Nuclear targeting of the parvoviral replicator molecule NS1: evidence for self-association prior to nuclear transport.
In order to investigate functions and properties of NS1, the major non-structural protein of the parvovirus minute virus of mice (MVM), we established an expression system in mammalian cells using recombinant vaccinia viruses. Using immunofluorescence and Western blots of subcellular fractions, nuclear localization of wildtype and mutant NS1 protein could be studied in the absence of other MVM proteins or viral DNA. Analysis of in-frame deletion and substitution mutants revealed that amino acid substitution in a triple lysine motif (residues 214-216) completely abrogated nuclear localization of the 672 amino acid NS1 polypeptide. In addition, substitution of a double lysine just upstream of this essential element also compromised nuclear localization, suggesting that the nuclear localization signal is at least bipartite. We also performed co-transport studies expressing cytoplasmic mutant NS1 with either wildtype or mutant nuclear NS1. Both wildtype and a C-terminally-deleted mutant NS1 were able to co-translocate cytoplasmic mutant NS1 polypeptides into the nucleus. Mutations introduced into the putative NTP-binding domain abolished this co-transport function, although these mutant NS1 proteins were themselves transported into the nucleus.
Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
J P
JP
Nüesch
P
P
Tattersall
D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008915:Minute Virus of Mice / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D014616:Vaccinia virus / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
C081367:NS1 protein, minute virus of mice; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins