Syazwani Izzati Siswanto, Yap Wei Boon, and Nur Firdaus Isa

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ECTOPIC EXPRESSION OF H5N1 NS1 AND MOLECULAR DOCKING INSIGHTS INTO TRIM25-MEDIATED MODULATION OF HOST ANTIVIRAL SIGNALING

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Abstract 

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The non-structural protein 1 (NS1) of H5N1 influenza A virus is a key immune antagonist that disrupts host antiviral signaling by inhibiting TRIM25-mediated activation of the RIG-I pathway. Despite biochemical and in silico evidence for this interaction, the precise contact residues and dynamic behavior of the NS1–TRIM25 complex remain incompletely defined. Site-specific approaches such as amber suppression technology, which enables incorporation of unnatural amino acids at defined positions to probe protein–protein interfaces, represent a promising future avenue for resolving this interaction at single-residue resolution, but a validated mammalian expression platform for H5N1 NS1 is a necessary first step toward this goal. In this study, we established and characterized a mammalian expression system for wild-type H5N1 NS1 using transient transfection in the bat lung epithelial cell line Tb1Lu. The NS1 gene was cloned into a pCMV6 vector, validated by colony PCR and Sanger sequencing, and expressed in Tb1Lu cells, with anti-FLAG immunoprecipitation and Western blotting confirming a ~26 kDa protein consistent with the predicted molecular weight of NS1. NS1 sequences from diverse H5N1 strains were aligned with Influenza A/Chicken/Malaysia/5858/2004, revealing a conserved RNA binding domain. Molecular docking of NS1 with TRIM25 using HADDOCK predicted a high-affinity interaction interface within this conserved region, generating a testable structural hypothesis for the mechanism of TRIM25 antagonism. Together, these findings establish a reliable NS1 ectopic expression platform and an in silico interaction model that lay the groundwork for future experimental validation including amber suppression-based mapping of the NS1–TRIM25 interface at single- residue resolution to advance understanding of influenza pathogenesis and inform antiviral target discovery.

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