Gaya K. Amarasinghe - US grants

DESCRIPTION (provided by applicant): Ebola virus is a category A priority pathogen and a causative agent of viral hemorrhagic fever. Enhanced pathogenicity displayed by the Ebola virus is achieved through simultaneous inhibition of host immune responses and enhanced production of viral proteins and RNA. However, the exact nature of the interactions between the Ebola virus and host cells that promote viral infection and propagation are poorly understood. Consequently, no vaccines or antiviral agents against Ebola are currently available. These factors combined, underscore the need for detailed structural and functional characterization of the Ebola viral components. Ebola VP35 protein is an important virulence factor required in several viral lifecycle stages, including viral assembly, genome replication, packaging, viral transcription, and antiviral activity toward the host innate immune system. Overall goal of the research program is to explore host-viral interactions that lead to immune evasion. The proposed study, using recombinantly expressed non-infectious VP35 protein, will examine the structure of VP35 and characterize its antiviral activity using biochemical methods. These studies will significantly improve our understanding of how VP35 functions to enhance viral pathogenesis and facilitate the design of novel diagnostic and therapeutic strategies to disrupt critical host-pathogen interactions. PUBLIC HEALTH RELEVANCE: Growing concerns of rare but increasing Ebola outbreaks among human populations, coupled with a rising potential of misuse in the form of bioterrorism, underscore the importance of developing a greater understanding of viral components that make Ebola virus a significant threat to global human health. Ebola VP35 is responsible for immune suppression and it can enhance viral replication. Work described in this proposal will characterize the structure and mechanistic basis for innate immune antagonism by Ebola viral VP35 protein, which will provide potential targets to therapeutic interventions and design of cell biological reagents.

? DESCRIPTION (provided by applicant): The filoviruses, Ebola and Marburg viruses (EBOV and MARV), are emerging, negative-strand RNA viruses associated with outbreaks of severe viral hemorrhagic fever. The virulence and emerging nature of these zoonotic pathogens makes them a significant threat to human health, potential agents of bioterrorism, and NIAID category A priority pathogens. Currently, no approved anti-filovirus therapeutics are available. Among the proteins encoded by EBOV, VP35 is of particular interest for antiviral development because it makes a critical contribution to pathogenesis through its role in viral replication and host immune suppression. The Basler lab first identified the VP35 protein as an EBOV-encoded inhibitor of the RIG-I signaling pathway, which normally functions to trigger IFN?/? production in response to RNAs produced during viral infection. VP35 also functions as a co-factor in the filoviral replication complex. Important insight into the molecular basis by which VP35 IFN inhibitory domain (IID) participates in the viral RNA polymerase complex and inhibits the RIG-I pathway have recently been provided by structural studies from the Amarasinghe laboratory. Our recent collaborative studies have identified a novel interaction between Ebola VP35 and NP proteins that will be exploited for high-throughput screen (HTS) development. Since we are targeting a protein-protein interface, we will use fragment screens to validate the interface. Studies here are aimed to provide highly sensitive assays that can be converted to HTS assays in order to identify replication inhibitors.

Abstract: Project Overview for Structural and Functional Studies of Ebola Virus RNA synthesis Ebolaviruses (EBOVs) are non-segmented negative-strand RNA viruses (NNSVs) and Category A Priority biodefense pathogens that cause frequent lethal hemorrhagic fever, including the devastating and ongoing outbreak in West Africa. Approved anti-EBOV therapeutics are lacking. EBOV replicates with high efficiency in vivo while effectively evading host innate antiviral immunity. The unrestrained replication and associated inflammation leads to death. A complete understanding of EBOV pathogenesis therefore requires understanding how the viral RNA dependent RNA polymerase (RDRP) complex interacts with host factors. The overarching goal of this program project proposal is to understand the mechanisms of RDRP function by defining the interactions among its components and with the host proteome and by defining signals that regulate its function. Toward this goal Project 1 will identify cis- and trans-acting factors that impact EBOV RDRP activity. Project 2 will define a structural basis for the EBOV RDRP complex, identify regulatory mechanisms, and determine species and strain specificities, and Project 3 will use genome-wide RNAi and proteomic strategies to identify host factors that modulate RDRP function. Project 1 provides functional context into which a subset of Project 3 ?hits? can be interpreted, while Project 3 will likely identify host factors relevant to the findings of Project 1. Project 2 will provide a structural framework that will be used to integrate findings from both Projects 1 and 3. Together, these studies will provide mechanistic insights into how protein-protein and protein-vRNA interactions control EBOV RDRP function. Research projects are supported by an Administrative Core, a Protein Production and Protein Interaction Core, and critically, a Biosafety Level 4 (BSL4) Core. Core B will generate unique reagents, including monoclonal and synthetic antibodies. Core C (BSL4) will provide unique capabilities to obtain materials from and perform tests using wild type and mutant EBOVs as well as pathogenesis evaluation. The work will be performed by highly productive and collaborative investigators with expertise in every aspect of the proposed studies, including biochemistry, EBOV pathogenesis, high throughput screening and data analysis, immunology, proteomics, structural biology, and virology. These studies will provide unprecedented insights into the components of the EBOV RDRP and in its interaction with the host as well as species and strain differences that affect the RDRP complex. We also expect to identify specific viral and host factor interactions as novel therapeutic targets.

Ebola viruses are important emerging and biodefense pathogens for which there are no approved efficacious therapeutics. The overarching goal of the center will be to study Ebola viral RNA-dependent RNA polymerase complex and its interaction with proteins and RNA using structural and functional studies and to identify novel host factors that are critical for viral replication. The characterization of the viral RNA synthesis machinery by this interdisciplinary project will provide insight in to Ebola virus biology and identify new therapeutic targets. This is a multi-investigator collaborative research center with research conducted at several sites. The Administrative Core will coordinate advances in the research among the scientific components (Projects 1, 2, and 3; Core B and C). To ensure that the progress of this integrated research program will be maximized, the Administrative Core will be responsible for the following roles: Coordinate: weekly (at a minimum) teleconferences between the PIs of the research projects and cores; monthly (at a minimum) scheduled web- based interlab meetings to exchange and critique data; and annual Meeting among members of all participating groups, to be held at one of the participating sites. These meetings will facilitate reagent and scientific exchanges between the different laboratories. The Administrative Core will also coordinate annual meetings between the investigators and the External Advisors to assess scientific progress and to receive critical evaluations on the performance and future directions of the P01 project. In addition, the Administration will coordinate budget usage and establish a ?Core Usage Committee? to ensure that the research projects are well supported by the scientific cores. It will assist the investigators with preparation and submission of annual progress reports to the NIH; maintain a web site for data dissemination in connection with this project. The Administrative Core will provide support for efforts to recruit and educate research fellows, graduate and medical students in the field of virus-host interactions.

The recent Ebola virus (EBOV) outbreak in Western Africa, which introduced the virus to non-African nations including the United States, highlights the imminent threat to global health posed by filoviruses and the urgent need for basic and translational efforts. A critical step in the viral lifecycle of filoviruses is the replication of the non-segmented, negative strand RNA genome by the viral RNA dependent RNA polymerase (RDRP) complex. The EBOV RDRP complex is composed of the large protein (L), nucleoprotein (NP), viral protein 35 (VP35) and viral protein 30 (VP30) and is essential for transcription of viral mRNA and replication of antigenomic and genomic RNA. During RNA synthesis, components of the RDRP complex undergo multiple structural rearrangements to make the NP-associated RNA template accessible to the L polymerase while protecting the NP-RNA template from cellular nucleases. Despite the importance of the EBOV RDRP complex to pathogenesis and the potential as therapeutic targets, insights into RDRP complex assembly and its regulatory interactions with host or viral proteins are lacking. We will address these longstanding mechanistic questions in this PPG. Project 2 will use biochemical and hybrid structural methods that will combine results from NMR, X-ray crystallography, small angle X-ray scattering (SAXS) and cryo-electron microscopy (cryoEM) in order to define the molecular mechanism of the EBOV RDRP complex assembly and function, and to define its regulatory mechanisms and structural dynamics. Our strong preliminary results support a working model where conformational remodeling of the RDRP complex is controlled by cis- and trans-acting factors. We have assembled a highly productive and collaborative team of investigators with complementary expertise to perform the following Aims: 1. Define the molecular mechanism by which EBOV VP35 facilitates delivery of NP in the RNA-free form. 2. Elucidate the internal dynamics of the NP protein and define the role of different NP conformations in viral RNA synthesis. 3. Determine the molecular architecture of the EBOV RDRP complex by hybrid structural methods. 4. Determine the structural basis for cis-acting RNA elements identified in Project 1 and novel host factors from Project 3/Core B that regulate EBOV RDRP complex. Completion of these studies will address longstanding and critical mechanistic questions in EBOV biology that are also central to a better understanding of non-segmented negative sense RNA viruses (NNSVs). These insights are also expected to yield new opportunities for antiviral development.

Nipah virus (NiV) and Hendra virus (HeV) are related highly pathogenic zoonotic henipaviruses in the paramyxovirus family that use bats from the Pteropus genus as reservoir hosts. NiV exhibits an unusually broad host range for a paramyxovirus and infects pigs, dogs, and cats. Although first identified in an outbreak in Malaysia, near annual outbreaks in Bangladesh and India are now known to occur with average case fatality rates of 73%. HeV infections have occurred in Australia where infected horses transmitted the virus to seven humans of whom four died. Both NiV and HeV have also caused late-onset lethal encephalitis in humans. Because of their high lethality in humans, the absence of approved vaccines or treatments, and evidence of NiV human to human transmission, these viruses are NIAID Emerging Infectious Diseases/Pathogens Category C Priority Pathogens. In addition, henipaviruses can infect livestock and are serious threats to agriculture. Despite the potential for severe public health and economic consequences, research into these viruses has lagged, reflecting in part the need for biosafety level 4 containment to study replicating virus. Importantly, there are no drugs currently available to treat or prevent these infections. Recent studies have started to provide insight into the determinants of viral pathogenesis and to define at the atomic and molecular levels how transcription and replication activities are carried out by the viral RNA-dependent RNA polymerase complex (also known as the viral RDRP complex). The viral RDRP complex has obvious potential as a therapeutic target, but sensitive reliable screens and secondary assays are needed to identify and validate inhibitors of this complex. Our recent collaborative studies defined an analogous interaction between Ebola VP35 and NP proteins that is currently being developed as a therapeutic target. In order to address a major unmed need for NiV and HeV therapeutics, we will use this successful framework to develop in vitro and cell-based assays that target the interface between NiV and HeV N and P proteins. We expect to identify replication inhibitor leads targeting zoonotic henipaviruses that will facilitate biological probe and antiviral development.

ABSTRACT The filoviruses, Ebola and Marburg viruses (EBOV and MARV), are emerging, negative-strand RNA viruses associated with outbreaks of severe viral hemorrhagic fever. The virulence and emerging nature of these zoonotic pathogens makes them a significant threat to human health, potential agents of bioterrorism, and NIAID category A priority pathogens. Currently, no approved anti-filovirus therapeutics are available. Importantly, there is a major gap in our understanding with regard to the role of host factors at critical stages in the viral replication cycle. The overall goal of this revised R01 application is to characterize EBOV VP30 (eVP30), a key viral protein that facilitates viral transcription, and its interactions with host factors. Our plan builds on recent successes in structurally and functionally characterizing how eVP30 interacts with the viral nucleoprotein (NP) to modulate EBOV RNA synthesis and on a joint (Amarasinghe, Basler, and Krogan groups) unbiased proteomics screen using EBOV proteins as bait that uncovered 193 high-confidence EBOV- human protein-protein interactions (PPIs), including one between eVP30 and the host ubiquitin ligase RBBP6. A crystal structure of this complex revealed that RBBP6 and the viral NP compete for the same VP30 binding surface. Comparison of NP and RBBP6 peptides that bind eVP30 revealed a common PPxPxY motif that is necessary for the interaction. Whereas knockdown of endogenous RBBP6 stimulated viral transcription and increased EBOV infectivity, overexpression of RBBP6 or its peptide severely inhibited EBOV transcription and infection. Interestingly, at least two additional eVP30 interactors from our dataset (hnRNP L and hnRNP UL1) also possess PPxPxY motifs. Based on these findings, we propose a multidisciplinary approach to (1) Determine the structure of eVP30 N-terminus and define its association with RNA and protein ligands in the absence and presence of NP; (2) Determine the mechanisms by which eVP30-interacting proteins RBBP6, hnRNP L, and hnRNP UL1 modulate eVP30 function and RNA synthesis; and (3) Test the hypothesis that eVP30 modulates the function of host factors RBBP6, hnRNP L, and hnRNP UL1. These studies will characterize unique host interactions that negatively regulate EBOV replication with the goals of defining how EBOV manipulates host pathways and identifying novel therapeutic targets.

Project Summary Filoviruses, including the Ebola viruses, Marburg viruses, and Cueva virus, are a class of pathogens with enormous health implications. Filoviruses have been implicated in numerous outbreaks in Africa, including the ongoing outbreak in the Democratic Republic of Congo (DRC), potential risk as an agent of bioterrorism, and the potential risk of transmission to non-endemic countries such as the United States. Critical in both the management of outbreaks as well as the treatment of infected patients are specific and rapid diagnostic tests. Despite the critical health risk posed by these pathogens, current diagnostic techniques utilizing PCR and ELISAs have significant limitations, including the need for centralized laboratories, cold-chain custody, and limited diagnostic ability in the early stages of infection. This proposal seeks to fill this gap and leverage a highly sensitive class of optical sensors, whispering gallery mode (WGM) devices, for the rapid detection of filovirus glycoproteins (GP) and the ebolavirus soluble glycoprotein (sGP), two highly sensitive biomarkers for filoviruses. WGM sensors are a unique class of optical devices in which light is confined to a small volume, therefore leading to an enormous enhancement in signal response. In addition to their sensitivity, these devices also offer the advantages of inexpensive and scalable fabrication costs, the ability to be integrated with conventional electronics, and the potential for multiplexed measurements. In combination with these devices will be antibodies against filovirus GP and sGP, the latter an enormously powerful sentinel biomarker that is positive hours to days before presentation of symptoms in infected individuals. This academic-industry partnership will develop a rapid and sensitive diagnostic test for filovirus GP and sGP, and subsequently lay the foundation for the developing this technology into a powerful, field-deployable device for filovirus detection.

Project Summary/Abstract This R01 entitled, ?Development and characterization of engineered therapeutic antibodies against SARS-CoV- 2?, builds on our project infrastructure, expertise, and experience in characterizing viral-host factor interactions in negative strand RNA viruses. Since originating in China, SARS-CoV-2 has since rapidly spread and is now a global pandemic. Significant concerns are that humans are immunologically naïve, and there are no available therapies. In the US, the disease has already overwhelmed the healthcare system in some states and have a serious knock-on effect in exacerbating the standard of care for other diseases. At the time of writing, nearly 5 million cases and >160,000 deaths have been attributed to COVID-19. The virus replicates in the lungs and causes a severe respiratory disease, COVID-19, which is fatal in >2% of cases. Neutralizing antibodies (nAbs) generated by natural infection or vaccines is known to control many infections and early studies in the current COVID-19 pandemic, including studies to test convalescent plasma treatments, are promising. These studies highlight the potential significance of nAb-based therapy. While IgG format of nAbs have long been the most extensively used format, early studies, including our own suggest that additional multivalent formats of nAbs may be more effective. This provides an innovative method to develop nAbs while acquiring potential benefits from effective neutralization at lower doses and lower likelihood of the emergence of resistance mutants. SARS-CoV- 2 is a single stranded, non-segmented, enveloped RNA virus. Viral infection requires interaction of the spike glycoprotein receptor binding domain (RBD) to the host receptor ACE2. Here, we will build on newly developed and established approaches that have been optimized through our work other systems to generate antibodies targeting spike and spike RBD using phage display technology and characterize their physical properties. We will engineer antibodies with increased valency and test for potency in in vitro neutralization assays and in vivo efficacy in a mouse model. At the completion, we expect to provide innovative and unique multivalent nAb leads with unique characteristics that will rival the best in class IgG drugs.

Project Summary/Abstract Rift Valley fever virus (RVFV) is a phlebovirus that belongs to the Phenuiviridae (formerly Bunyaviridae) family of negative-sense RNA viruses. As an emerging mosquito-borne virus, the significance of RVFV is highlighted by its designation as a NIAID Category A pathogen and its inclusion on the WHO's Blueprint of Priority Diseases. Recently, the Coalition for Epidemic Preparedness Innovations (CEPI) has also included RVFV as a part of their emerging infectious diseases vaccine program, further emphasizing the potential impact of RVFV on the global health and economy. While RVFV is endemic throughout sub-Saharan Africa, competent mosquito vector species are found in North America, highlighting the potential for emergence of RVFV in non-endemic countries, including the United States. During outbreaks, RVFV causes severe disease in livestock, including sheep and cattle, which dramatically impact the socioeconomic framework in resource limited settings. Humans are spill- over hosts, where infections can result in severe consequences, including hepatic necrosis, hemorrhagic fever, encephalitis, and retinal vasculitis. Despite its significance to human health and the potential to negatively impact the socioeconomic fabric of resource-limited countries where the virus is endemic, there is a lack of safe and efficacious prophylactic and therapeutic treatment options. This gap is in part due to our lack of knowledge on host factors that contribute to RVFV infection. To address this need, we conducted a genomic screen that defined several critical factors, including a potential entry factor, which we will characterize by a multidisciplinary approach. In support, we provide compelling preliminary data, including in vitro validation in host factor sufficient and deficient cells, transcomplementation studies, direct interaction between RVFV glycoprotein Gn and the host proteins in vitro, inhibition of the entry factor by endogenous ligands in vitro in multiple cell lines from evolutionarily distinct hosts, and preliminary results of protection from RVFV infection in two conditional knock out mouse models. Importantly, we have generated many key reagents, including most cell lines and proteins, and knock-out mice supporting the feasibility. Importantly, this work will be performed by highly productive and collaborative investigators with expertise in every aspect of the proposed studies, including biochemistry, RVFV pathogenesis, immunology, proteomics, structural biology, and virology. Completion of the proposed studies will define novel host or entry factors for RVFV in target cells with tissue-specific relevance. As a specific receptor for RVFV has not previously been identified, these studies will provide important information for design of therapeutic interventions to prevent RVFV infection and disease. At the completion, we expect to fill a key gap in the field and to provide novel targets for therapeutic development.