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Identification of two essential aspartates for polymerase activity in parainfluenza virus L protein by a minireplicon system expressing secretory luciferase
Author(s) -
Matsumoto Yusuke,
Ohta Keisuke,
Yumine Natsuko,
Goto Hideo,
Nishio Machiko
Publication year - 2015
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/1348-0421.12329
Subject(s) - biology , polymerase , microbiology and biotechnology , luciferase , mutant , t7 rna polymerase , reporter gene , messenger rna , rna polymerase , gene expression , virology , gene , transfection , biochemistry , rna , escherichia coli , bacteriophage
Gene expression of nonsegmented negative‐strand RNA viruses (nsNSVs) such as parainfluenza viruses requires the RNA synthesis activity of their polymerase L protein; however, the detailed mechanism of this process is poorly understood. In this study, a parainfluenza minireplicon assay expressing secretory Gaussia luciferase (Gluc) was established to analyze large protein (L) activity. Measurement of Gluc expression in the culture medium of cells transfected with the minigenome and viral polymerase components enabled quick and concise calculation of L activity. By comparing the amino acid sequences in conserved region III (CRIII), a putative polymerase‐active domain of the L protein, two strictly conserved aspartates were identified in all families of nsNSV. A series of L mutants from human parainfluenza virus type 2 and parainfluenza virus type 5 showed that these aspartates are necessary for reporter gene expression. It was also confirmed that these aspartates are important for the production of viral mRNA and antigenome cRNA, but not for a polymerase‐complex formation. These findings suggest that these two aspartates are key players in the nucleotidyl transfer reaction using two metal ions.