Title

Characterization of the role of the zinc finger protein, NS53, in rotavirus replication

Date of Award

1994

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Microbiology and Immunology

First Committee Member

John T. Patton, Committee Chair

Abstract

NS53 (NSP1), the gene 5 product of the group A rotaviruses, is now identified as a minor inner core structural protein of 486 to 495 amino acids (aa) which binds zinc and contains an amino-terminal highly conserved cysteine-rich region that may form two zinc fingers. NS53 is unique among the proteins of the group A rotaviruses in that it is extremely variable among the species but is relatively conserved within the species.To study the structure-function of the gene 5 product, an RNA-capture assay was developed to access the RNA-binding function of NS53. Wild-type (wt) and mutant forms of NS53 were produced using a recombinant baculovirus expression system and a recombinant vaccinia virus/T7 RNA polymerase (vTF7-3) expression system. Analysis of the RNA-binding activity of wt 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. The target-site on viral mRNA might form a stem-loop structure. 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 highly conserved C$\sb4$-C$\sb3$H zinc fingers within this region of the protein are essential for the activity. The C$\sb4$-C$\sb3$H motif suggests that NS53 belongs to the "Ring Finger" family of zinc finger proteins, a class which binds nucleic acid. Analysis of several cysteine to serine (Cys -$>$ Ser) mutants within the C$\sb4$-C$\sb3$H motif by the RNA-capture assay have shown that both zinc fingers are crucial for the RNA-binding.Gel electrophoresis and Western immunoblot analyses of lysates 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. The double staining of cells with antibodies specific for the NS53 and for the intermediate filament, vimentin, has showed that NS53 co-localizes with vimentin.The genome of the bovine rotavirus variant, brvA, lacks a normal segment 5 but includes a novel dsRNA (gene A) of approximately 2600 basepairs (bp) that contains segment 5-specific sequences (111). To gain information about the nature of the rearrangement in gene A and its capacity to encode a protein product, we prepared and sequenced complementary (c)DNA of the gene A RNA. The results showed that gene A is 2693 bp in size and contains a head-to-tail duplication of 1112 bp that originates from the open reading frame (ORF) of gene 5. Gene A contains a point mutation at nt 808 which results in the presence of a nonsense codon near the middle of the ORF for NS53. Thus the predicted product of gene A is a truncated NS53 of 258 aa (31 kDa). Examination of lysates from brvA-infected cells by western blot assay using an NS53-specific antibody confirmed that the variant encodes only a truncated gene 5 product. Together, these data show that the RNA-binding domain and the intracellular localization domain lie upstream from the region of NS53 determined not to be essential for replication of rotaviruses in cell cultures.

Keywords

Biology, Molecular; Biology, Microbiology

Link to Full Text

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