Deletion mapping of the rotavirus metalloprotein NS53 (NSP1): the conserved cysteine-rich region is essential for virus-specific RNA binding.
NS53 (NSP1), the gene 5 product of the group A rotaviruses, is a minor nonstructural protein of 486 to 495 amino acids which binds zinc and contains an amino-terminal highly conserved cysteine-rich region that may form one or two zinc fingers. To study the structure-function of the gene 5 product, wild-type and mutant forms of NS53 were produced by using a recombinant baculovirus expression system and a recombinant vaccinia virus/T7 (vTF7-3) expression system. Analysis of the RNA-binding activity of the wild-type NS53 immobilized onto protein A-Sepharose beads with NS53-specific antiserum showed that the protein exhibited specific affinity for all 11 rotavirus mRNAs. The use of short virus-specific RNA probes indicated that NS53 specifically recognizes an element located near the 5' ends of viral mRNAs. Analysis of the RNA-binding activity of deletion mutants of NS53 showed that the RNA-binding domain resides within the first 81 amino acids of the protein and that the highly conserved cysteine-rich region within this region of the protein is essential for the activity. Gel electrophoresis and Western immunoblot analyses of intracellular fractions derived from infected cells revealed that large amounts of NS53 were present in the cytosol and in association with the cytoskeletal matrix. Indirect immunofluorescence analysis of cells programmed to transiently express mutant forms of NS53 using vTF7-3 indicated that the intracellular localization domain resides between amino acids 84 and 176 of NS53. Together, these data show that the RNA-binding domain and the intracellular localization domain lie upstream from the region of NS53 previously determined not to be essential for replication of rotaviruses in cell culture (J. Hua and J. T. Patton, Virology 198:567-576, 1994).
Hua
J
J
Department of Microbiology and Immunology, University of Miami School of Medicine, Florida 33101.
Chen
X
X
Patton
J T
JT
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D017124:Conserved Sequence; D003545:Cysteine / Q000235:genetics; D003599:Cytoskeleton / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics; D005455:Fluorescent Antibody Technique; D008667:Metalloproteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D012401:Rotavirus / Q000235:genetics* / Q000378:metabolism*; D017384:Sequence Deletion; D013347:Subcellular Fractions / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism*; D016335:Zinc Fingers / Q000235:genetics
D004279:DNA, Viral; D008667:Metalloproteins; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D017361:Viral Nonstructural Proteins; C104282:nsp1 protein, Rotavirus; D003545:Cysteine