Mutational analysis of conserved tyrosines in the NS-1 protein of the parvovirus minute virus of mice.
The NS-1 gene of the parvovirus minute virus of mice encodes a multifunctional protein essential for viral DNA replication and gene expression. In addition to possessing DNA helicase and ATPase activities, NS-1 forms a covalent linkage with the 5' ends of viral DNA and is a strong candidate for the site-specific nicking-closing enzyme postulated to be involved in the resolution of concatemers and terminal hairpin structures that arise during parvoviral DNA replication. Since the covalent linkage between NS-1 and the 5' terminus of MVM DNA resists alkali and mild acid treatment, a tyrosine phosphodiester is likely to be involved. To map domains responsible for this activity, mutations converting tyrosine to phenylalanine were introduced into the NS-1 gene using oligonucleotide-directed mutagenesis and their effect on the DNA replication and transcriptional activation functions of NS-1 was examined in transient in vivo transfection assays. Replacement of Tyr-188, Tyr-197, Tyr-210, Tyr-310, Tyr-422, or Tyr-550 with phenylalanine greatly reduced the ability of NS-1 to complement the replication of the target genome ins 20B in COS-7 cells. However, a Ser-545 to Thr-545 substitution in the Phe-550 mutant restored DNA replication activity. Replacement of 5 other tyrosines in NS-1 with phenylalanine either enhanced (Phe-6), had a moderate inhibitory effect (Phe-209) or had no effect (Phe-47, Phe 227 and Phe-543) on its DNA replication activity. Two of the 11 phenylalanine substitution mutations, Phe-188 and Phe-197, also greatly reduced the ability of NS-1 to transactivate the p38 promoter and displayed a dominant negative phenotype with respect to transactivation. Since the remaining tyrosines in MVM NS-1, Tyr-152, Tyr-252, Tyr-374, and Tyr-595, are not conserved among the NS-1 proteins encoded by porcine and feline parvoviruses, they are presumed to be nonessential for the normal functioning of NS-1. The results point to a role for either Tyr-188, Tyr-197, Tyr-210, Tyr-310, or Tyr-422 in forming a covalent linkage with viral DNA and further suggest a regulatory role for several tyrosines in other DNA replication and transcriptional activation functions of NS-1.
Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia.
M H
MH
Skiadopoulos
E A
EA
Faust
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D004261:DNA Replication; D008915:Minute Virus of Mice / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D015533:Transcriptional Activation; D014162:Transfection; D014443:Tyrosine / Q000235:genetics*; D017361:Viral Nonstructural Proteins / Q000235:genetics*
C081367:NS1 protein, minute virus of mice; D017361:Viral Nonstructural Proteins; D014443:Tyrosine; D010649:Phenylalanine