Biochemical and genetic analyses of the interaction between the helicase-like and polymerase-like proteins of the brome mosaic virus.
Replication of the three positive-strand genomic RNAs of brome mosaic virus requires the activities of the helicase-like 1a and the polymerase-like 2a proteins. One hundred fifteen amino acids of the 2a N-terminus and the 1a helicase-like region of over 50 kDa are both necessary and sufficient for 1a-2a interaction. Requirement of the large size of the 1a helicase-like domain suggests that higher order structures might be necessary for the protein's interaction with 2a. To explore the structural properties of 1a, we used limited proteolysis of in vitro-translated 1a protein. Treatment of 1a and its deletion derivatives with papain or trypsin revealed that the C-terminal helicase-like segment of approximately 50-60 kDa is highly resistant under our assay conditions to proteolysis, while the N-terminus is rapidly degraded. All tested mutations in the helicase-like region that renders this region protease-sensitive have previously been found to be defective for RNA replication in vivo. To complement the in vitro studies, we examined the interaction of the 1a helicase-like domain and the 2a N-terminus in yeast using the two-hybrid system. Mutations previously known to disrupt 1a-2a interaction also prevented interaction in yeast. Furthermore, results from two-hybrid analysis suggest that the structural domain mapped in vitro is important for 1a-2a interaction. Finally, we found that the helicase-like proteins of three other tripartite RNA viruses also contain equivalently located protease-resistant domains.
Department of Biology, Indiana University, Bloomington 47405, USA.
E K
EK
O'Reilly
N
N
Tang
P
P
Ahlquist
C C
CC
Kao
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017795:Bromovirus / Q000201:enzymology*; D004265:DNA Helicases / Q000378:metabolism*; D017931:DNA Primers; D010450:Endopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010206:Papain / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D015183:Restriction Mapping; D013329:Structure-Activity Relationship; D015003:Yeasts / Q000378:metabolism
D017931:DNA Primers; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase; D010450:Endopeptidases; D010206:Papain; D004265:DNA Helicases