A peptide from autoantigen La blocks poliovirus and hepatitis C virus cap-independent translation and reveals a single tyrosine critical for La RNA binding and translation stimulation.
La, a 52-kDa autoantigen in patients with systemic lupus erythematosus, was one of the first cellular proteins identified to interact with viral internal ribosome entry site (IRES) elements and stimulate poliovirus (PV) and hepatitis C virus (HCV) IRES-mediated translation. Previous results from our laboratory have shown that a small, yeast RNA (IRNA) could selectively inhibit PV and HCV IRES-mediated translation by sequestering the La protein. Here we have identified an 18-amino-acid-long sequence from the N-terminal "La motif" which is required for efficient interaction of La with IRNA and viral 5' untranslated region (5'-UTR) elements. A synthetic peptide (called LAP, for La peptide) corresponding to this sequence (amino acids 11 to 28) of La was found to efficiently inhibit viral IRES-mediated translation in vitro. The LAP efficiently enters Huh-7 cells and preferentially inhibits HCV IRES-mediated translation programmed by a bicistronic RNA in vivo. The LAP does not bind RNA directly but appears to block La binding to IRNA and PV 5'-UTR. Competition UV cross-link and translation rescue experiments suggested that LAP inhibits IRES-mediated translation by interacting with proteins rather than RNA. Mutagenesis of LAP demonstrates that single amino acid changes in a highly conserved sequence within LAP are sufficient to eliminate the translation-inhibitory activity of LAP. When one of these mutations (Y23Q) is introduced into full-length La, the mutant protein is severely defective in interacting with the PV IRES element and consequently unable to stimulate IRES-mediated translation. However, the La protein with a mutation of the next tyrosine moiety (Y24Q) could still interact with PV 5'-UTR and stimulate viral IRES-mediated translation significantly. These results underscore the importance of the La N-terminal amino acids in RNA binding and viral RNA translation. The possible role of the LAP sequence in La-RNA binding and stimulation of viral IRES-mediated translation is discussed.
Izumi
Raquel E
RE
Department of Microbiology, Immunology and Molecular Genetics, UCLA School of Medicine, University of California-Los Angeles, Los Angeles, California 90095, USA.
Das
Saumitra
S
Barat
Bhaswati
B
Raychaudhuri
Santanu
S
Dasgupta
Asim
A
D020121:5' Untranslated Regions / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001324:Autoantigens / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002457:Cell Extracts / Q000494:pharmacology; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D002474:Cell-Free System; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D006367:HeLa Cells; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D017955:Poliovirus / Q000187:drug effects* / Q000235:genetics; D014176:Protein Biosynthesis / Q000187:drug effects*; D012315:RNA Caps / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D017384:Sequence Deletion / Q000235:genetics; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D000097585:SS-B Antigen
D020121:5' Untranslated Regions; D001324:Autoantigens; D002457:Cell Extracts; D010446:Peptide Fragments; D012315:RNA Caps; D012367:RNA, Viral; D012261:Ribonucleoproteins; D014443:Tyrosine; D000097585:SS-B Antigen