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Journal of Virology, August 2002, p. 7987-7995, Vol. 76, No. 16
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.16.7987-7995.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Given the Opportunity, the Sendai Virus RNA-Dependent RNA Polymerase Could as Well Enter Its Template Internally

Diane Vulliémoz and Laurent Roux*

Department of Genetics and Microbiology, University of Geneva Medical School, 1211 Geneva 4, Switzerland

Received 18 January 2002/ Accepted 15 May 2002

The negative-stranded RNA viral genome is an RNA-protein complex of helicoidal symmetry, resistant to nonionic detergent and high salt, in which the RNA is protected from RNase digestion. The 15,384 nucleotides of the Sendai virus genome are bound to 2,564 subunits of the N protein, each interacting with six nucleotides so tightly that the bases are poorly accessible to soluble reagents. With such a uniform structure, the question of template recognition by the viral RNA polymerase has been raised. In a previous study, the N-phase context has been proposed to be crucial for this recognition, a notion referring to the importance of the position in which the nucleotides interact with the N protein. The N-phase context ruled out the role of the template 3'-OH congruence, a feature resulting from the obedience to the rule of six that implies the precise interaction of the last six 3'-OH nucleotides with the last N protein. The N-phase context then allows prediction of the recognition by the RNA polymerase of a replication promoter sequence even if internally positioned, a promoter which normally lies at the template extremity. In this study, with template minireplicons bearing tandem replication promoters separated by intervening sequences, we present data that indeed show that initiation of RNA synthesis takes place at the internal promoter. This internal initiation can best be interpreted as the result of the polymerase entering the template at the internal promoter. In this way, the data are consistent with the importance of the N-phase context in template recognition. Moreover, by introducing between the two promoters a stretch of 10 A residues which represent a barrier for RNA synthesis, we found that the ability of the RNA polymerase to cross this barrier depends on the type of replication promoter, strong or weak, that the RNA polymerase starts on, a sign that the RNA polymerase may be somehow imprinted in its activity by the nature of the promoter on which it starts synthesis.


* Corresponding author. Mailing address: Department of Genetics and Microbiology, University of Geneva Medical School, CMU, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland. Phone: (44 22) 702 56 83. Fax: (44 22) 702 57 02. E-mail: Laurent.Roux{at}medecine.unige.ch.


Journal of Virology, August 2002, p. 7987-7995, Vol. 76, No. 16
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.16.7987-7995.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.




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