Significance: During the ongoing COVID-19 pandemic, protein engineering offers a rapid and powerful approach for building therapeutics to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. We use computational design, affinity maturation, and fusion to dimerization domains to engineer “receptor traps” based on wild-type angiotensin-converting enzyme II (ACE2), the target for viral spike-mediated SARS-CoV-2 entry into cells. The optimized ACE2 receptor traps neutralize authentic SARS-CoV-2 infections as effectively as high-affinity antibodies isolated from convalescent patients and also bind viral spike proteins from other coronaviruses known to cause respiratory diseases. ACE2 receptor traps have large binding interfaces and block the entire receptor binding interface, limiting the potential impact of viral escape mutations.

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The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by the acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) poses a persistent threat to global public health. Although primarily a respiratory illness, extrapulmonary manifestations of COVID-19 include gastrointestinal, cardiovascular, renal and neurological diseases. Recent studies suggest that dysfunction of the endothelium during COVID-19 may exacerbate these deleterious events by inciting inflammatory and microvascular thrombotic processes. Although controversial, there is evidence that SARS-CoV-2 may infect endothelial cells by binding to the angiotensin-converting enzyme 2 (ACE2) cellular receptor using the viral Spike protein. In this review, we explore current insights into the relationship between SARS-CoV-2 infection, endothelial dysfunction due to ACE2 downregulation, and deleterious pulmonary and extra-pulmonary immunothrombotic complications in severe COVID-19. We also discuss preclinical and clinical development of therapeutic agents targeting SARS-CoV-2-mediated endothelial dysfunction. Finally, we present evidence of SARS-CoV-2 replication in primary human lung and cardiac microvascular endothelial cells. Accordingly, in striving to understand the parameters that lead to severe disease in COVID-19 patients, it is important to consider how direct infection of endothelial cells by SARS-CoV-2 may contribute to this process.

Zika virus (ZIKV), a member of theFlaviviridaefamily, has recently emerged as an important human pathogen with increasing economic and health impact worldwide. Because of its teratogenic nature and association with the serious neurological condition Guillain-Barré syndrome, a tremendous amount of effort has focused on understanding ZIKV pathogenesis. To gain further insights into ZIKV interaction with host cells, we investigated how this pathogen affects stress response pathways. While ZIKV infection induces stress signaling that leads to phosphorylation of eIF2α and cellular translational arrest, stress granule (SG) formation was inhibited. Further analysis revealed that the viral proteins NS3 and NS4A are linked to translational repression, whereas expression of the capsid protein, NS3/NS2B-3, and NS4A interfered with SG formation. Some, but not all, flavivirus capsid proteins also blocked SG assembly, indicating differential interactions between flaviviruses and SG biogenesis pathways. Depletion of the SG components G3BP1, TIAR, and Caprin-1, but not TIA-1, reduced ZIKV replication. Both G3BP1 and Caprin-1 formed complexes with capsid, whereas viral genomic RNA stably interacted with G3BP1 during ZIKV infection. Taken together, these results are consistent with a scenario in which ZIKV uses multiple viral components to hijack key SG proteins to benefit viral replication.IMPORTANCEThere is a pressing need to understand ZIKV pathogenesis in order to advance the development of vaccines and therapeutics. The cellular stress response constitutes one of the first lines of defense against viral infection; therefore, understanding how ZIKV evades this antiviral system will provide key insights into ZIKV biology and potentially pathogenesis. Here, we show that ZIKV induces the stress response through activation of the UPR (unfolded protein response) and PKR (protein kinase R), leading to host translational arrest, a process likely mediated by the viral proteins NS3 and NS4A. Despite the activation of translational shutoff, formation of SG is strongly inhibited by the virus. Specifically, ZIKV hijacks the core SG proteins G3BP1, TIAR, and Caprin-1 to facilitate viral replication, resulting in impaired SG assembly. This process is potentially facilitated by the interactions of the viral RNA with G3BP1 as well as the viral capsid protein with G3BP1 and Caprin-1. Interestingly, expression of capsid proteins from several other flaviviruses also inhibited SG formation. Taken together, the present study provides novel insights into how ZIKV modulates cellular stress response pathways during replication.

Argonaute proteins are effectors of RNA interference that function in the context of cytoplasmic ribonucleoprotein complexes to regulate gene expression. Processing bodies (PBs) and stress granules (SGs) are the two main types of ribonucleoprotein complexes with which Argonautes are associated. Targeting of Argonautes to these structures seems to be regulated by different factors. In the present study, we show that heat-shock protein (Hsp) 90 activity is required for efficient targeting of hAgo2 to PBs and SGs. Furthermore, pharmacological inhibition of Hsp90 was associated with reduced microRNA- and short interfering RNA-dependent gene silencing. Neither Dicer nor its cofactor TAR RNA binding protein (TRBP) associates with PBs or SGs, but interestingly, protein activator of the double-stranded RNA-activated protein kinase (PACT), another Dicer cofactor, is recruited to SGs. Formation of PBs and recruitment of hAgo2 to SGs were not dependent upon PACT (or TRBP) expression. Together, our data suggest that Hsp90 is a critical modulator of Argonaute function. Moreover, we propose that Ago2 and PACT form a complex that functions at the level of SGs.