Recent advances in functional studies of coronavirus NSP13 helicase and challenges in inhibitor development

Host protein cleavage by Dengue and Zika virus NS3 proteases: from substrate identification to potential biological consequences

Dengue virus (DENV) and Zika virus (ZIKV) are medically important orthoflaviviruses that utilize the multifunctional NS3 protease, in complex with its cofactor NS2B, for viral replication and host modulation. Here, we summarize current knowledge of host proteins targeted by NS3 proteases and discuss recent advances in proteomic and computational approaches for identifying these substrates. We further discuss evidence showing that NS2B3-mediated cleavage alters innate immune signaling, autophagy, protein translation, and cytoskeletal dynamics. In addition, we compare the host substrate specificities of DENV and ZIKV proteases, emphasizing both shared mechanisms and virus-specific differences that may contribute to their distinct disease manifestations. A deeper understanding of NS3-mediated host protein cleavage will provide critical insights into orthoflavivirus biology and further establish NS3 as a promising target for antiviral intervention.

Structure-inspired design of Nsp8-based protein inhibitors to suppress SARS-CoV-2 replication

SARS-CoV-2 relies on a conserved RNA-dependent RNA polymerase (RdRp) complex composed of nsp12 and its cofactors nsp7 and nsp8 to replicate its RNA genome. Whereas most antiviral strategies target viral enzymes or surface proteins directly, an alternative approach is to disrupt the assembly or function of an essential viral molecular machine using a defective component derived from the pathogen itself. Here, guided by structural analyses of the nsp12–nsp7–nsp8 replication complex, we designed truncated nsp8 proteins that retain the ability to associate with nsp12 but are defective in engaging RNA. Using a purified nsp12–nsp7–nsp8 system capable of RNA primer extension, we show that selected truncated nsp8 variants inhibit polymerase activity when introduced into an otherwise functional complex. These results are consistent with a competitive mechanism in which the defective nsp8 variants associate with nsp12 and interfere with incorporation or function of wild-type nsp8, thereby compromising formation of a productive replication complex. To further explore this strategy, we used structure-guided in silico analysis of the nsp8– nsp12 interface to identify interaction hotspots and screened corresponding single-amino-acid substitutions. Several variants exhibited enhanced inhibitory activity in the reconstituted polymerase assay. Together, these findings establish a proof-of-concept strategy in which a structurally engineered, pathogen-derived protein can act as a dominant-negative inhibitor of an essential viral replication machinery. This approach provides a framework for developing protein- or peptide-based inhibitors that target conserved protein–protein interactions within viral replication complexes.

Targeting Picornavirus Nonstructural 2C Protein: Structure, Function, and Antiviral Discovery

Picornaviruses cause substantial human and veterinary disease, yet direct-acting antivirals remain limited. The nonstructural protein 2C is among the most conserved picornaviral proteins and has ATPase and helicase-like activities. 2C is involved in membrane remodeling, RNA replication, encapsidation, morphogenesis, and multiple virus–host interactions. In this review, we summarize the domain architecture and structural biology of 2C, highlighting the N-terminal membrane-binding region, the ATPase core, the C-terminal helical domain, and accessory motifs that regulate oligomerization and ligand recognition. We further review experimentally supported roles of 2C throughout the viral replication cycle and compare known inhibitors, resistance mutations, and emerging design strategies. Collectively, current evidence supports 2C as a tractable yet still underexploited antiviral target for medically important picornaviruses.

Coronavirus Nsp15 endoribonuclease: linking viral RNA regulation to immune evasion and viral fitness

ABSTRACT Coronavirus nonstructural protein 15 (Nsp15) is a conserved uridine-preferring endoribonuclease (EndoU). Studies using mouse hepatitis virus (MHV), SARS-CoV-2, and other coronaviruses have shown that Nsp15 associates with replication–transcription complexes (RTCs) and contributes to viral immune evasion. Structural studies of alpha- and beta-coronavirus Nsp15 proteins reveal a hexameric enzyme that engages viral RNA substrates and cleaves at unpaired uridines through an RNase A-like, largely metal-independent mechanism stimulated by divalent cations. The Nsp15 hexamer functions as a dynamic, cooperative platform capable of accommodating extended double-stranded and structured RNA substrates. Genetic studies in several coronaviruses indicate that EndoU activity is dispensable for viral RNA synthesis in cell culture, but critical for suppressing host antiviral responses. Loss of EndoU activity promotes accumulation of immunostimulatory RNA species and activation of dsRNA-sensing pathways, including MDA5-dependent interferon signaling, PKR-mediated translational arrest, and the OAS/RNase L system. Mechanistically, Nsp15 is proposed to suppress these responses by selectively processing uridine-rich and structurally accessible regions in viral RNA, including poly(U)-containing negative-strand RNAs, and elements within untranslated regions and transcription regulatory sequences. Beyond catalysis, Nsp15 may contribute to RTC organization and regulate viral RNA recombination or defective viral genome formation, although these roles remain less well-defined and may vary among coronavirus species. Together, these findings support a model in which Nsp15 functions as a regulator of viral RNA composition and immunogenicity rather than solely as a degradative nuclease. This review summarizes recent advances in Nsp15 structure, RNA processing, immune evasion, and antiviral targeting, and highlights key unresolved questions.

Structural Overview of Herpesvirus Tegument Proteins

Structural and biochemical analysis of the IBV nsp15 endoribonuclease reveals the necessity of peripheral site residues for activity

Infectious bronchitis virus (IBV) is a member of the Gammacoronavirus genus responsible for respiratory illness and weakened eggshells in infected chickens, adversely impacting the poultry industry. Escaping innate immune detection during infection is crucial for coronavirus proliferation in the host. The production of double-stranded RNA during coronavirus replication triggers innate immune sensors to create an antiviral state within infected cells. To counter this response, coronaviruses employ nonstructural protein 15 (nsp15) endoribonuclease to degrade double-stranded RNA. Here, we use cryo-electron microscopy and biochemistry to characterize IBV nsp15’s interactions with RNA. While the overall structure and active site of IBV nsp15 strongly resemble previous studies of nsp15 from other coronaviral genera, we note that double-stranded RNA contacts several non-conserved residues peripheral to the enzyme active site. Our data show that these residue positions can have strong impacts on RNA cleavage suggesting unique solutions for RNA engagement across coronavirus species. We also demonstrate a preference for IBV nsp15 to cleave double-stranded RNA over single-stranded RNA and observe nsp15 hexamers with two double-stranded RNAs bound simultaneously. This study reenforces the need to study diverse coronavirus species to identify distinct viral enzyme characteristics. GRAPHICAL ABSTRACT