The identification and elucidation of host pathways for viral infection are critical for understanding the viral infection processes and novel therapeutics development. Here, for the first time, we discover that the human SUMOylation pathway is essential for the IBV viral life cycle. First, IBV viruses were completely inhibited by a novel SUMOylation specific inhibitor, STE025, discovered from our FRET-based high-throughput screening, and the inhibition was very potent, with IC50~ 0.1 µM in an IBV-induced cell death rescue assay; Second, we determined that the IBV M1 protein was SUMOylated, which was mediated by the SUMOylation E2 conjugation enzyme and the E3 ligase enzyme at very high affinities, of 0.20 µM and 0.22 µM, respectively; Third, the mutation of the IBV M1 SUMOylation site, K21R, completely abolished the viral particle generation, strongly suggesting the requirement of SUMOylation for the IBV life cycle. These results suggest that the blockage of the host human SUMOylation pathway is very effective for IBV inhibition. We therefore propose that the host SUMOylation pathway is a critical host factor for the IBV virus life cycle. The identification and inhibition of critical host factor(s) provide a novel strategy for future anti-viral therapeutics development, such as IBV and other viruses.

The influenza A virus (IAV) is able to infect multiple mammalian and avian species, and in humans IAV is responsible for annual seasonal epidemics and occasional pandemics of respiratory disease with significant health and economic impacts. Studying IAV involves laborious secondary methodologies to identify infected cells. Therefore, to circumvent this requirement, in recent years, multiple replication-competent infectious IAV expressing traceable reporter genes have been developed. These IAVs have been very useful for in vitro and/or in vivo studies of viral replication, identification of neutralizing antibodies or antivirals, and in studies to evaluate vaccine efficacy, among others. In this report, we describe, for the first time, the generation and characterization of two replication-competent influenza A/Puerto Rico/8/1934 H1N1 (PR8) viruses where the viral non-structural protein 1 (NS1) was substituted by the monomeric (m)Cherry fluorescent or the NanoLuc luciferase (Nluc) proteins. The ΔNS1 mCherry was able to replicate in cultured cells and in Signal Transducer and Activator of Transcription 1 (STAT1) deficient mice, although at a lower extent than a wild-type (WT) PR8 virus expressing the same mCherry fluorescent protein (WT mCherry). Notably, expression of either reporter gene (mCherry or Nluc) was detected in infected cells by fluorescent microscopy or luciferase plate readers, respectively. ΔNS1 IAV expressing reporter genes provide a novel approach to better understand the biology and pathogenesis of IAV, and represent an excellent tool to develop new therapeutic approaches against IAV infections.

Live visualization of influenza A virus (IAV) structural proteins during viral infection in cells is highly sought objective to study different aspects of the viral replication cycle. To achieve this, we engineered an IAV to express a Tetra Cysteine tag (TC tag) from hemagglutinin (HA), which allows intracellular labeling of the engineered HA protein with biarsenic dyes and subsequent fluorescence detection. Using such constructs, we rescued a recombinant IAV with TC tag inserted in HA, in A/Puerto Rico/8/1934(H1N1) background (HA-TC). This recombinant HA-TC tag reporter IAV was replication-competent; however, as compared to wild type PR8 IAV, it was attenuated in multicycle replication. We confirmed expression of TC tag and biarsenical labeling of HA by immunofluorescence assay in cells infected with an HA-TC tag reporter IAV. Further, we used this reporter virus to visualize HA expression and translocation in IAV infected cells by live confocal imaging. We also tested the utility of the HA-TC IAV in testing chemical inhibitors of the HA translocation. Overall, HA-TC IAV is a versatile tool that will be useful for studying viral life cycle events, virus-host interactions, and anti-viral testing.