# COMPOSITION, ASSEMBLY AND REPLICATION OF RNA VIRUSES

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA-IRVINE · 1993 · $269,918

## Abstract

Our laboratory will study the mechanism(s) by which the influenza A virus
RNA-dependent RNA polymerase is modified to switch from transcription
(synthesis of virus-specific mRNAs which are capped, methylated and
polyadenylated) to replication (synthesis of full length (+) and (-) sense
RNAs).  We will take advantage of a recent major breakthrough which should
enable us to reconstitute an in vitro RNA synthesis system containing
influenza RNA (full length and truncated T7 transcripts) assembled with NP
protein which serves as template for influenza transcriptase subunits
purified from either virion RNPs or from Sf9 cells with high levels of
expression from baculovirus recombinants.  We will study the biochemical
functions of the isolated purified polymerase subunits (PB1, PB2, PA), to
assign cap endonuclease, casein kinase, ATPase, GTP-binding, initiation and
elongation, and promoter binding functions.  We will also study the
interaction of the holoenzyme with the 3' termini of (+) and (-) sense
influenza templates using synthetic oligoribonucleotides and reconstituted
RNP complexes, and initiation and elongation by the enzyme.  By using
chimeric influenza RNA constructs containing the 3' untranslated region of
one genome segment and the downstream coding sequence from another we will
examine influenza transcriptional and translational controls in vitro.
This reconstituted system for influenza RNA synthesis will also enable us
to explore the error frequency of the influenza polymerase, and the
specificities of influenza A and B which prohibit their reassortment.

Using reconstituted RNPs which contain the influenza-specific 3' and 5'
termini and an inserted reporter gene (luciferase) as ssRNA (-) sense
constructs in RNP complexes we will transfect influenza virus (wt) infected
cells to see if this reporter gene-containing construct can be transcribed,
replicated and packaged into virions.

Nuclear extracts from influenza virus infected-cells, treated with either
cycloheximide or alpha-amanatin to inhibit replication or transcription
respectively, and anti-sera against polymerase subunits and influenza
nonstructural proteins will be utilized in an attempt to determine the
nature of the enzyme modification(s) involved in the switch from
transcription to replication and the possible involvement of cellular
factors in this switch.

## Key facts

- **NIH application ID:** 3125164
- **Project number:** 5R01AI012316-17
- **Recipient organization:** UNIVERSITY OF CALIFORNIA-IRVINE
- **SAM.gov UEI:** MJC5FCYQTPE6
- **Principal Investigator:** DONALD F SUMMERS
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1993
- **Award amount:** $269,918
- **Award type:** 5
- **Project period:** 1975-05-01 → 1995-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3125164

## Citation

> US National Institutes of Health, RePORTER application 3125164, COMPOSITION, ASSEMBLY AND REPLICATION OF RNA VIRUSES (5R01AI012316-17). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3125164. Licensed CC0.

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