Research
The Menachery Lab seeks to define virus/host interactions that dictate disease outcomes by taking advantage of cutting-edge technologies and platforms. Described below, our tools and projects provide a window into our approach and identify critical areas of exploration that we believe have implications for understanding coronavirus (CoV) infection, emergence, and disease. Tools to study CoV infection. Tools are needed to explore CoV infection and pathogenesis including robust reverse genetic systems, relevant in vivo models, and novel molecular approaches. Over the past decade, our group has pioneered novel tools and new approaches to advance our understanding of CoVs. • Reverse Genetics. We have developed a series of tools to manipulate and characterize CoVs. Building on prior systems (Nature Med. 2015, PNAS 2016, JVI 2020), we published the first reverse genetic system for SARS-CoV-2 (Cell Host Microbe 2020, Nat. Protocols 2021). We subsequently used this system to generate reporter viruses and developed rapid neutralization/drug efficacy assays (Nat. Com. 2020, Cell Rep. Med. 2020). We have also advanced attenuated SARS-CoV-2 platforms for use at lower biocontainment including a single-cycle virus and BSL2 virus (Cell 2021, Nat. Com. 2022). Our current work expands to other CoVs by developing new infectious clones, reporter viruses, and attenuated strains to facilitate therapeutic testing. Together, these systems provide tools necessary to explore CoV biology, test vaccines, and evaluate therapeutics. • Animal Models. A key element of understanding CoV pathogenesis is robust, physiologically relevant animal models. For SARS-CoV-2, we were among the first to use the golden Syrian hamster model to define changes in pathogenesis and transmission due to specific mutations. In addition, we developed a mouse-adapted SARS-CoV-2 which induces significant disease and has mutations that correspond to variants of concern (PlosBiology 2021). Importantly, we also employ unique and diverse animal models to probe the role of host morbidities including age, genetic diversity, and immune status on CoV infection. Moving forward, we will leverage these models to define viral and host elements that contribute to infection, disease, and transmission. • RNA Sequencing & Mass Spectrometry. Understanding the mechanisms that facilitate CoV infection requires exploration at the molecular level for both viral RNA and protein. Our group has developed novel approaches to explore both. Working with Andrew Routh, we have developed the Tiled-Clickseq approach for SARS-CoV- 2 that provides complete genome coverage including 5'UTR, at high depth for both short and long read NGS platforms (E-Life 2022). Importantly, the work allows analysis and exploration of the biology of CoV recombination without the bias of two primer based approaches. Similarly, we have developed virion purification and mass spectrometry protocols to explore post-translational modifications on viral proteins and their impact on SARS-CoV-2 infection. Our work shows that virus diversity extends beyond viral sequence alone and may be shaped by the host, tissue, or individual that the CoV infects. Ongoing work seeks to expand to RNA SHAPE, phosphoproteomics, and glycosylation analysis to identify key molecular motifs driving changes in infection, pathogenesis, and transmission. The tools described above represent our most common approaches to study CoV infection and builds on more than a decade of experience in virology, immunology, and systems biology. Importantly, we are always looking for new ways to dissect the host and virus interaction that dictate infection outcomes. This has and will lead us to add to and refine our approaches as we move forward on the following projects: Identify key traits necessary for emergence and fitness of novel CoVs. The outbreaks of SARS-CoV, MERS-CoV, and SARS-CoV-2 underscore the need for surveillance of zoonotic viruses and understanding factors that drive emergence. In these studies, we examine epidemic and zoonotic strains for traits that predict the likelihood of emergence, pathogenic potential, and efficacy of current therapeutics. • CoV Spike. While most spike research focuses on the receptor binding domain, our group is specifically interested in proteolytic cleavage. Examining the highly conserved C-terminal of S1 (CTS1), we believe this region dictates spike processing. Our CTS1 work defined the role of the furin cleavage site (FCS) in SARS- CoV-2 infection (Nature 2021); in addition, we have shown that loop length and glycosylation upstream of the FCS are key for protease utilization and spike cleavage (PNAS 2022). Our ongoing SARS-CoV-2 work has identified additional amino acid changes and post-translational modifications in the CTS1 that impact proteolytic processing in variants of concern (alpha, delta, omicron). For zoonotic CoVs, we have explored how amino acid differences in the CTS1 distinguish them from closely related epidemic strains. While spike proteins of WIV1- and SHC014-CoVs were capable of robust replication in human cells, these chimeric viruses were attenuated in vivo (Nat. Med 2015, PNAS 2016). We have subsequently found the CTS1, not the NTD/RBD portions, is responsible for this attenuation due to reduced airway infection. Ongoing work explores specific amino acid changes that drives this deficit. Similarly, we found that the barrier to MERS-like PDF2180-CoV spike is not receptor binding, but host protease use; addition of trypsin rescued replication of multiple MERS- like zoonotic CoVs (JVI 2020). Together, the work shows that the CTS1 contains motifs that dictate CoV infectivity and emergence. • CoV Nucleocapid. Several hotspots exist for SARS-CoV-2 mutations outside the spike. We have identified positive selection at several locations in the nucleocapsid (N), including at position 203-205 in the SR domain. Two mutations (R203K/G204R & R203M) have been found in dominant variants including alpha, delta, and omicron. Notably, we generated a R203K/G204R mutant in the WA1 backbone finding augmented infection and pathogenesis; we subsequently showed increased N phosphorylation corresponding to increased viral RNA (PlosPathogens 2022). Ongoing work indicates similar mutations at T205 occur not only in SARS-CoV-2 variants, but in the original SARS-CoV and other zoonotic CoVs. These data suggest selection in the N protein play a role in emergence and fitness in a new host. Examine viral RNA elements that impact CoV infection. While amino acid changes are often the research focus, we also know that RNA sequence, structure, and recombination play a major, but poorly understood role in CoV infection. In these studies, we partner with our collaborator Andrew Routh to explore how RNA dynamics influence and drive changes in CoV infection and pathogenesis. • 5' and 3'UTR. A significant advantage of the Tiled-Clickseq approach is the capacity to capture the true sequence of the 5' and 3'UTR of novel coronaviruses. We have recently identified changes in the 5'UTR of several SARS-CoV-2 variants of concern derived from human samples. Using our reverse genetic system, we incorporated the changes and found rapid reversion or attenuation in cell culture models. Ongoing work seeks to further characterize how these changes impact infection. We are also examining how these changes alter the RNA SHAPE of the 5'UTR using DMS-seq. We will also extend these 5'UTR analyses to other emergent human and zoonotic CoVs. • Recombination. RNA recombination is thought to be critical for CoV diversity and emergence; it is also required for efficient CoV replication. In these studies, we explore the underlying mechanisms governing CoV recombination both in vitro and in vivo. Initial results from the Routh lab found that specific U-tracts associated with recombination in SARS-CoV-2. Importantly, we have identified a role for viral endonuclease NSP15 in contributing to recombination rates. This ongoing work seeks to explore hotspots for recombination and genome variability. Develop broad spectrum coronavirus vaccines, antivirals, and therapeutics. Over the past two decades, CoVs have caused three distinct outbreaks in humans. In addition, several animal CoVs have also emerged leading to significant economic losses. This reality underscores the need to prepare for future emergence events by developing vaccines, drugs, and therapeutics that can be rapidly deployed. • Drug Discovery. A long pursued goal in antiviral research has been to have "on the shelf" drugs and therapeutics to respond to an outbreak. In collaboration with Novartis, our Antiviral Drug Discovery (NIAID AViDD-U19) Center seeks to develop and expand upon CoV antivirals. Our current work builds on FDA- approved drugs against the CoV 3CL-protease and polymerase. In addition, we have developed new targets including viral methyltransferase and other highly conserved domains in CoV proteins. Our goal is to develop several broad-based CoV INDs by the end of the 5-year project. Similarly, we have collaborated with groups pursuing monoclonal antibody treatments by testing breadth and efficacy of standard and novel formulations. • Vaccines. The success of COVID-19 mRNA vaccines illustrates the power of vaccination as a tool to mitigate outbreaks. Our group has been able to use our tools to collaborate with industry partners and validate the efficacy of these vaccines. In addition, we have helped generate new platforms for live-attenuated and single- cycle vaccines. While the utility of these are limited in humans, the potential exists for these platforms to be deployed for animal pathogens. We have also begun to evaluate the capacity for pan-CoV vaccines exploring cross-protection between SARS-CoV and SARS-CoV-2. Combined with our work on zoonotic CoVs, this research could form a foundation for protection against future emergent CoVs. Define how age and genetic diversity impact viral infection and disease. Susceptibility to CoV infection is governed by many host co-morbidities including genetics, age, and immune status. In our studies, we take advantage of unique animal models that capture elements of these co-morbidities and evaluate how these host factors change viral infection and disease outcomes. • Aging. SARS-CoV, SARS-CoV-2 and MERS-CoV induce more severe infection and increased mortality in aged humans. Notably, this phenotype is recapitulated in young and aged mouse models of SARS-CoV and SARS-CoV-2. However, the increase in susceptibility is not due to increased viral titer; rather, the host responses vary between young and old driving changes in disease. Our current and ongoing studies seek to identify, confirm, and validate host changes in pathway activation between young and aged animals. We also want to develop treatment approaches to mitigate disease in the aged hosts in response to respiratory virus infection. • Genetic Diversity. Host genetic diversity plays a critical role in the response to CoV infection. Employing the Collaborative Cross (CC), a panel of recombinant inbred mice that captures genetic diversity similar to humans, we observe a wide spectrum of phenotypes . These readouts can be dysregulated from each other, allowing fine mapping to define specific genetic components that drive phenotypic responses. Ongoing work in our lab uses an F2 mapping approach to define quantitative trait loci associated with a low titer QTL for CoVs and genetic correlates of age-dependent susceptibility. Overall, the underlying thread of the Menachery Lab has been using highly successful viruses to examine critical aspects of the immunity. By employing coronaviruses (CoV), we have been able to probe and identify host-virus interactions that dictate disease outcomes. These insights extend into immunology, host genetic variation, and viral processes that are critical to understand and ameliorate human disease.