Role of a highly conserved NH(2)-terminal domain of the human parainfluenza virus type 3 RNA polymerase.
The RNA polymerase complex of human parainfluenza virus type 3 (HPIV 3), a member of the family Paramyxoviridae, is composed of two virally encoded polypeptides: a multifunctional large protein (L, 255 kDa) and a phosphoprotein (P, 90 kDa). From extensive deduced amino acid sequence analyses of the cDNA clones of a number of L proteins of nonsegmented negative-strand RNA viruses, a cluster of high-homology sequence segments have been identified within the body of the L proteins. Here, we have focused on the NH(2)-terminal domain of HPIV 3 L protein that is also highly conserved. Following mutational analyses within this domain, we examined the ability of the mutant L proteins to (i) transcribe an HPIV 3 minireplicon, (ii) transcribe the viral RNA in vitro using the HPIV 3 nucleocapsid RNA template, and (iii) interact with HPIV 3 P protein. Our results demonstrate that the first 15 amino acids of the NH(2)-terminal domain spanning a highly conserved motif is directly involved in transcription of the genome RNA and in forming a functional complex with the P protein. Substitution of eight nonconserved amino acids within this domain by the corresponding Sendai virus L protein residues yielded mutants with variable transcriptional activities. However, one mutant in which all eight amino acids were replaced with the corresponding residues of Sendai virus L protein failed to both transcribe the minireplicon and interact with HPIV 3 P and the Sendai virus P protein. The possible functional significance of the NH(2)-terminal domain of paramyxovirus L protein is discussed.
Department of Virology, Lerner Research Institute, Cleveland Clinic Foundation, Ohio 44195, USA.
Achut G
AG
Malur
Suresh K
SK
Choudhary
Bishnu P
BP
De
Amiya K
AK
Banerjee
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002460:Cell Line; D017124:Conserved Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010224:Parainfluenza Virus 3, Human / Q000201:enzymology* / Q000235:genetics; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
C095185:P protein, Human parainfluenza virus 3; C055859:P protein, Sendai virus; D010750:Phosphoproteins; D014764:Viral Proteins; C056831:parainfluenza 3 virus, L Protein; D008156:Luciferases; D012321:DNA-Directed RNA Polymerases