Monkeypox virus (MPXV) infections in humans have historically been restricted to regions of endemicity in Africa. However, in 2022, an alarming number of MPXV cases were reported globally, with evidence of person-to-person transmission. Because of this, the World Health Organization (WHO) declared the MPXV outbreak a public health emergency of international concern. The supply of MPXV vaccines is limited, and only two antivirals, tecovirimat and brincidofovir, approved by the U.S. Food and Drug Administration (FDA) for the treatment of smallpox, are currently available for the treatment of MPXV infection. Here, we evaluated 19 compounds previously shown to inhibit different RNA viruses for their ability to inhibit orthopoxvirus infections. We first used recombinant vaccinia virus (rVACV) expressing fluorescence (mScarlet or green fluorescent protein [GFP]) and luciferase (Nluc) reporter genes to identify compounds with antiorthopoxvirus activity. Seven compounds from the ReFRAME library (antimycin A, mycophenolic acid, AVN-944, pyrazofurin, mycophenolate mofetil, azaribine, and brequinar) and six compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) showed inhibitory activity against rVACV. Notably, the anti-VACV activity of some of the compounds in the ReFRAME library (antimycin A, mycophenolic acid, AVN-944, mycophenolate mofetil, and brequinar) and all the compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) were confirmed with MPXV, demonstrating their inhibitory activity in vitro against two orthopoxviruses. IMPORTANCE Despite the eradication of smallpox, some orthopoxviruses remain important human pathogens, as exemplified by the recent 2022 monkeypox virus (MPXV) outbreak. Although smallpox vaccines are effective against MPXV, access to those vaccines is limited. In addition, current antiviral treatment against MPXV infections is limited to the use of the FDA-approved drugs tecovirimat and brincidofovir. Thus, there is an urgent need to identify novel antivirals for the treatment of MPXV infection and other potentially zoonotic orthopoxvirus infections. Here, we show that 13 compounds, derived from two different libraries, previously found to inhibit several RNA viruses, also inhibit VACV. Notably, 11 compounds also displayed inhibitory activity against MPXV.
Publications by Year: 2023
2023
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, drastically modifies infected cells to optimize virus replication. One such modification is the activation of the host p38 mitogen-activated protein kinase (MAPK) pathway, which plays a major role in inflammatory cytokine production, a hallmark of severe COVID-19. We previously demonstrated that inhibition of p38/MAPK activity in SARS-CoV-2-infected cells reduced both cytokine production and viral replication. Here, we combined quantitative genetic screening, genomics, proteomics, and phosphoproteomics to better understand mechanisms underlying the dependence of SARS-CoV-2 on the p38 pathway. We found that p38β is a critical host factor for SARS-CoV-2 replication in multiple relevant cell lines and that it functions at a step after viral mRNA expression. We identified putative host and viral p38β substrates in the context of SARS-CoV-2 infection and found that most host substrates have intrinsic antiviral activities. Taken together, this study reveals a unique proviral function for p38β and supports exploring p38β inhibitor development as a strategy toward creating a new class of COVID-19 therapies. IMPORTANCE SARS-CoV-2 is the causative agent of the COVID-19 pandemic that has claimed millions of lives since its emergence in 2019. SARS-CoV-2 infection of human cells requires the activity of several cellular pathways for successful replication. One such pathway, the p38 MAPK pathway, is required for virus replication and disease pathogenesis. Here, we applied systems biology approaches to understand how MAPK pathways benefit SARS-CoV-2 replication to inform the development of novel COVID-19 drug therapies.
Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the therapeutic potential of Mac1 inhibition, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by 10-fold, mutations to aspartic acid (N40D) reduced activity by 100-fold relative to wildtype. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, N40D replicated at >1000-fold lower levels compared to the wildtype virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection and showed no signs of lung pathology. Our data validate the SARS-CoV-2 NSP3 Mac1 domain as a critical viral pathogenesis factor and a promising target to develop antivirals.
INTRODUCTION: The coronavirus disease 2019 (COVID-19) pandemic has demonstrated the need for novel, affordable, and efficient reagents to help reduce viral transmission, especially in high-risk environments including medical treatment facilities, close quarters, and austere settings. We examined transition-metal nanozeolite suspensions and quaternary ammonium compounds as an antiviral surface coating for various textile materials.
METHODS: Zeolites are crystalline porous aluminosilicate materials, with the ability of ion-exchanging different cations. Nanozeolites (30 nm) were synthesized and then ion-exchanged with silver, zinc and copper ions. Benzalkonium nitrate (BZN) was examined as the quaternary ammonium ion (quat). Suspensions of these materials were tested for antiviral activity towards SARS-CoV-2 using plaque assay and immunostaining. Suspensions of the nanozeolite and quat were deposited on polyester and cotton fabrics and the ability of these textiles towards neutralizing SARS-CoV-2 was examined.
RESULTS: We hypothesized that transition metal ion containing zeolites, particularly silver and zinc (AM30) and silver and copper (AV30), would be effective in reducing the infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Additionally, AM30 and AV30 antiviral potency was tested when combined with a quaternary ammonium carrier, BZN. Our results indicate that exposure of SARS-CoV-2 to AM30 and/or AV30 suspensions reduced viral loads with time and exhibited dose-dependence. Antiviral activities of the combination of zeolite and BZN compositions were significantly enhanced. When used in textiles, AM30 and AV30-coated cotton and polyester fabrics alone or in combination with BZN exhibited significant antiviral properties, which were maintained even after various stress tests, including washes, SARS-CoV-2-repeated exposures, or treatments with soil-like materials.
CONCLUSION: This study shows the efficacy of transition metal nanozeolite formulations as novel antiviral agents and establishes that nanozeolite with silver and zinc ions (AM30) and nanozeolite with silver and copper ions (AV30) when combined with benzalkonium nitrate (BZN) quickly and continuously inactivate SARS-CoV-2 in suspension and on fabric materials.
The COVID-19 pandemic has underscored the importance of swift responses and the necessity of dependable technologies for vaccine development. Our team previously developed a fast cloning system for the modified vaccinia virus Ankara (MVA) vaccine platform. In this study, we reported on the construction and preclinical testing of a recombinant MVA vaccine obtained using this system. We obtained recombinant MVA expressing the unmodified full-length SARS-CoV-2 spike (S) protein containing the D614G amino-acid substitution (MVA-Sdg) and a version expressing a modified S protein containing amino-acid substitutions designed to stabilize the protein a in a pre-fusion conformation (MVA-Spf). S protein expressed by MVA-Sdg was found to be expressed and was correctly processed and transported to the cell surface, where it efficiently produced cell-cell fusion. Version Spf, however, was not proteolytically processed, and despite being transported to the plasma membrane, it failed to induce cell-cell fusion. We assessed both vaccine candidates in prime-boost regimens in the susceptible transgenic K18-human angiotensin-converting enzyme 2 (K18-hACE2) in mice and in golden Syrian hamsters. Robust immunity and protection from disease was induced with either vaccine in both animal models. Remarkably, the MVA-Spf vaccine candidate produced higher levels of antibodies, a stronger T cell response, and a higher degree of protection from challenge. In addition, the level of SARS-CoV-2 in the brain of MVA-Spf inoculated mice was decreased to undetectable levels. Those results add to our current experience and range of vaccine vectors and technologies for developing a safe and effective COVID-19 vaccine.
Molecular clock REV-ERBα is central to regulating lung injuries, and decreased REV-ERBα abundance mediates sensitivity to pro-fibrotic insults and exacerbates fibrotic progression. In this study, we determine the role of REV-ERBα in fibrogenesis induced by bleomycin and Influenza A virus (IAV). Bleomycin exposure decreases the abundance of REV-ERBα, and mice dosed with bleomycin at night display exacerbated lung fibrogenesis. Rev-erbα agonist (SR9009) treatment prevents bleomycin induced collagen overexpression in mice. Rev-erbα global heterozygous (Rev-erbα Het) mice infected with IAV showed augmented levels of collagens and lysyl oxidases compared with WT-infected mice. Furthermore, Rev-erbα agonist (GSK4112) prevents collagen and lysyl oxidase overexpression induced by TGFβ in human lung fibroblasts, whereas the Rev-erbα antagonist exacerbates it. Overall, these results indicate that loss of REV-ERBα exacerbates the fibrotic responses by promoting collagen and lysyl oxidase expression, whereas Rev-erbα agonist prevents it. This study provides the potential of Rev-erbα agonists in the treatment of pulmonary fibrosis.
Coronaviruses (CoV) are enveloped, positive-sense, single-stranded RNA viruses responsible for causing seasonal, mild respiratory disease in humans [...].
How SARS-CoV-2 penetrates the airway barrier of mucus and periciliary mucins to infect nasal epithelium remains unclear. Using primary nasal epithelial organoid cultures, we found that the virus attaches to motile cilia via the ACE2 receptor. SARS-CoV-2 traverses the mucus layer, using motile cilia as tracks to access the cell body. Depleting cilia blocks infection for SARS-CoV-2 and other respiratory viruses. SARS-CoV-2 progeny attach to airway microvilli 24 h post-infection and trigger formation of apically extended and highly branched microvilli that organize viral egress from the microvilli back into the mucus layer, supporting a model of virus dispersion throughout airway tissue via mucociliary transport. Phosphoproteomics and kinase inhibition reveal that microvillar remodeling is regulated by p21-activated kinases (PAK). Importantly, Omicron variants bind with higher affinity to motile cilia and show accelerated viral entry. Our work suggests that motile cilia, microvilli, and mucociliary-dependent mucus flow are critical for efficient virus replication in nasal epithelia.