Role of V protein RNA binding in inhibition of measles virus minigenome replication.
Measles virus V protein represses genome replication through a poorly understood mechanism, which led us to investigate whether V protein might be an RNA-binding modulatory factor. Recombinant V protein, expressed from transfected HEp-2 cells or E. coli, formed protein-RNA complexes with poly-guanosine (poly-G) or poly-U linked to agarose beads. RNA binding was not exclusive to ribonucleotide homopolymers as complex formation between V protein and an RNA molecule equivalent to the 3' terminal 107 bases of the measles virus genome was observed with an electrophoretic mobility shift assay (EMSA). The interaction with poly-G was used to further examine the RNA binding properties of V demonstrating that protein-RNA complex formation was dependent upon the unique Cys-rich carboxy terminus, a region also required to induce maximal repression of minireplicon-encoded reporter gene expression in transient assays. Surprisingly, two mutant proteins that contained Cys-to-Ala substitutions in the C-terminus were found to retain their ability to bind poly-G binding and repress minireplicon reporter gene expression indicating that neither activity was dependent on the integrity of all 7 C-terminal Cys residues. Additional genetic analysis revealed that amino acids 238-266 were necessary for efficient RNA binding and overlapped with residues (238-278) required for maximal repression induced by the C-terminal domain. In addition, a 10 amino acid deletion was identified (residues 238-247) that blocked RNA binding and repression indicating that these two activities were related.
Parks
Christopher L
CL
Wyeth Vaccines Research, 401 North Middletown Road, Pearl River, NY 10965, USA. parksc@wyeth.com
Witko
Susan E
SE
Kotash
Cheryl
C
Lin
Shuo L
SL
Sidhu
Mohinder S
MS
Udem
Stephen A
SA
D019943:Amino Acid Substitution; D019677:Artificial Gene Fusion; D002460:Cell Line; D003545:Cysteine / Q000235:genetics / Q000502:physiology; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D008459:Measles virus / Q000502:physiology*; D020125:Mutation, Missense; D010750:Phosphoproteins / Q000235:genetics / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D012313:RNA / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology*; D014779:Virus Replication
D010750:Phosphoproteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C069181:V protein, measles virus; D014764:Viral Proteins; D012313:RNA; D008156:Luciferases; D003545:Cysteine