Vaccinia Virus Gene Expression

Vaccinia virus (Fig. 1) is a double stranded DNA containing virus that replicates in the cytoplasm of infected cells.  In order to conduct this unusual life cycle, vaccinia contains the genes needed to produce the enzymes responsible for gene transcription, mRNA modification and DNA replication.  Viral gene expression is divided into three temporal classes.  Early genes are transcribed in the virus core.  Intermediate and late genes employ a replicating DNA template.  Each class has its own set of transcription regulatory factors.  Only early genes are subject to signal dependent transcription termination.  Early gene transcription termination requires several factors including: the virion form of RNA polymerase containing the Rap94 subunit, NPH I, which provides energy from the hydrolysis of ATP, VTF the vaccinia termination factor and the signal UUUUUNU in the nascent mRNA.  We are investigating the interplay among these factors and searching for a UUUUUNU recognition protein, in order to elucidate the mechanism of viral transcription termination.  We have demonstrated that the addition of an oligonucleotide containing the UUUUUNU sequence to an in vitro transcription reaction stimulates transcription termination, in trans.  Based on this observation, we are investigating the possibility that chimeric UUUUUNU containing oligonucleotides can be developed as efficacious anti-poxvirus agents.

 Fig. 1.  EM of the surface and cross section of a poxvirus.  Note the viral core, which contains the template DNA, various enzymes and is the site of early gene transcription in the infected cell.

Fig. 2  Early Gene Transcription: P, promoter; T, terminator; VETF, vaccinia early transcription factor; CE, mRNA capping enzyme; Rap94, early gene specific RNA polymerase subunit; NPH I, ssDNA dependent nucleoside triphosphate phosphohydrolase I; VTF, vaccinia termination factor.

Fig. 3.  Early Gene Transcription Termination.  For termination to occur, NPH I must be activated by binding to ssDNA found in the transcription bubble.  NPH I must also bind to the Rap94 subunit of the RNA polymerase.  UUUUUNU must be outside of the RNA polymerase and interact with an essential UUUUUNU recognition factor.  The role of VTF has not been defined. 

Recent Publications:

Mohamed, M.R., Latner, D., Condit, R.C., and Niles, E.G. (2001)  The vaccinia virus mRNA processing protein J3R interacts with the H4L subunit of the virion RNA polymerase.  Virology 230: 143-152.

Osman, A., Niles, E.G. and LoVerde, P.T. (2001)  Identification and characterization of a Schistosoma mansoni Smad2 homologue; a TGFb responder.  J. Biol. Chem. 276: 10072-10082.

Mohamed, M.R. and Niles, E.G. (2001) The Viral RNA Polymerase H4L Subunit is Required for Vaccinia Virus Early Gene Transcription Termination.J. Biol. Chem. 276: 20758-20765.

Fantappie, M.R., Freebern, W.J., Osman, A., LaDuca, J., Niles, E.G., and LoVerde, P.T. (2001) Characterization of Schistosoma mansoni retinoid X receptors: Evaluation of SmRXR1 and SmRXR2 activity and tissue distribution. Molecular and Biochemical Parasitology 115: 87-99.

Romeo, A.M., Christen, L.A., Niles, E.G. and Kosman, D.J. (2001) Intracellular chelation of iron by bipyridyl inhibits DNA virus replication: Ribonucleotide reductase maturation as a probe of intracellular iron pools.  J. Biol. Chem. 276: 26,301-26,308.

Condit, R.C. and Niles, E.G. (2002) Regulation of viral transcription elongation and termination in a vaccinia virus infection.  Biochem. Biophys Acta 1577: 325-336. 

Mohamed, M.R., Christen, L.A. and Niles, E.G. (2002).  Antibodies directed against an epitope in the N-terminal region of the H4L subunit of the vaccinia virus RNA polymerase inhibit both transcription initiation and transcription termination, in vitro.  Virology 299: 142-153.

Piacente. S.C., Christen, L.A., Mohamed, M.R. and Niles, E.G. (2003) Effect of Selected Mutations in the C-terminal Region of the Vaccinia Virus Nucleoside Triphosphate Phosphohydrolase I (NPH I) on Binding to the H4L Subunit of the Viral RNA Polymerase and Early Gene Transcription Termination, in vitro. Virology 310: 109-117.

Mohamed, M.R. and Niles, E.G. (2003) UUUUUNU oligonucleotide stimulation of vaccinia virus early gene transcription termination, in trans. J. Biol. Chem. 278: 11,794-801.

Fantappie, M.R., Osman, A., Ericsson, C. Niles, E.G. and LoVerde, P.T. (2003) Cloning of Schistosoma mansoni Seven in Absentia (SmSINA) Homologue cDNA, a gene Involved in Ubiquitination of SmRXR1 and smRXR2. Molecular and Biochemical Parasitology 131; 45-54.

Shalaby, K.A., Yin, L., Thakur, A., Christen, L., Niles, E.G. and LoVerde, P.T. (2003) Protection against Schistosoma mansoni utilizing DNA vaccination with genes encoding Cu/Zn cytosolic superoxide dismutase, signal peptide-containing superoxide dismutase and glutathione peroxidase enzymes. Vaccine 22: 130-136.

Mohamed, M.R and Niles, E.G. (2003). UUUUUNU Stimulation of Vaccinia Virus Early Gene Transcription Termination: Oligonucleotide Sequence and Structural Requirements for Stimulation of Premature Termination, in vitro. J. Biol. Chem. 278: 39534-39541.

Osman, A., Niles, E.G., LoVerde, P.T. (2004) Expression of functional Schistosoma mansoni Smad4: Role in Erk-mediated transforming growth factor-beta (TGF-b) down regulation. J. Biol. Chem. 279: 6474-6486.

LoVerde, P.T., Niles, E.G., Osman, A. and Wu, W. (2004) Schistosoma mansoni Male-Female Interactions. Canadian Journal of Zoology 82: 357-374.

Mohamed, M.R. and Niles, E.G. (2004) Transient and Inducible Expression of Vaccinia/T7 Rebombinant Viruses. In Methods in Molecular Biology, Vaccinia Virus and Poxvirology: Methods and Protocols 269: 41-50. ed.

Stuart N. Isaacs (Humana Press, Totowa, N.J.). Mohamed, M.R. and Niles, E.G. (2004). UUUUUNU oligonucleotide inhibition of RNA synthesis in vaccinia virus cores. Virology 324: 493-500.

Wu, W., Niles , E.G., El-Sayed, N., Berriman, M. and Loverde, P.T. (2006). Schistosoma mansoni nuclear receptors: sixteen new members and a novel subfamily. Gene 366: 303-315.

Mohamed, M.R., Piacente , S.C. , Dickerman, B.K. and Niles , E.G. (2006). Effect of UTP Sugar and Base Modifications on Vaccinia Virus Early Gene Transcription Virology, 349: 359-370.

Hu, Rong, Wu, W, Niles , E.G. and LoVerde, P.T. (2006). Isolation and characterization of Schistosoma mansoni constitutive androstane receptor. Molecular & Biochemical Parasitology 148: 31-43.  

Egiza, A., Niles , E.G., Verjovski-Almeida, S. and LoVerde, P.T. (2006) Schistosoma mansoni TGF- b Receptor II: Role in host ligand-induced Regulation of a Schistosome Target Gene. PLoS Pathogens 2 (6) June e54.  

Lu, C., Wu, W., Niles , E.G. and LoVerde, P.T. (2006) Identification and characterization of a novel fush tarazu factor 1 (FTZ-F1) nuclear receptor in Schistosoma mansoni . Molecular and Biochemical Parasitology, in press .  

Hu, R., Wu, W., Niles , E.G. and LoVerde, P.T (2006) SmTR2/4, a Schistosoma mansoni homologue of TR2/TR4 orphan nuclear receptor. International Journal for Parasitology, in press .

Lu, C., Niles , E.G. and LoVerde, P.T. (2006). Characterization of the DNA-binding properties and the transactivation activity of Schistosoma mansoni nuclear receptor fushi tarazu-factor 1 a SmFTZ-F1 a . Molecular and Biochemical Parasitology, in press .

Hu, Rong, Niles , E.G. and LoVerde, P.T. (2006). DNA binding and transactivation properties of the Schistosoma mansoni constitutive androstane receptor homologue. Molecular & Biochemical Parasitology , in press.

Wu W. Niles EG. Hirai H. LoVerde PT. Evolution of a novel subfamily of nuclear receptors with members that each contain two DNA binding domains. BMC Evolutinary Biology. 7:27, 2007.

Wu W. Niles EG. Hirai H. LoVerde PT. Identification and characterization of a nuclear receptor subfamily I member in the Playthelminth Schistosoma mansoni (SmNR1). FEBS Journal. 274(2):390-405, 2007 Jan.

Hu R. Wu W. Niles, EG LoVerde PT. SmTR2/4 , a Schistosoma mansoni homologue of TR2/TR4 orphan nuclear receptor. International Journal for Parasitology. 36(10-11):1113-22, 2006 Sep.

Carlo JM. Osman A. Niles EG. Wu W. Fantappie MR. Oliveira FM. LoVerde PT. Identification and characterization of an R-Smad ortholog (SmSmad1B) from Schistosoma mansoni. FEBS Journal. 274(16):4075-93, 2007 Aug.