Biology subjects

Stewart, C. M.

Publications and source records attributed to Stewart, C. M..

4 recordsLinked to original sources

bioRxiv2022

Deep mutational scans for ACE2 binding, RBD expression, and antibody escape in the SARS-CoV-2 Omicron BA.1 and BA.2 receptor-binding domains

SARS-CoV-2 continues to acquire mutations in the spike receptor-binding domain (RBD) that impact ACE2 receptor binding, folding stability, and antibody recognition. Deep mutational scanning prospectively characterizes the impacts of mutations on these biochemical properties, enabling rapid assessment of new mutations seen during viral surveillance. However, the effects of mutations can change as the virus evolves, requiring updated deep mutational scans. We determined the impacts of all amino acid mutations in the Omicron BA.1 and BA.2 RBDs on ACE2-binding affinity, RBD folding, and escape from binding by the LY-CoV1404 (bebtelovimab) monoclonal antibody. The effects of some mutations in Omicron RBDs differ from those measured in the ancestral Wuhan-Hu-1 background. These epistatic shifts largely resemble those previously seen in the Beta variant due to the convergent epistatically modifying N501Y substitution. However, Omicron variants show additional lineage-specific shifts, including examples of the epistatic phenomenon of entrenchment that causes the Q498R and N501Y substitutions present in Omicron to be more favorable in that background than in earlier viral strains. In contrast, the Omicron substitution Q493R exhibits no sign of entrenchment, with the derived state, R493, being as unfavorable for ACE2 binding in Omicron RBDs as in Wuhan-Hu-1. Likely for this reason, the R493Q reversion has occurred in Omicron sub-variants including BA.4/BA.5 and BA.2.75, where the affinity buffer from R493Q reversion may potentiate concurrent antigenic change. Consistent with prior studies, we find that Omicron RBDs have reduced expression, and identify candidate stabilizing mutations that ameliorate this deficit. Last, our maps highlight a broadening of the sites of escape from LY-CoV1404 antibody binding in BA.1 and BA.2 compared to the ancestral Wuhan-Hu-1 background. These BA.1 and BA.2 deep mutational scanning datasets identify shifts in the RBD mutational landscape and inform ongoing efforts in viral surveillance. Author SummarySARS-CoV-2 evolves in part through mutations in its spike receptor-binding domain. As these mutations accumulate in evolved variants, they shape the future evolutionary potential of the virus through the phenomenon of epistasis. We characterized the functional impacts of mutations in the Omicron BA.1 and BA.2 receptor-binding domains on ACE2 receptor binding, protein folding, and recognition by the clinical LY-CoV1404 antibody. We then compared the measurements to prior data for earlier variants. These comparisons identify patterns of epistasis that may alter future patterns of Omicron evolution, such as turnover in the availability of specific affinity-enhancing mutations and an expansion in the number of paths of antibody escape from a key monoclonal antibody used for therapeutic treatment of COVID-19. This work informs continued efforts in viral surveillance and forecasting.

microbiology↗

bioRxiv2022

Sphingosine kinases promote Ebola virus infection and can be targeted to inhibit filoviruses, coronaviruses, and arenaviruses using late endocytic trafficking to enter cells

Entry of enveloped viruses in host cells requires the fusion of the viral and host cell membranes, a process that is facilitated by viral fusion proteins protruding from the viral envelope. For fusion, viral fusion proteins need to be triggered by host factors and for some viruses, such as Ebola virus (EBOV) and Lassa fever virus, this event occurs inside endosomes and/or lysosomes. Consequently, these late-penetrating viruses must be internalized and delivered to entry-conducive intracellular vesicles. Because endocytosis and vesicular trafficking are tightly regulated cellular processes, late penetrating viruses also depend on specific host factors, such as signaling molecules, for efficient viral delivery to the site of fusion, suggesting that these could be targeted for antiviral therapy. In this study, we investigated a role for sphingosine kinases (SKs) in viral entry and found that chemical inhibition of sphingosine kinase 1 (SK1) and/or SK2 and knockdown of SK1 or SK2, inhibited entry of EBOV into host cells. Mechanistically, inhibition of SK1 and/or SK2 prevented EBOV from reaching late-endosomes and lysosomes that are positive for the EBOV receptor, Niemann Pick C1 (NPC1). Furthermore, we present evidence that suggests the trafficking defect caused by SK1/2 inhibition occurs independently of S1P signaling through cell-surface S1PRs. Lastly, we found that chemical inhibition of SKs prevents entry of other late-penetrating viruses, including arenaviruses and coronaviruses, in addition to inhibiting infection by replication competent EBOV and SARS-CoV-2 in Huh7.5 cells. In sum, our results highlight an important role played by SKs in endocytic trafficking which can be targeted to inhibit entry of late-penetrating viruses. SK inhibitors could serve as a starting point for the development of broad-spectrum antiviral therapeutics.

microbiology↗

bioRxiv2022

Female investment in terminal reproduction or somatic maintenance depends on infection dose

Immune responses are energetically costly to produce and often trade off with investment in reproduction and offspring provisioning. We predicted that hosts can interpret the magnitude of the infection threat from cues like initial bacterial load to adjust reproductive investment into offspring quantity and quality. To test this prediction, we exposed female flour beetles (Tribolium castaneum) to naive or sterile media controls or one of three increasing doses of heat-killed Bacillus thuringiensis (Bt). To estimate offspring quantity, we measured the number of eggs and hatched larvae from each maternal treatment group. To estimate offspring quality, we measured egg protein content, development to pupation, pupal weight, and offspring survival against Bt infection. Compared to naive controls, low and intermediate bacterial doses resulted in lower female fecundity, suggesting a shift to a somatic maintenance strategy. Meanwhile, in the highest-dose group, fecundity was negatively correlated with egg protein content, suggesting a trade-off between offspring quality and quantity that could mask terminal investment based on metrics of quantity alone. Our results underscore the need to account for the magnitude of environmental cue when quantifying plasticity and trade-offs among life history traits, and provide new insight into the trans-generational effects of immune responses.

evolutionary biology↗

bioRxiv2022

Identification of a SARS-CoV-2 host metalloproteinase-dependent entry pathway differentially used by SARS-CoV-2 and variants of concern Alpha, Delta, and Omicron

To infect cells, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) binds to angiotensin converting enzyme 2 (ACE2) via its spike glycoprotein (S), delivering its genome upon S-mediated membrane fusion. SARS-CoV-2 uses two distinct entry pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In investigating serine protease-independent cell-cell fusion, we found that the matrix metalloproteinases (MMPs), MMP2/9, can activate SARS-CoV-2 S fusion activity, but not that of SARS-CoV-1. Importantly, metalloproteinase activation of SARS-CoV-2 S represents a third entry pathway in cells expressing high MMP levels. This route of entry required cleavage at the S1/S2 junction in viral producer cells and differential processing of variants of concern S dictated its usage. In addition, metalloproteinase inhibitors reduced replicative Alpha infection and abrogated syncytia formation. Finally, we found that the Omicron S exhibit reduced metalloproteinase-dependent fusion and viral entry. Taken together, we identified a MMP2/9-dependent mode of activation of SARS-CoV-2 S. As MMP2/9 are released during inflammation and severe COVID-19, they may play important roles in SARS-CoV-2 S-mediated cytopathic effects, tropism, and disease outcome.

microbiology↗