Structural Basis of SARS-CoV-2- and SARS-CoV-Receptor Binding and Small-Molecule Blockers as Potential Therapeutics.

Over the past two decades, deadly coronaviruses, with the most recent being the severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) 2019 pandemic, have majorly challenged public health. The path for virus invasion into humans and other hosts is mediated by host-pathogen interactions, specifically virus-receptor binding. An in-depth understanding of the virus-receptor binding mechanism is a prerequisite for the discovery of vaccines, antibodies, and small-molecule inhibitors that can interrupt this interaction and prevent or cure infection. In this review, we discuss the viral entry mechanism, the known structural aspects of virus-receptor interactions (SARS-CoV-2 S/humanACE2, SARS-CoV S/humanACE2, and MERS-CoV S/humanDPP4), the key protein domains and amino acid residues involved in binding, and the small-molecule inhibitors and other drugs that have (as of June 2020) exhibited therapeutic potential. Specifically, we review the potential clinical utility of two transmembrane serine protease 2 (TMPRSS2)-targeting protease inhibitors, nafamostat mesylate and camostat mesylate, as well as two novel potent fusion inhibitors and the repurposed Ebola drug, remdesivir, which is specific to RNA-dependent RNA polymerase, against human coronaviruses, including SARS-CoV-2.

Structural Basis of SARS-CoV-2 and SARS-CoV Antibody Interactions.

The 2019 coronavirus pandemic remains a major public health concern. Neutralizing antibodies (nAbs) represent a cutting-edge antiviral strategy. We focus here on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-CoV, and discuss current progress in antibody research against rampant SARS-CoV-2 infections. We provide a perspective on the mechanisms of SARS-CoV-2-derived nAbs, comparing these with existing SARS-CoV-derived antibodies. We offer insight into how these antibodies cross-react and cross-neutralize by analyzing available structures of spike (S) glycoprotein-antibody complexes. We also propose ways of adopting antibody-based strategies - such as cocktail antibody therapeutics against SARS-CoV-2 - to overcome the possible resistance of currently identified mutants and mitigate possible antibody-dependent enhancement (ADE) pathologies. This review provides a platform for the progression of antibody and vaccine design against SARS-CoV-2, and possibly against future coronavirus pandemics.

Effect of the COVID-19 Pandemic on Respiratory Diseases and Their Economic Impacts.

COVID-19 is an airborne respiratory disease that mainly affects the lungs. To date, COVID-19 has infected 580 million people with a mortality of approximately 7 million people worldwide. The emergence of COVID-19 has also affected the infectivity, diagnosis, and disease outcomes of existing diseases such as influenza, TB, and asthma in human populations. These are airborne respiratory diseases with symptoms and mode of transmission similar to those of COVID-19. It was speculated that the protracted nature of the COVID-19 pandemic coupled with vaccination could impact other respiratory diseases and mortality. In this study, we analyzed the impact of COVID-19 on flu, tuberculosis (TB), and asthma. Our analyses suggest that COVID-19 has a potential impact on the mortality of flu, TB, and asthma. These impacts vary across before the COVID-19 era, during the peak period of the pandemic, and after vaccinations/preventive measures were implemented, as well as across different regions of the world. Overall, the spread of flu generally reduced during the pandemic, resulting in a reduced expenditure on flu-related hospitalizations, although there were sporadic spikes at setting times. In contrast, TB deaths generally increased perhaps due to the disruption in access to TB services and reduction in resources. Asthma deaths, on the other hand, only marginally varied. Collectively, the emergence of COVID-19 added extra cost to the overall expenditure on some respiratory infectious diseases, while the cost for other infectious diseases was either reduced or somewhat unaffected.

Recognition of Aedes aegypti Mosquito Saliva Protein LTRIN by the Human Receptor LTßR for Controlling the Immune Response.

Salivary proteins from mosquitoes have received significant attention lately due to their potential to develop therapeutic treatments or vaccines for mosquito-borne diseases. Here, we report the characterization of LTRIN (lymphotoxin beta receptor inhibitor), a salivary protein known to enhance the pathogenicity of ZIKV by interrupting the LTßR-initiated NF-κB signaling pathway and, therefore, diminish the immune responses. We demonstrated that the truncated C-terminal LTRIN (ΔLTRIN) is a dimeric protein with a stable alpha helix-dominant secondary structure, which possibly aids in withstanding the temperature fluctuations during blood-feeding events. ΔLTRIN possesses two Ca2+ binding EF-hand domains, with the second EF-hand motif playing a more significant role in interacting with LTßR. Additionally, we mapped the primary binding regions of ΔLTRIN on LTßR using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and identified that 91QEKAHIAEHMDVPIDTSKMSEQELQFHY118 from the N-terminal of ΔLTRIN is the major interacting region. Together, our studies provide insight into the recognition of LTRIN by LTßR. This finding may aid in a future therapeutic and transmission-blocking vaccine development against ZIKV.

Structural and functional characterization of Aedes aegypti pupal cuticle protein that controls dengue virus infection.

The pupal cuticle protein from Aedes aegypti (AaPC) inhibits dengue virus (DENV) infection; however, the underlying mechanism of this inhibition remains unknown. Here, we report that AaPC is an intrinsically disordered protein and interacts with domain I/II of the DENV envelope protein via residues Asp59, Asp61, Glu71, Asp73, Ser75, and Asp80. AaPC can directly bind to and cause the aggregation of DENV, which in turn blocks virus infection during the virus-cell fusion stage. AaPC may also influence viral recognition and attachment by interacting with human immune receptors DC-SIGN and CD4. These findings enhance our understanding of the role of AaPC in mitigating viral infection and suggest that AaPC is a potential target for developing inhibitors or antibodies to control dengue virus infection.

Identification of Aedes aegypti salivary gland proteins interacting with human immune receptor proteins.

Mosquito saliva proteins modulate the human immune and hemostatic systems and control mosquito-borne pathogenic infections. One mechanism through which mosquito proteins may influence host immunity and hemostasis is their interactions with key human receptor proteins that may act as receptors for or coordinate attacks against invading pathogens. Here, using pull-down assays and proteomics-based mass spectrometry, we identified 11 Ae. aegypti salivary gland proteins (SGPs) (e.g., apyrase, Ae. aegypti venom allergen-1 [AaVA-1], neutrophil stimulating protein 1 [NeSt1], and D7 proteins), that interact with one or more of five human receptor proteins (cluster of differentiation 4 [CD4], CD14, CD86, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin [DC-SIGN], and Toll-like receptor 4 [TLR4]). We focused on CD4- and DC-SIGN-interacting proteins and confirmed that CD4 directly interacts with AaVA-1, D7, and NeST1 recombinant proteins and that AaVA-1 showed a moderate interaction with DC-SIGN using ELISA. Bacteria responsive protein 1 (AgBR1), an Ae. aegypti saliva protein reported to enhance ZIKV infection in humans but that was not identified in our pull-down assay moderately interacts with CD4 in the ELISA assay. Functionally, we showed that AaVA-1 and NeST1 proteins promoted activation of CD4+ T cells. We propose the possible impact of these interactions and effects on mosquito-borne viral infections such as dengue, Zika, and chikungunya viruses. Overall, this study provides key insight into the vector-host (protein-protein) interaction network and suggests roles for these interactions in mosquito-borne viral infections.

Structure of Aedes aegypti procarboxypeptidase B1 and its binding with Dengue virus for controlling infection.

Metallocarboxypeptidases play critical roles in the development of mosquitoes and influence pathogen/parasite infection of the mosquito midgut. Here, we report the crystal structure of Aedes aegypti procarboxypeptidase B1 (PCPBAe1), characterized its substrate specificity and mechanism of binding to and inhibiting Dengue virus (DENV). We show that the activated PCPBAe1 (CPBAe1) hydrolyzes both Arg- and Lys-substrates, which is modulated by residues Asp251 and Ser239 Notably, these residues are conserved in CPBs across mosquito species, possibly required for efficient digestion of basic dietary residues that are necessary for mosquito reproduction and development. Importantly, we characterized the interaction between PCPBAe1 and DENV envelope (E) protein, virus-like particles, and infectious virions. We identified residues Asp18A, Glu19A, Glu85, Arg87, and Arg89 of PCPBAe1 are essential for interaction with DENV. PCPBAe1 maps to the dimeric interface of the E protein domains I/II (Lys64-Glu84, Val238-Val252, and Leu278-Leu287). Overall, our studies provide general insights into how the substrate-binding property of mosquito carboxypeptidases could be targeted to potentially control mosquito populations or proposes a mechanism by which PCPBAe1 binds to and inhibits DENV.

Structure of Aedes aegypti carboxypeptidase B1-inhibitor complex uncover the disparity between mosquito and non-mosquito insect carboxypeptidase inhibition mechanism.

Metallocarboxypeptidases (MCPs) in the mosquito midgut play crucial roles in infection, as well as in mosquito dietary digestion, reproduction, and development. MCPs are also part of the digestive system of plant-feeding insects, representing key targets for inhibitor development against mosquitoes/mosquito-borne pathogens or as antifeedant molecules against plant-feeding insects. Notably, some non-mosquito insect B-type MCPs are primarily insensitive to plant protease inhibitors (PPIs) such as the potato carboxypeptidase inhibitor (PCI; MW 4 kDa), an inhibitor explored for cancer treatment and insecticide design. Here, we report the crystal structure of Aedes aegypti carboxypeptidase-B1 (CPBAe1)-PCI complex and compared the binding with that of PCI-insensitive CPBs. We show that PCI accommodation is determined by key differences in the active-site regions of MCPs. In particular, the loop regions α6-α7 (Leu242 -Ser250 ) and ß8-α8 (Pro269 -Pro280 ) of CPBAe1 are replaced by α-helices in PCI-insensitive insect Helicoverpa zea CPBHz. These α-helices protrude into the active-site pocket of CPBHz, restricting PCI insertion and rendering the enzyme insensitive. We further compared our structure with the only other PCI complex available, bovine CPA1-PCI. The potency of PCI against CPBAe1 (Ki  = 14.7 nM) is marginally less than that of bovine CPA1 (Ki  = 5 nM). Structurally, the above loop regions that accommodate PCI binding in CPBAe1 are similar to that of bovine CPA1, although observed changes in proteases residues that interact with PCI could account for the differences in affinity. Our findings suggest that PCI sensitivity is largely dictated by structural interference, which broadens our understanding of carboxypeptidase inhibition as a mosquito population/parasite control strategy.