Paul W. Rothlauf

Arenaviruses are divided into Old World (OW) and New World (NW) groups. OW arenaviruses enter cells through a pH dependent receptor switch from a plasma membrane factor to an endolysosomal receptor for subsequent membrane fusion, whereas clade B NW arenaviruses use transferrin receptor 1 without a secondary receptor. Using a vesicular stomatitis virus (VSV) chimera expressing the glycoprotein complex (GPC) of the clade A NW arenavirus Pichindé virus, we performed a genome wide CRISPR loss of function screen and identified the endolysosomal sialomucin CD164 as an essential host factor. CD164 knockout cells were resistant to VSV chimeras bearing the GPCs of Pichindé, Paraná, and Flexal viruses, and to authentic Pichindé and Paraná virus, with susceptibility restored by complementation. The requirement mapped to the cysteine rich domain of CD164, which bound GP1 in a pH dependent manner through main-chain interactions. These findings define CD164 as an endolysosomal receptor for clade A NW arenaviruses expanding the receptor switching paradigm.

Infection by yellow fever virus (YFV), the prototype Orthoflavivirus, induces a febrile syndrome in humans that can progress to liver failure, haemorrhage and death¹. Despite decades of study, the entry receptors for YFV remain unclear. Here, using a surface protein-targeted CRISPR–Cas9 screen, we identified LRP4, a low-density lipoprotein receptor (LDLR) family member, as a candidate entry receptor for YFV. Genetic ablation of LRP4 impaired YFV infection of cells and, reciprocally, complementation or ectopic expression of LRP4 increased infection. Related viruses in the YFV antigenic complex also showed LRP4-dependent infection. LRP4 promoted YFV entry into cells through LDLR type A (LA) domain binding to domain III of the YFV envelope protein. Soluble LRP4–Fc decoy receptors neutralized YFV infection in cell culture and reduced viral burden in vivo. As we observed residual YFV infection in LRP4-deficient cells, we evaluated whether other LDLR family members promote YFV entry. This approach identified LRP1 and VLDLR as additional receptors for YFV infection in cell culture. LRP1–Fc, LRP4–Fc and VLDLR–Fc decoys protected mice from YFV challenge, and LRP1–Fc decoys inhibited YFV infection and liver pathogenesis in mice engrafted with human hepatocytes. A genetic deficiency of LRP1 in primary human hepatocyte cultures also resulted in reduced YFV infection. Our findings establish a role for multiple LDLR family members in YFV entry, infection and pathogenesis, which has implications for receptor use and countermeasure development for multiple emerging orthoflaviviruses.

The majority of viruses classified as pandemic threats are enveloped viruses which enter the cell through receptor-mediated endocytosis and take advantage of endosomal acidification to activate their fusion machinery. Here we report that the endosomal fusion of low pH-requiring viruses is highly dependent on TRPM7, a widely expressed TRP channel that is located on the plasma membrane and in intracellular vesicles. Using several viral infection systems expressing the envelope glycoproteins of various viruses, we find that loss of TRPM7 protects cells from infection by Lassa, LCMV, Ebola, Influenza, MERS, SARS-CoV-1, and SARS-CoV-2. TRPM7 ion channel activity is intrinsically necessary to acidify virus-laden endosomes but is expendable for several other endosomal acidification pathways. We propose a model wherein TRPM7 ion channel activity provides a countercurrent of cations from endosomal lumen to cytosol necessary to sustain the pumping of protons into these virus-laden endosomes. This study demonstrates the possibility of developing a broad-spectrum, TRPM7-targeting antiviral drug to subvert the endosomal fusion of low pH-dependent enveloped viruses.

The global emergence of many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants jeopardizes the protective antiviral immunity induced after infection or vaccination. To address the public health threat caused by the increasing SARS-CoV-2 genomic diversity, the National Institute of Allergy and Infectious Diseases within the National Institutes of Health established the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme. This effort was designed to provide a real-time risk assessment of SARS-CoV-2 variants that could potentially affect the transmission, virulence, and resistance to infection- and vaccine-induced immunity. The SAVE programme is a critical data-generating component of the US Government SARS-CoV-2 Interagency Group to assess implications of SARS-CoV-2 variants on diagnostics, vaccines and therapeutics, and for communicating public health risk. Here we describe the coordinated approach used to identify and curate data about emerging variants, their impact on immunity and effects on vaccine protection using animal models. We report the development of reagents, methodologies, models and notable findings facilitated by this collaborative approach and identify future challenges. This programme is a template for the response to rapidly evolving pathogens with pandemic potential by monitoring viral evolution in the human population to identify variants that could reduce the effectiveness of countermeasures.

Significance: Vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. Currently available vaccines require cold storage and sophisticated manufacturing capacity, complicating their distribution, especially in less developed countries. We report a protein-based SARS-CoV-2 vaccine that directly and specifically targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (Spike _RBD ) fused to a nanobody that recognizes class II major histocompatibility complex antigens (VHH _MHCII ). Our vaccine elicits robust humoral (high-titer binding and neutralizing antibodies) and cellular immunity against SARS-CoV-2 and its variants in both young and aged mice. VHH _MHCII -Spike _RBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy, desirable attributes for logistical reasons.

The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC₅₀ (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC₅₀ (half-maximal effective concentration) of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV-SARS-CoV-2 chimeric virus, and an EC₅₀ of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East respiratory syndrome coronavirus (MERS-CoV) cell entry with an EC₅₀ of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice, with a half-life of 8.6 h in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment. Significance MM3122 represents an advanced lead candidate for clinical development as a novel antiviral drug for COVID-19. In addition to being novel drugs, these selective TMRSS2 inhibitors can be used as valuable chemical probes to help elucidate mechanisms of viral pathogenesis. Since TMPRSS2 plays a key role as a viral protein processing protease in the pathogenesis of other coronaviruses (SARS-CoV, MERS-CoV) as well as influenza viruses, MM3122 and this class of TMPRSS2 inhibitors hold much promise as new drugs to not only treat SARS-CoV-2 infections but also potentially represent broad-spectrum antivirals.

Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an IFN-stimulated gene screen against vesicular stomatitis virus (VSV)-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of SARS-CoV-2 replication. Internalized 25HC accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion via blockade of cholesterol export. Our results highlight one of the possible antiviral mechanisms of 25HC and provide the molecular basis for its therapeutic development. Significance The novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiological agent of coronavirus disease-2019 (COVID-19), has swept the world in unprecedented speed. In a few months, SARS-CoV-2 has infected millions of people and caused tens of thousands of deaths. There are no Food and Drug Administration-approved antivirals or vaccines yet available and clinical treatments are limited to supportive therapies that help alleviate the symptoms. Thus, there is an urgent need to identify effective antivirals as countermeasures before safe and effective vaccines are developed, tested, and then produced on a large scale. Our approach is to harness the germline-encoded interferon antiviral response to inhibit SARS-CoV-2 replication thereby limiting its pathogenicity.

Although neutralizing antibodies against the SARS-CoV-2 spike (S) protein are a goal of COVID-19 vaccines and have received emergency use authorization as therapeutics, viral escape mutants could compromise their efficacy. To define the immune-selected mutational landscape in S protein, we used a VSV-eGFP-SARS-CoV-2-S chimeric virus and 19 neutralizing monoclonal antibodies (mAbs) against the receptor-binding domain (RBD) to generate 50 different escape mutants. The variants were mapped onto the RBD structure and evaluated for cross-resistance to mAbs and convalescent human sera. Each mAb had a unique resistance profile, although many shared residues within an epitope. Some variants ( e.g ., S477N) were resistant to neutralization by multiple mAbs, whereas others ( e.g ., E484K) escaped neutralization by convalescent sera, suggesting some humans induce a narrow repertoire of neutralizing antibodies. Comparing the antibody-mediated mutational landscape in S with sequence variation in circulating SARS-CoV-2, we define substitutions that may attenuate neutralizing immune responses in some humans.

Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and make a major contribution to the neutralizing antibody response elicited by infection. Here, we describe a deep mutational scanning method to map how all amino-acid mutations in the RBD affect antibody binding, and apply this method to 10 human monoclonal antibodies. The escape mutations cluster on several surfaces of the RBD that broadly correspond to structurally defined antibody epitopes. However, even antibodies targeting the same RBD surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies, and enable us to design escape-resistant antibody cocktails–including cocktails of antibodies that compete for binding to the same surface of the RBD but have different escape mutations. Therefore, complete escape-mutation maps enable rational design of antibody therapeutics and assessment of the antigenic consequences of viral evolution.