FORMATION OF BROME MOSAIC-VIRUS RNA-DEPENDENT RNA-POLYMERASE IN YEASTREQUIRES COEXPRESSION OF VIRAL-PROTEINS AND VIRAL-RNA

Citation
R. Quadt et al., FORMATION OF BROME MOSAIC-VIRUS RNA-DEPENDENT RNA-POLYMERASE IN YEASTREQUIRES COEXPRESSION OF VIRAL-PROTEINS AND VIRAL-RNA, Proceedings of the National Academy of Sciences of the United Statesof America, 92(11), 1995, pp. 4892-4896
Citations number
27
Categorie Soggetti
Multidisciplinary Sciences
ISSN journal
00278424
Volume
92
Issue
11
Year of publication
1995
Pages
4892 - 4896
Database
ISI
SICI code
0027-8424(1995)92:11<4892:FOBMRR>2.0.ZU;2-3
Abstract
In this report we show that yeast expressing brome mosaic virus (BMV) replication proteins 1a and 2a and replicating a BMV RNA3 derivative c an be extracted to yield a template-dependent BMV RNA-dependent RNA po lymerase (RdRp) able to synthesize (-)-strand RNA from BMV (+)-strand RNA templates added in vitro, This virus-specific yeast-derived RdRp m irrored the template selectivity and other characteristics of RdRp fro m BMV-infected plants. Equivalent extracts from yeast expressing 1a an d 2a but lacking RNA3 contained normal amounts of 1a and 2a but had no RdRp activity on BMV RNAs added in vitro, To determine which RNA3 seq uences were required in vivo to yield RdRp activity, we tested deletio ns throughout RNA3, including the 5', 3', and intercistronic noncoding regions, which contain the cis-acting elements required for RNA3 repl ication in vivo, RdRp activity was obtained only from cells expressing 1a, 2a, and RNA3 derivatives retaining both 3' and intercistronic non coding sequences, Strong correlation between extracted RdRp activity a nd BMV (-)-strand RNA accumulation in vivo was found for all RNA3 deri vatives tested, Thus, extractable in vitro RdRp activity paralleled fo rmation of a complex capable of viral RNA synthesis in vivo. The resul ts suggest that assembly of active RdRp requires not only viral protei ns but also viral RNA, either to directly contribute some nontemplate function or to recruit essential host factors into the RdRp complex an d that sequences at both the 3'-terminal initiation site and distant i nternal sites of RNA3 templates may participate in RdRp assembly and i nitiation of(-)-strand synthesis.