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
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.