Sequence-function analysis of the Sendai virus L protein domain VI.
The large (about 2200 amino acids) L polymerase protein of nonsegmented negative-strand RNA viruses (order Mononegavirales) has six conserved sequence regions ("domains") postulated to constitute the specific enzymatic activities involved in viral mRNA synthesis, 5'-end capping, cap methylation, 3' polyadenylation, and genomic RNA replication. Previous studies with vesicular stomatitis virus identified amino acid residues within the L protein domain VI required for mRNA cap methylation. In our recent study we analyzed four amino acid residues within domain VI of the Sendai virus L protein and our data indicated that there could be differences in L protein sequence requirements for cap methylation in two different families of Mononegavirales - rhabdoviruses and paramyxoviruses. In this study, we conducted a more comprehensive mutational analysis by targeting the entire SeV L protein domain VI, creating twenty-four L mutants, and testing these mutations for their effects on viral mRNA synthesis, cap methylation, viral genome replication and virus growth kinetics. Our analysis identified several residues required for successful cap methylation and virus replication and clearly showed the importance of the K-D-K-E tetrad and glycine-rich motif in the SeV cap methylation. This study is the first extensive sequence analysis of the L protein domain VI in the family Paramyxoviridae, and it confirms structural and functional similarity of this domain across different families of the order Mononegavirales.
Murphy
Andrea M
AM
Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223, USA.
Moerdyk-Schauwecker
Megan
M
Mushegian
Arcady
A
Grdzelishvili
Valery Z
VZ
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism; D008969:Molecular Sequence Data; D018096:Mononegavirales / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D012315:RNA Caps / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D029082:Sendai virus / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000502:physiology*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D014709:Vero Cells; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D012315:RNA Caps; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; D008780:Methyltransferases; C064365:L protein, Sendai virus; D012321:DNA-Directed RNA Polymerases