Biology subjects

Almasy, K. M.

Publications and source records attributed to Almasy, K. M..

3 recordsLinked to original sources

bioRxiv2021

Comparative host interactomes of the SARS-CoV-2 nonstructural protein 3 and human coronavirus homologs

Human coronaviruses have become an increasing threat to global health; three highly pathogenic strains have emerged since the early 2000s, including most recently SARS-CoV-2, the cause of COVID-19. A better understanding of the molecular mechanisms of coronavirus pathogenesis is needed, including how these highly virulent strains differ from those that cause milder, common-cold like disease. While significant progress has been made in understanding how SARS-CoV-2 proteins interact with the host cell, non-structural protein 3 (nsp3) has largely been omitted from the analyses. Nsp3 is a viral protease with important roles in viral protein biogenesis, replication complex formation, and modulation of host ubiquitinylation and ISGylation. Herein, we use affinity purification-mass spectrometry to study the host-viral protein-protein interactome of nsp3 from five coronavirus strains: pathogenic strains SARS-CoV-2, SARS-CoV, and MERS-CoV; and endemic common-cold strains hCoV-229E and hCoV-OC43. We divide each nsp3 into three fragments and use tandem mass tag technology to directly compare the interactors across the five strains for each fragment. We find that few interactors are common across all variants for a particular fragment, but we identify shared patterns between select variants, such as ribosomal proteins enriched in the N-terminal fragment (nsp3.1) dataset for SARS-CoV-2 and SARS-CoV. We also identify unique biological processes enriched for individual homologs, for instance nuclear protein important for the middle fragment of hCoV-229E, as well as ribosome biogenesis of the MERS nsp3.2 homolog. Lastly, we further investigate the interaction of the SARS-CoV-2 nsp3 N-terminal fragment with ATF6, a regulator of the unfolded protein response. We show that SARS-CoV-2 nsp3.1 directly binds to ATF6 and can suppress the ATF6 stress response. Characterizing the host interactions of nsp3 widens our understanding of how coronaviruses co-opt cellular pathways and presents new avenues for host-targeted antiviral therapeutics.

biochemistry↗

bioRxiv2020

Comparative multiplexed interactomics of SARS-CoV-2 and homologous coronavirus non-structural proteins identifies unique and shared host-cell dependencies

Human coronaviruses (hCoV) have become a threat to global health and society, as evident from the SARS outbreak in 2002 caused by SARS-CoV-1 and the most recent COVID-19 pandemic caused by SARS-CoV-2. Despite high sequence similarity between SARS-CoV-1 and -2, each strain has distinctive virulence. A better understanding of the basic molecular mechanisms mediating changes in virulence is needed. Here, we profile the virus-host protein-protein interactions of two hCoV non-structural proteins (nsps) that are critical for virus replication. We use tandem mass tag-multiplexed quantitative proteomics to sensitively compare and contrast the interactomes of nsp2 and nsp4 from three betacoronavirus strains: SARS-CoV-1, SARS-CoV-2, and hCoV-OC43 - an endemic strain associated with the common cold. This approach enables the identification of both unique and shared host cell protein binding partners and the ability to further compare the enrichment of common interactions across homologs from related strains. We identify common nsp2 interactors involved in endoplasmic reticulum (ER) Ca2+ signaling and mitochondria biogenesis. We also identifiy nsp4 interactors unique to each strain, such as E3 ubiquitin ligase complexes for SARS-CoV-1 and ER homeostasis factors for SARS-CoV-2. Common nsp4 interactors include N-linked glycosylation machinery, unfolded protein response (UPR) associated proteins, and anti-viral innate immune signaling factors. Both nsp2 and nsp4 interactors are strongly enriched in proteins localized at mitochondrial-associated ER membranes suggesting a new functional role for modulating host processes, such as calcium homeostasis, at these organelle contact sites. Our results shed light on the role these hCoV proteins play in the infection cycle, as well as host factors that may mediate the divergent pathogenesis of OC43 from SARS strains. Our mass spectrometry workflow enables rapid and robust comparisons of multiple bait proteins, which can be applied to additional viral proteins. Furthermore, the identified common interactions may present new targets for exploration by host-directed anti-viral therapeutics.

biochemistry↗

bioRxiv2020

Dengue and Zika virus infection are impaired by small molecule ER proteostasis regulator 147 in an ATF6-independent manner

Flaviviruses, including Dengue and Zika, are widespread human pathogens, however, no broadly active therapeutics exist to fight infection. Here, we establish the recently discovered pharmacologic modulator of ER proteostasis 147 as an effective host-centered antiviral strategy. Compound 147 reduces infection by attenuating viral replication without causing toxicity in host cells. 147 is a preferential activator of the ATF6 pathway of the unfolded protein response, which requires targeting of cysteine residues primarily on protein disulfide isomerases (PDIs). We find that the antiviral activity of 147 is independent of ATF6 induction but does require modification of reactive thiols on protein targets. Targeting PDIs using RNAi and other PDI small molecule inhibitors was unable to recapitulate the antiviral effects, suggesting additional identified protein targets of 147 may mediate the activity. Importantly, 147 can impair infection of multiple strains of Dengue and Zika virus, indicating that it is suitable as a broad-spectrum antiviral agent.

biochemistry↗