNA substrates, revealing two spatially distinct RNA-binding sites. A single-stranded RNA (ssRNA) engages the nsP2 helicase and extends toward a pore formed at the nsP1-nsP4 interface, whereas a double-stranded RNA (dsRNA) is accommodated within the central catalytic pocket of the nsP4 polymerase in an elongation configuration. Structural analysis suggests a helicase-assisted RNA threading model in which the 5' end of the viral genome is guided toward the spherule lumen through the RC pore. Concurrently, the nsP4 polymerase engages dsRNA in a manner consistent with RNA synthesis. This work provides a structural framework for understanding RNA trafficking and enzymatic coordination in viral replication organelles. Significance StatementLike many positive-sense RNA viruses, alphavirus replication occurs within membrane-bound spherules, but how viral RNA is delivered into these compartments and coupled to RNA synthesis has remained unclear. Here, we show that the CHIKV RC core contains two functionally distinct RNA-engagement modules: an nsP2-associated ssRNA path directed toward the nsP1-nsP4 pore, and an nsP4-bound duplex RNA positioned in an elongation-like polymerase channel. These observations support a model in which alphavirus RCs integrate genome import and RNA synthesis within a single membrane-associated molecular machine. This work provides a structural framework for understanding RNA trafficking, replication-organelle function, and potential antiviral targeting of alphavirus replication.

microbiology↗

bioRxiv2025

Discovery of a potent anti-Zika virus benzamide series targeting the viral protein NS4B

1.Zika virus (ZIKV), a member of the Flaviviridae family, causes significant public health concerns through congenital Zika syndrome and Guillain-Barre syndrome, yet no effective anti-ZIKV drugs or vaccines are available. To address this critical need, we conducted phenotypic, cytopathic effect-based, high-throughput screening followed by medicinal chemistry optimization and discovered novel benzamide anti-ZIKV leads. Current best compounds demonstrated superior potency (EC50 values 40-400 nM, CC50 > 50 {micro}M) compared to NITD-008, the most potent known anti-ZIKV agent. Time-of-addition assays, resistant virus selection studies, and biophysical binding experiments confirmed that NS4B interference constitutes the primary antiviral mechanism. Notably, resistance mutations mapped to the C-terminus of NS4B, distinct from other flavivirus NS4B inhibitors targeting dengue or yellow fever viruses, revealing novel insights into a critical function of the region. These findings establish NS4B as an Achilles heel for flaviviruses and support the development of pan-flavivirus therapeutics targeting this essential viral protein. 2. AUTHOR SUMMARYSince its pandemic spread in 2015-2016, Zika virus infection remains a significant public health threat worldwide. The virus can cause severe brain damage in developing babies and serious neurological complications like Guillain-Barre syndrome in adults. Despite these devastating consequences, we currently lack effective medicines or vaccines to prevent the virus from spreading through communities or from mothers to their unborn children. To address this critical gap, we conducted a large-scale screening of chemical compounds and discovered a promising new class of molecules that can effectively stop Zika virus from replication. Using medicinal chemistry techniques, we were able to make these compounds even more potent against the virus. In follow-up studies, we found that our compounds work by interfering with a specific viral protein called NS4B, which the virus needs to replicate its genome within the cell. Remarkably, other research teams studying related other flaviviruses (e.g., dengue and yellow fever virus) have independently discovered that this same protein is a vulnerable target. Our findings suggest that NS4B represents a universal weakness across the entire flavivirus family, making it an attractive target for developing broad-spectrum antiviral treatments.

microbiology↗

bioRxiv2025

Saddle Curvature Association of nsP1 Facilitates the Replication Complex Assembly of Chikungunya Virus in Cells

The replication of positive-sense RNA viruses, including the pathogenic SARS-CoV-1 and -2, DENV, and ZIKV, often takes place in curved cellular membrane compartments of tens to hundreds of nanometers in diameter within host cells. The non-structural proteins (nsPs) of viruses are found to critically control the formation and maintenance of such unique nanoscale membrane structures. However, the molecular mechanism underlying the association and assembly of nsPs around curved membranes has remained elusive, due to the technical difficulties of imaging the nanoscale interaction in live cells. In this study, we leveraged vertically aligned nanostructures to define membrane curvatures in live cells and investigated the assembly of viral nsPs on these curved membranes. Using Chikungunya virus as a model system, we found that its nsP1 was preferentially bound to and stabilized around positively curved membrane sites and the preference became more apparent as the radius of curvature decreased to 150 nm or smaller. This preferential accumulation was mainly attributed to the hydrophobic residues on recently identified membrane association loops (MA loops) of each nsP1 monomer. Molecular dynamics simulations further confirmed the improved binding kinetics and stability of nsP1 on positively curved membranes, especially when a 12-mer ring was formed by the nsP1 dodecamer. More interestingly, a saddle curvature association was enabled by the 3D coordination of nsP1 monomers along the dodecamer ring, where the MA loops of individual nsP1 monomers sensed a wide range of the positively curved membrane in the x-z plane while the rigid nsP1 dodecamer ring stabilized the negative curvature in the x-y plane. Its strong coupling to the saddle curvature fulfilled the need to constrain the neck of the membrane spherule for viral replication. Strikingly, productive CHIKV replication exhibited strong enrichment on patterned nanostructure array, proving the effectiveness of membrane curvature-guided assembly of the functional replication complex. Our findings revealed that the cell membrane facilitated the local enrichment of viral nsPs in a curvature- dependent manner. It opens up membrane curvature modulation as a new dimension to dissect the formation and regulation of membrane compartments for viral replication.

biophysics↗

bioRxiv2022

Molecular Architecture of the Chikungunya Virus Replication Complex

All positive-strand (+) RNA viruses assemble membrane-associated replication complexes (RCs) for viral RNA synthesis in virus-infected cells. However, how these multi-component RCs assemble and function in synthesizing, processing, and transporting viral RNAs to the cytosol remains poorly defined. Here, we determined both the structure of the core RNA replicase of chikungunya virus (family Togaviridae) at a near-atomic level and the native RC architecture in its cellular context at the subnanometer resolution, using in vitro reconstitution and in situ electron cryotomography, respectively. Within the core RNA replicase (nsP1+2+4), the viral RNA-dependent RNA polymerase nsP4, in complex with nsP2 helicase-protease, was found to co-fold with the membrane-anchored nsP1 RNA-capping dodecameric ring and is located asymmetrically within nsP1 central pore. This complex forms the minimal core RNA replicase, while the addition of a large cytoplasmic ring next to the C-terminus of nsP1 forms the holo-RNA-RC as observed at the neck of spherules formed in virus-infected cells. These results represent a major conceptual advance in elucidating the molecular mechanisms of RNA virus replication and the principles underlying the molecular architecture of RCs, likely to be shared with many pathogenic (+) RNA viruses. At last, our study will direct the needed development of antiviral therapies targeting RCs of pathogenic viruses. SummaryCryoEM structure of the chikungunya virus replication complex reveals a multicomponent RNA synthesis nanomachine embedded in the plasma membrane of the host cell.

microbiology↗

bioRxiv2021

A crystal structure of alphavirus nonstructural protein 4 (nsP4) reveals an intrinsically dynamic RNA-dependent RNA polymerase

Alphaviruses such as Ross River virus (RRV), chikungunya virus, Sindbis virus (SINV), and Venezuelan equine encephalitis virus are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report crystal structures of the RdRp domain of nsP4 from both RRV and SINV determined at resolutions of 2.6 and 1.9 [A]. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses, and picornaviruses. Hydrogendeuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods, showed that in solution, nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were capable to catalyze RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication. Key PointsO_LICrystal structures of alphavirus nsP4 RNA polymerase domain from RRV and SINV. C_LIO_LInsP4 protein is highly dynamic with an intrinsically disordered N-terminal domain. C_LIO_LIOptimized RNA elongation activity assay to facilitate antiviral discovery. C_LI

molecular biology↗