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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI012316-17
Application #
3125164
Study Section
Experimental Virology Study Section (EVR)
Project Start
1975-05-01
Project End
1995-03-31
Budget Start
1993-04-01
Budget End
1994-03-31
Support Year
17
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of California Irvine
Department
Type
Schools of Medicine
DUNS #
161202122
City
Irvine
State
CA
Country
United States
Zip Code
92697
Szewczyk, B; Pilat, Z; Bienkowska-Szewczyk, K et al. (1998) Elution of glycoproteins from replicas of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Electrophoresis 19:220-3
Peng, Q; Galarza, J M; Shi, L et al. (1996) Influenza A virus RNA-dependent RNA polymerase cleaves influenza mRNA in vitro. Virus Res 42:149-58
Shi, L; Galarza, J M; Summers, D F (1996) Recombinant-baculovirus-expressed PB2 subunit of the influenza A virus RNA polymerase binds cap groups as an isolated subunit. Virus Res 42:1-9
Shi, L; Summers, D F; Peng, Q et al. (1995) Influenza A virus RNA polymerase subunit PB2 is the endonuclease which cleaves host cell mRNA and functions only as the trimeric enzyme. Virology 208:38-47
Neufeld, K L; Galarza, J M; Richards, O C et al. (1994) Identification of terminal adenylyl transferase activity of the poliovirus polymerase 3Dpol. J Virol 68:5811-8
Galarza, J M; Sowa, A; Hill, V M et al. (1992) Influenza A virus NP protein expressed in insect cells by a recombinant baculovirus is associated with a protein kinase activity and possesses single-stranded RNA binding activity. Virus Res 24:91-106
Hill, V M; Summers, D F (1990) A minor microtubule-associated protein is responsible for the stimulation of vesicular stomatitis virus transcription in vitro. J Gen Virol 71 ( Pt 2):289-98
Szewczyk, B; Laver, W G; Summers, D F (1988) Purification, thioredoxin renaturation, and reconstituted activity of the three subunits of the influenza A virus RNA polymerase. Proc Natl Acad Sci U S A 85:7907-11
Szewczyk, B; Summers, D F (1987) Fluorescent staining of proteins transferred to nitrocellulose allowing for subsequent probing with antisera. Anal Biochem 164:303-6
Hill, V M; Harmon, S A; Summers, D F (1986) Stimulation of vesicular stomatitis virus in vitro RNA synthesis by microtubule-associated proteins. Proc Natl Acad Sci U S A 83:5410-3

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