Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the newly emergent causative agent of coronavirus disease-19 (COVID-19), has resulted in more than two million deaths worldwide since it was first detected in 2019. There is a critical global need for therapeutic intervention strategies that can be deployed to safely treat COVID-19 disease and reduce associated morbidity and mortality. Increasing evidence shows that both natural and synthetic antimicrobial peptides (AMPs), also referred to as Host Defense Proteins/Peptides (HDPs), can inhibit SARS-CoV-2, paving the way for the potential clinical use of these molecules as therapeutic options. In this manuscript, we describe the potent antiviral activity exerted by brilacidin—a de novo designed synthetic small molecule that captures the biological properties of HDPs—on SARS-CoV-2 in a human lung cell line (Calu-3) and a monkey cell line (Vero). These data suggest that SARS-CoV-2 inhibition in these cell culture models is likely to be a result of the impact of brilacidin on viral entry and its disruption of viral integrity. Brilacidin demonstrated synergistic antiviral activity when combined with remdesivir. Collectively, our data demonstrate that brilacidin exerts potent inhibition of SARS-CoV-2 against different strains of the virus in cell culture.

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family of receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, ErbB2, and ErbB4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2–induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, proinflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production, and disruption of blood-brain barrier integrity in microfluidics-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof of principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

The blood brain barrier (BBB) is a multicellular microenvironment that plays an important role in regulating bidirectional transport to and from the central nervous system (CNS). Infections by many acutely infectious viruses such as alphaviruses and flaviviruses are known to impact the integrity of the endothelial lining of the BBB. Infection by Venezuelan Equine Encephalitis Virus (VEEV) through the aerosol route causes significant damage to the integrity of the BBB, which contributes to long-term neurological sequelae. An effective therapeutic intervention strategy should ideally not only control viral load in the host, but also prevent and/or reverse deleterious events at the BBB. Two dimensional monocultures, including trans-well models that use endothelial cells, do not recapitulate the intricate multicellular environment of the BBB. Complex in vitro organ-on-a-chip models (OOC) provide a great opportunity to introduce human-like experimental models to understand the mechanistic underpinnings of the disease state and evaluate the effectiveness of therapeutic candidates in a highly relevant manner. Here we demonstrate the utility of a neurovascular unit (NVU) in analyzing the dynamics of infection and proinflammatory response following VEEV infection and therapeutic effectiveness of omaveloxolone to preserve BBB integrity and decrease viral and inflammatory load.

Background: Although multiple studies have demonstrated a role for exosomes during virus infections, our understanding of the mechanisms by which exosome exchange regulates immune response during viral infections and affects viral pathogenesis is still in its infancy. In particular, very little is known for cytoplasmic single-stranded RNA viruses such as SARS-CoV-2 and Rift Valley fever virus (RVFV). We have used RVFV infection as a model for cytoplasmic single-stranded RNA viruses to address this gap in knowledge. RVFV is a highly pathogenic agent that causes RVF, a zoonotic disease for which no effective therapeutic or approved human vaccine exist. Results: We show here that exosomes released from cells infected with RVFV (designated as EXi-RVFV) serve a protective role for the host and provide a mechanistic model for these effects. Our results show that treatment of both naïve immune cells (U937 monocytes) and naïve non-immune cells (HSAECs) with EXi-RVFV induces a strong RIG-I dependent activation of IFN-B. We also demonstrate that this strong anti-viral response leads to activation of autophagy in treated cells and correlates with resistance to subsequent viral infection. Since we have shown that viral RNA genome is associated with EXi-RVFV, RIG-I activation might be mediated by the presence of packaged viral RNA sequences. Conclusions: Using RVFV infection as a model for cytoplasmic single-stranded RNA viruses, our results show a novel mechanism of host protection by exosomes released from infected cells (EXi) whereby the EXi activate RIG-I to induce IFN-dependent activation of autophagy in naïve recipient cells including monocytes. Because monocytes serve as reservoirs for RVFV replication, this EXi-RVFV-induced activation of autophagy in monocytes may work to slow down or halt viral dissemination in the infected organism. These findings offer novel mechanistic insights that may aid in future development of effective vaccines or therapeutics, and that may be applicable for a better molecular understanding of how exosome release regulates innate immune response to other cytoplasmic single-stranded RNA viruses.

Venezuelan equine encephalitis virus (VEEV) is a new world alphavirus and a category B select agent. Currently, no FDA-approved vaccines or therapeutics are available to treat VEEV exposure and resultant disease manifestations. The C-terminus of the VEEV non-structural protein 3 (nsP3) facilitates cell-specific and virus-specific host factor binding preferences among alphaviruses, thereby providing targets of interest when designing novel antiviral therapeutics. In this study, we utilized an overexpression construct encoding HA-tagged nsP3 to identify host proteins that interact with VEEV nsP3 by mass spectrometry. Bioinformatic analyses of the putative interactors identified 42 small molecules with the potential to inhibit the host interaction targets, and thus potentially inhibit VEEV. Three inhibitors, tomatidine, citalopram HBr, and Z-VEID-FMK, reduced replication of both the TC-83 strain and the Trinidad donkey (TrD) strain of VEEV by at least 10-fold in astrocytoma, astroglial, and microglial cells. Further, these inhibitors reduced replication of the related New World (NW) alphavirus Eastern equine encephalitis virus (EEEV) in multiple cell types, thus demonstrating broad-spectrum antiviral activity. Time-course assays revealed all three inhibitors reduced both infectious particle production and positive-sense RNA levels post-infection. Further evaluation of the putative host targets for the three inhibitors revealed an interaction of VEEV nsP3 with TFAP2A, but not eIF2S2. Mechanistic studies utilizing siRNA knockdowns demonstrated that eIF2S2, but not TFAP2A, supports both efficient TC-83 replication and genomic RNA synthesis, but not subgenomic RNA translation. Overall, this work reveals the composition of the VEEV nsP3 proteome and the potential to identify host-based, broad spectrum therapeutic approaches to treat new world alphavirus infections.

New World alphaviruses including Venezuelan Equine Encephalitis Virus (VEEV) and Eastern Equine Encephalitis Virus (EEEV) are mosquito-transmitted viruses that cause disease in humans and equines. There are currently no FDA-approved therapeutics or vaccines to treat or prevent exposure-associated encephalitic disease. The ubiquitin proteasome system (UPS)-associated signaling events are known to play an important role in the establishment of a productive infection for several acutely infectious viruses. The critical engagement of the UPS-associated signaling mechanisms by many viruses as host–pathogen interaction hubs led us to hypothesize that small molecule inhibitors that interfere with these signaling pathways will exert broad-spectrum inhibitory activity against alphaviruses. We queried eight inhibitors of the UPS signaling pathway for antiviral outcomes against VEEV. Three of the tested inhibitors, namely NSC697923 (NSC), bardoxolone methyl (BARM) and omaveloxolone (OMA) demonstrated broad-spectrum antiviral activity against VEEV and EEEV. Dose dependency and time of addition studies suggest that BARM and OMA exhibit intracellular and post-entry viral inhibition. Cumulatively, our studies indicate that inhibitors of the UPS-associated signaling pathways exert broad-spectrum antiviral outcomes in the context of VEEV and EEEV infection, supporting their translational application as therapeutic candidates to treat alphavirus infections.