uu.seUppsala University Publications
Change search
ReferencesLink to record
Permanent link

Direct link
Template-dependent initiation of Sindbis virus RNA replication in vitro
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
1998 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 72, no 8, 6546-6553 p.Article in journal (Refereed) Published
Abstract [en]

Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of minus-strand RNA synthesis. Based on this observation, we have succeeded in reconstituting an in vitro system for template-dependent initiation of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and assayed by the addition of exogenous template RNAs. Extracts from cells expressing P123C>S, a protease-defective P123 polyprotein, and nsP4 synthesized a genome-length minus-sense RNA product. Replicase activity was dependent upon addition of exogenous RNA and was specific for alphavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23C>S, and nsP4. However, extracts from cells expressing nsP4 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity. In addition, a P123 polyprotein containing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These results provide further evidence that processing of the polyprotein inactivates the minus-strand initiation complex. Finally, RNA synthesis was detected when soluble nsP4 was added to a membrane fraction containing P123C>S, thus providing a functional assay for purification of the nsP4 RNA polymerase.

Place, publisher, year, edition, pages
1998. Vol. 72, no 8, 6546-6553 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-52722ISI: 000074730200034PubMedID: 9658098OAI: oai:DiVA.org:uu-52722DiVA: diva2:80632
Available from: 2008-10-17 Created: 2008-10-17 Last updated: 2010-10-26Bibliographically approved

Open Access in DiVA

No full text


Search in DiVA

By author/editor
Bergqvist, Anders
By organisation
Department of Medical Biochemistry and Microbiology
In the same journal
Journal of Virology
Medical and Health Sciences

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 135 hits
ReferencesLink to record
Permanent link

Direct link