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Principal Investigator: Bo Liang
Organization: EMORY UNIVERSITY
Fiscal Year: 2024
Award: $391,250
Funding agency: National Institute of General Medical Sciences
PROJECT ABSTRACT
The long-term goal of our project is to elucidate the molecular basis of the poorly understood mechanism of
the RNA synthesis machinery of NNS RNA viruses and to determine the structures of key protein complexes
involved in the RSV RNA synthesis, facilitating the development of antiviral drugs. Despite several decades of
research, the molecular mechanism of RNA synthesis by NNS RNA viruses remains elusive. The catalytic core
of the RNA synthesis machine of NNS RNA viruses is the RNA-dependent RNA polymerase (RdRP) that
comprises a large 250 kDa protein (L) and a cofactor phosphoprotein (P). The L protein catalyzes three
enzymatic activities: nucleotide polymerization, cap addition, and cap methylation. P is essential for the full
activity of L to act on the viral genome. In some cases, additional viral proteins (VP30 in Ebola and M2-1 in RSV)
are necessary for full RdRP processivity. Several important and long-standing questions for RNA synthesis
by NNS RNA viruses remain unanswered - First, how does L carry out the catalytic reactions of the
phosphodiester bond formation of the first two nucleotides (de novo RNA synthesis), RNA elongation (RNA
extension), and RNA polyadenylation (shutter mechanism)? Second, how does a single protein L coordinate
three distinct enzymatic activities (makes, caps, and methylates RNA), and what determines the switch to the
next process? Third, how do the cofactor P and transcription factor M2-1 regulate the activities of the L protein?
The lack of clear answers to these critical questions represents a major knowledge gap in our understanding
of the mechanism of NNS RNA synthesis. We propose to tackle these challenging questions with a novel hybrid
approach that integrates biochemistry, enzymology, mutagenesis, virology, genetics, crystallography, and cryo-
EM methods. Guided by our preliminary data, we hypothesize that the catalytic activities of RNA polymerization,
cap addition, and cap methylation reside within RSV L and that L requires a dynamic assembly with P and M2-
1 to coordinate these activities during RNA synthesis. To test this hypothesis, we will investigate the functional
interplay between the multi-functional enzyme L, cofactor P, and transcription factor M2-1. We will seek to
provide clear answers to the long-standing questions regarding the mechanisms governing RNA synthesis of
RSV and other NNS RNA viruses. The proposed research is significant and groundbreaking because the novel
knowledge and structures obtained from this proposed research will significantly advance our understanding of
RSV and NNS RNA synthesis. Ultimately, such knowledge will provide a framework for developing novel
antivirals to treat RSV infections.
Terms: <0-11 years old><Active Sites><Anti-viral Agents><Area><Assay><Basal Transcription Factor><Basal transcription factor genes><Binding><Bioassay><Biochemical><Biochemistry><Biologic Models><Biological Assay><Biological Chemistry><Biological Models><Catalytic Core><Catalytic Domain><Catalytic Region><Catalytic Site><Catalytic Subunit><Child><Child Youth><Childhood><Children (0-21)><Classification><Cryo-electron Microscopy><Cryoelectron Microscopy><Crystallographies><Crystallography><DNA-Dependent RNA Polymerases><DNA-Directed RNA Polymerase><Data><Dissection><EC 2.1.1><EC 2.7.7.48><Ebola><Electron Cryomicroscopy><Ensure><Enzymatic Biochemistry><Enzyme Gene><Enzymes><Enzymology><Funding><Gene Transcription><General Transcription Factor Gene><General Transcription Factors><Genetic><Genetic Transcription><Genetics-Mutagenesis><Glycine cleavage system P-protein><Glycine decarboxylase><Goals><Grant><Hybrids><In Vitro><Knowledge><Legal patent><Lower Respiratory Tract Infection><Lower respiratory infection><Measles><Methods><Methylation><Methyltransferase><Mission><Model System><Molecular><Molecular Interaction><Mutagenesis><Mutagenesis Molecular Biology><NIH><National Institutes of Health><Non-Polyadenylated RNA><Nucleotides><P-protein><Patents><Pathogenicity><Phosphoproteins><Polyadenylation><Polymerase><Polymers><Process><Proteins><Public Health><Publications><RNA><RNA Expression><RNA Gene Products><RNA Polyadenylation><RNA Polymerases><RNA Replicase><RNA Seq><RNA Viruses><RNA chemical synthesis><RNA methylation><RNA sequencing><RNA synthesis><RNA-Dependent RNA Polymerase><RNA-Directed RNA Polymerase><RNAseq><RSV infection><Rabies><Reaction><Regulation><Reporting><Research><Respiratory Disease><Respiratory Syncytial Virus Infections><Respiratory System Disease><Respiratory System Disorder><Respiratory syncytial virus><Ribonucleic Acid><Role><Rubeola><Scientific Publication><Solid><Structure><Surface><Systematics><Testing><Transcription><Transcription Factor Proto-Oncogene><Transcription factor genes><United States National Institutes of Health><VSV><Vesicular Stomatitis Virus><Vesicular stomatitis Indiana virus><Viral><Viral Gene Products><Viral Gene Proteins><Viral Genome><Viral Proteins><Virus><anti-viral compound><anti-viral development><anti-viral drug development><anti-viral drugs><anti-viral medication><anti-viral therapeutic><anti-viral therapeutic development><anti-viral therapy development><anti-virals><antiviral development><antiviral drug development><antiviral therapeutic development><antiviral therapy development><cofactor><cryo-EM><cryoEM><cryogenic electron microscopy><developing anti-viral agent><developing anti-viral drug><developing anti-viral therapeutic><developing anti-viral therapy><developing antiviral agent><developing antiviral drug><developing antiviral therapeutic><developing antiviral therapy><disability><glycine decarboxylase P-protein><kids><lyssa><methylase><morbilli><novel><older adult><older adulthood><pediatric><phosphodiester><polymer><polymeric><polymerization><promoter><promotor><protein complex><reconstitute><reconstitution><social role><success><transcription factor><transcriptome sequencing><transcriptomic sequencing><transmethylase><virology><virus genome><virus protein><youngster>