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Principal Investigator: Sean PJ Whelan
Organization: WASHINGTON UNIVERSITY
Fiscal Year: 2023
Award: $561,330
Funding agency: National Institute of Allergy and Infectious Diseases
This application is to extend a MERIT award to study RNA synthesis in minus-strand RNA viruses. Nonsegmented
negative-sense (NNS) RNA viruses include some of the most significant human pathogens that are an ongoing threat
to US public health. For measles, mumps and rabies there are licensed vaccines, but for most NNS RNA viruses there
are no vaccines and no antiviral drugs. Our long-term objective is to understand the mechanisms by which the
replication machinery of vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, functions. VSV is the
ideal choice for such studies because it is the only NNS RNA virus for which robust transcription can be reconstituted
in vitro from purified recombinant components. The catalytic core of the RNA synthesis machinery is a 241 kDa large
protein (L) that contains an RNA dependent RNA polymerase (RdRP), a polyribonucleotidyltransferase (PRNTase)
that caps the mRNA, and a dual specificity mRNA cap methyltransferase (MTase). During mRNA synthesis, those
activities are coordinated so that the nascent mRNA is capped, methylated and polyadenylated. Although L contains
all the enzymatic activities for RNA synthesis, it requires a 29 kDa phosphoprotein (P) that bridges interactions
between L and the nucleocapsid protein (N) that completely coats the RNA template. Since the last competing
renewal of this grant, we have obtained an atomic model of the VSV L protein. That structure profoundly changes our
understanding of the RNA synthesis machinery of the NNS RNA viruses revealing that dynamic inter domain
rearrangements in L protein must occur during RNA synthesis, identifying an intimate linkage between capping and
RNA synthesis and identifying key P-L interactions that likely facilitate the inter domain rearrangements. Our
underlying hypothesis is that the catalytic activities of L in RNA polymerization, mRNA cap addition and cap
methylation which reside within structurally separate domains are coordinated by the presence of the P and the
template associated N to regulate their activities during mRNA synthesis, and to downregulate them during assembly
by complex formation with the viral matrix protein (M). A major gap in understanding the machinery of RNA synthesis
is the existing structures of L likely represent the pre-initiation form of the transcriptase. We have in hand an
interpretable density map of a rabies virus L-P complex from cryo EM. During the next funding period, we will continue
to use cryo electron microscopy (EM), negative-stain EM, X-ray crystallography, in vitro biochemistry of polymerase
and molecular virology to provide unique structural and functional insights into VSV and rabies virus polymerases
during distinct stages of RNA synthesis and assembly. The successful completion of this work will provide an atomic
level structure of an NNS RNA virus polymerase complex and new mechanistic insights into the function and
regulation of this RNA synthesis machine during transcription, replication and assembly. Those results may aid efforts
toward rational attenuation of NNS RNA viruses for vaccine purposes, and the development of antiviral therapeutics.
RELEVANCE (See instructions):
The L polymerase protein of nonsegmented negative-strand (NNS) RNA viruses contains an RNA dependent RNA
polymerase and a suite of unconventional mRNA capping enzymes including a polyribonucleotidyltransferase and a
dual-specificity cap methyltransfease. Understanding how those activities are controlled to ensure that the viral
genome is expressed is of intrinsic interest and has the potential to impact the development of antiviral drugs and live
attenuated vaccines. Here we will compare structural and functional insights into this protein for a prototype NNS RNA
virus, vesicular stomatitis virus and the human pathogen rabies.
Terms: <Antiviral Agents><Antiviral Drugs><Antivirals><Attenuated Vaccines><Award><Biochemistry><Biological Chemistry><Catalytic Core><Catalytic Domain><Catalytic Region><Catalytic Site><Catalytic Subunit><Complex><Cryo-electron Microscopy><Cryoelectron Microscopy><EC 2.1.1><EC 2.7.7.48><Electron Cryomicroscopy><Electron Microscopy><Epidemic Parotitis><Funding><GTP mRNA guanylyltransferase><Gene Transcription><Genetic Transcription><Grant><Hand><In Vitro><Instruction><Licensing><Live-attenuated Vaccine><Maps><Measles><Messenger RNA><Methylation><Methyltransferase><Modeling><Molecular Virology><Mumps><Negative Staining><Non-Polyadenylated RNA><Nucleocapsid Proteins><Phosphoproteins><Poly A><Poly(rA)><Polyadenylation><Polymerase><Polymers><Proteins><Public Health><RNA><RNA Expression><RNA Gene Products><RNA Polyadenylation><RNA Processing><RNA Replicase><RNA Viruses><RNA chemical synthesis><RNA guanylyltransferase><RNA synthesis><RNA-Dependent RNA Polymerase><RNA-Directed RNA Polymerase><Rabies><Rabies virus><Recombinants><Regulation><Ribonucleic Acid><Rubeola><Single Crystal Diffraction><Specificity><Structure><Transcriptase><Transcription><VSV><Vaccines><Vesicular Stomatitis Virus><Vesicular stomatitis Indiana virus><Viral Genome><Viral M Proteins><Viral Matrix Proteins><Viral Membrane Proteins><Work><X Ray Crystallographies><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray/Neutron Crystallography><Xray Crystallography><anti-viral agents><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 compound><antiviral development><antiviral drug development><antiviral medication><antiviral therapeutic><antiviral therapeutic development><antiviral therapy development><attenuation><cryo-EM><cryoEM><cryogenic electron microscopy><density><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><epidemic parotiditis><hands><human pathogen><insight><interest><live vaccine><live vaccines><lyssa><m-RNA Guanylyltransferase><mRNA><mRNA 5'-guanylyltransferase><mRNA capping><mRNA capping enzyme><mRNA guanylyltransferase><methylase><morbilli><polyadenylate><polymer><polymeric><polymerization><prototype><reconstitute><reconstitution><transmethylase><vesicular stomatitis virus L protein><virus genome>