# RNA Processing in Non-Segmented Minus-Strand RNA Viruses

> **NIH NIH R37** · WASHINGTON UNIVERSITY · 2021 · $561,330

## Abstract

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, replic...

## Key facts

- **NIH application ID:** 10126795
- **Project number:** 5R37AI059371-17
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** Sean PJ Whelan
- **Activity code:** R37 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $561,330
- **Award type:** 5
- **Project period:** 2005-03-01 → 2025-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10126795

## Citation

> US National Institutes of Health, RePORTER application 10126795, RNA Processing in Non-Segmented Minus-Strand RNA Viruses (5R37AI059371-17). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10126795. Licensed CC0.

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