Gili, S. | bioRxiv Science

bioRxiv2022

Structural features stabilized by divalent cation coordination within hepatitis E virus ORF1 are critical for viral replication

Hepatitis E virus (HEV) is an RNA virus responsible for over 20 million infections annually. HEVs open reading frame (ORF)1 polyprotein is essential for genome replication, though it is unknown how the different subdomains function within a structural context. Our data show that ORF1 operates as a multifunctional protein, which is not subject to proteolytic processing. Supporting this model, scanning mutagenesis performed on the putative papain-like cysteine protease (pPCP) domain revealed six cysteines essential for viral replication. Our data are consistent with their role in divalent metal ion coordination, which governs local and interdomain interactions that are critical for the overall structure of ORF1; further, the "pPCP" domain can only rescue viral genome replication in trans when expressed in the context of the full-length ORF1 protein but not as an individual subdomain. Taken together our work provides a comprehensive model of the structure and function of HEV ORF1. Significance StatementThe development of non-teratogenic and potent antiviral therapies against HEV have been hindered by an incomplete understanding of the viral replication cycle. Our work provides a mechanistic insight into the complex replicative cycle of this understudied human pathogen and identifies a novel domain-domain interaction that is vital for replicative fitness.

microbiology↗

bioRxiv2022

Stable Potassium Isotopes (41K/39K) Track Transcellular and Paracellular Potassium Transport in Biological Systems

As the most abundant cation in archaeal, bacterial, and eukaryotic cells, potassium (K+) is an essential element for life. While much is known about the machinery of transcellular and paracellular K transport - channels, pumps, co-transporters, and tight-junction proteins - many quantitative aspects of K homeostasis in biological systems remain poorly constrained. Here we present measurements of the stable isotope ratios of potassium (41K/39K) in three biological systems (algae, fish, and mammals). When considered in the context of our current understanding of potential mechanisms of K isotope fractionation and K+ transport in these biological systems, our results provide evidence that the fractionation of K isotopes depends on transport pathway and transmembrane transport machinery. Specifically, we find that passive transport of K+ down its electrochemical potential through channels and pores in tight-junctions at favors 39K, a result which we attribute to a kinetic isotope effect associated with dehydration and/or size selectivity at the channel/pore entrance. In contrast, we find that transport of K+ against its electrochemical gradient via pumps and co-transporters is associated with less/no isotopic fractionation, a result that we attribute to small equilibrium isotope effects that are expressed in pumps/co-transporters due to their slower turnover rate and the relatively long residence time of K+ in the ion pocket. These results indicate that stable K isotopes may be able to provide quantitative constraints on transporter-specific K+ fluxes (e.g. the fraction of K efflux from a tissue by channels vs. co-transporters) and how these fluxes change under different physiological states. In addition, precise determination of K isotope effects associated with K+ transport through channels, pumps, and co-transporters may provide unique constraints on the mechanisms of K transport that could be tested with steered molecular dynamic simulations.

physiology↗