R01AI161278

Structure, Function, and Inhibition of the SARS-CoV-2 Replication-Transcription Complex - Project Summary

COVID-19, caused by the coronavirus SARS-CoV-2, continues to devastate the world. In less than a year, there have been more than 20 million cases with over 700,000 deaths. The viral RNA-dependent RNA polymerase (RdRp) is the central enzyme responsible for transcription and replication of the viral RNA genome. This enzyme is also a target for the current antiviral, remdesivir, used to ameliorate the severity and duration of this disease. The virus also encodes several nucleic acid processing enzymes, in addition to the RdRp, including a helicase, an endonuclease, an exonuclease, and methyltransferases. However, it is unknown how these enzymes coordinate to transcribe and replicate the viral genome.

This proposal builds upon preliminary data of the structure of the helicase, NSP13, in complex with the RdRp and a primed substrate RNA (NSP13-replication/transcription complex or NSP13-RTC). The aims here include completing the structural analysis of this complex by utilizing additional data collected. The result of this aim will provide higher resolution (better than 2.7 Å in some parts of the RdRp), providing a rich basis for the development of antiviral inhibitors. Also, having this structure in hand allows for the collaboration with expert developers of antimicrobials, also part of the aims, including the investigation of the structural details of the pre-incorporation state of remdesivir and antivirals produced by human microbiome.

The models resulting from the structure of NSP13-RTC serve as foundations to test how the helicase and exonuclease function together with the RdRp. Specifically, real-time fluorescence assays, single-molecule fluorescence resonance energy transfer (FRET), and multi-color fluorescence microscopy will be used to probe the role of the helicase and the exonuclease in unwinding substrate RNA, backtracking, and proofreading.

Another aim applies the pipeline used to characterize the NSP13-RTC assembly, which yielded a high-resolution structure of the complex, to other RTC assemblies. Specifically, native electrophoretic mobility assays will be used as a starting point to probe larger assemblies of the RTC. Native mass-spectrometry will then be used to determine the composition and stoichiometry of the complexes. Finally, cryo-EM will be applied to solve the structures of these macromolecular machines. The resulting structures will provide a starting point to elucidate the coordinated functions of these enzymes, provide insight into their mechanisms, and establish novel targets for therapeutics.

In summary, this proposal aims to understand at the molecular and structural level how the SARS-CoV-2 nucleic acid processing enzymes coordinate to replicate and transcribe the viral genome, and to provide structure-guided targets for drug discovery, with the ultimate goal of providing relief for the COVID-19 pandemic.

Rockefeller University

TO ASSIST PUBLIC AND PRIVATE NONPROFIT INSTITUTIONS AND INDIVIDUALS TO ESTABLISH, EXPAND AND IMPROVE BIOMEDICAL RESEARCH AND RESEARCH TRAINING IN INFECTIOUS DISEASES AND RELATED AREAS, TO CONDUCT DEVELOPMENTAL RESEARCH, TO PRODUCE AND TEST RESEARCH MATERIALS. TO ASSIST PUBLIC, PRIVATE AND COMMERCIAL INSTITUTIONS TO CONDUCT DEVELOPMENTAL RESEARCH, TO PRODUCE AND TEST RESEARCH MATERIALS, TO PROVIDE RESEARCH SERVICES AS REQUIRED BY THE AGENCY FOR PROGRAMS IN INFECTIOUS DISEASES, AND CONTROLLING DISEASE CAUSED BY INFECTIOUS OR PARASITIC AGENTS, ALLERGIC AND IMMUNOLOGIC DISEASES AND RELATED AREAS. PROJECTS RANGE FROM STUDIES OF MICROBIAL PHYSIOLOGY AND ANTIGENIC STRUCTURE TO COLLABORATIVE TRIALS OF EXPERIMENTAL DRUGS AND VACCINES, MECHANISMS OF RESISTANCE TO ANTIBIOTICS AS WELL AS RESEARCH DEALING WITH EPIDEMIOLOGICAL OBSERVATIONS IN HOSPITALIZED PATIENTS OR COMMUNITY POPULATIONS AND PROGRESS IN ALLERGIC AND IMMUNOLOGIC DISEASES. BECAUSE OF THIS DUAL FOCUS, THE PROGRAM ENCOMPASSES BOTH BASIC RESEARCH AND CLINICAL RESEARCH. SMALL BUSINESS INNOVATION RESEARCH (SBIR) PROGRAM EXPANDS AND IMPROVES PRIVATE SECTOR PARTICIPATION IN BIOMEDICAL RESEARCH. THE SBIR PROGRAM INTENDS TO INCREASE AND FACILITATE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT, TO INCREASE SMALL BUSINESS PARTICIPATION IN FEDERAL RESEARCH AND DEVELOPMENT, AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION. THE SMALL BUSINESS TECHNOLOGY TRANSFER (STTR) PROGRAM STIMULATES AND FOSTERS SCIENTIFIC AND TECHNOLOGICAL INNOVATION THROUGH COOPERATIVE RESEARCH AND DEVELOPMENT CARRIED OUT BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS, TO FOSTER TECHNOLOGY TRANSFER BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS, TO INCREASE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT, AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION. RESEARCH CAREER DEVELOPMENT AWARDS SUPPORT THE DEVELOPMENT OF SCIENTISTS DURING THE FORMATIVE STAGES OF THEIR CAREERS. INDIVIDUAL NATIONAL RESEARCH SERVICE AWARDS (NRSAS) ARE MADE DIRECTLY TO APPROVE APPLICANTS FOR RESEARCH TRAINING IN SPECIFIED BIOMEDICAL SHORTAGE AREAS. IN ADDITION, INSTITUTIONAL NATIONAL RESEARCH SERVICE AWARDS ARE MADE TO ENABLE INSTITUTIONS TO SELECT AND MAKE AWARDS TO INDIVIDUALS TO RECEIVE TRAINING UNDER THE AEGIS OF THEIR INSTITUTIONAL PROGRAM.

93.855 - Allergy and Infectious Diseases Research

National Institute of Allergy and Infectious Diseases (NIAID) [HHS - NIH]

New York, New York 100656307 United States

Single Zip Code

Emergency Awards: Rapid Investigation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and Coronavirus Disease 2019 (COVID-19) (R01 Clinical Trial Not Allowed) (PAR-20-178)

COVID-19 $1,241,086 (40%) percent of this Project Grant was funded by COVID-19 emergency acts including the 2020 Coronavirus Preparedness and Response Supplemental Appropriations Act and the American Rescue Plan Act of 2021.
Amendment Since initial award the total obligations have increased 389% from $638,072 to $3,120,244.