ABSTRACT
Influenza A virus (IAV) infection leads to variable and imperfectly understood pathogenicity. We report that segment 3 of the virus contains a second open reading frame ("X-ORF"), accessed via ribosomal frameshifting. The frameshift product, termed PA-X, comprises the endonuclease domain of the viral PA protein with a C-terminal domain encoded by the X-ORF and functions to repress cellular gene expression. PA-X also modulates IAV virulence in a mouse infection model, acting to decrease pathogenicity. Loss of PA-X expression leads to changes in the kinetics of the global host response, which notably includes increases in inflammatory, apoptotic, and T lymphocyte-signaling pathways. Thus, we have identified a previously unknown IAV protein that modulates the host response to infection, a finding with important implications for understanding IAV pathogenesis.
Subject(s)
Frameshifting, Ribosomal , Influenza A Virus, H1N1 Subtype/genetics , Influenza A virus/genetics , Open Reading Frames , Orthomyxoviridae Infections/virology , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Codon , Conserved Sequence , Female , Gene Expression Regulation , Genome, Viral , HEK293 Cells , Humans , Influenza A Virus, H1N1 Subtype/growth & development , Influenza A Virus, H1N1 Subtype/pathogenicity , Influenza A virus/metabolism , Lung/pathology , Lung/virology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Mutation , Orthomyxoviridae Infections/genetics , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/pathology , Protein Interaction Domains and Motifs , Proteome , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/chemistry , Reassortant Viruses/genetics , Repressor Proteins/chemistry , Viral Nonstructural Proteins/chemistry , Viral Proteins/biosynthesis , Viral Proteins/chemistry , Virus ReplicationABSTRACT
In previous studies, a lutropin receptor mRNA binding protein implicated in the hormonal regulation of lutropin receptor mRNA stability was identified. This protein, termed LRBP-1, was shown by RNA gel electrophoretic mobility shift assay to specifically interact with lutropin receptor RNA sequences. The present studies have examined the specificity of lutropin receptor mRNA recognition by LRBP-1 and mapped the contact site by RNA footprinting and by site-directed mutagenesis. LRBP-1 was partially purified by cation-exchange chromatography, and the mRNA binding properties of the partially purified LRBP-1 were examined by RNA gel electrophoretic mobility shift assay and hydroxyl-radical RNA footprinting. These data showed that the LRBP-1 binding site is located between nucleotides 203 and 220 of the receptor open reading frame, and consists of the bipartite polypyrimidine sequence 5'-UCUC-X(7)-UCUCCCU-3'. Competition RNA gel electrophoretic mobility shift assays demonstrated that homoribopolymers of poly(rC) were effective RNA binding competitors, while poly(rA), poly(rG), and poly(rU) showed no effect. Mutagenesis of the cytidine residues contained within the LRBP-1 binding site demonstrated that all the cytidines in the bipartite sequence contribute to LRBP-1 binding specificity. Additionally, RNA gel electrophoretic mobility supershift analysis showed that LRBP-1 was not recognized by antibodies against two well-characterized poly(rC) RNA binding proteins, alphaCP-1 and alphaCP-2, implicated in the regulation of RNA stability of alpha-globin and tyrosine hydroxylase mRNAs. In summary, we show that partially purified LRBP-1 binds to a polypyrimidine sequence within nucleotides 203 and 220 of lutropin receptor mRNA with a high degree of specificity which is indicative of its role in posttranscriptional control of lutropin receptor expression.
Subject(s)
Cytidine/metabolism , Open Reading Frames , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Receptors, LH/genetics , Receptors, LH/metabolism , Ribonucleoproteins/metabolism , 3' Untranslated Regions/analysis , Animals , Base Sequence , Binding Sites/genetics , Chromatography, Ion Exchange , DNA Mutational Analysis , Electrophoresis, Polyacrylamide Gel , Female , Humans , Hydroxyl Radical/metabolism , RNA/analysis , RNA-Binding Proteins/genetics , RNA-Binding Proteins/isolation & purification , Rats , Rats, Sprague-Dawley , Ribonucleoproteins/genetics , Ribonucleoproteins/isolation & purificationABSTRACT
To elucidate the molecular events associated with the regulation of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor mRNA stability during hCG-induced receptor down-regulation, we have identified an LH/hCG receptor-specific mRNA binding protein. Proteins were isolated from control and down-regulated rat ovary and were incubated with in vitro transcribed RNAs corresponding to the full-length LH/hCG receptor, as well as 5'- and 3'-truncated receptor forms. Resultant ribonucleoprotein complexes were analyzed by RNA gel mobility shift. A prominent Mr 50,000 ribonucleoprotein complex was identified with the following characteristics: 1) specificity for LH/hCG receptor open reading frame sequences located between nucleotides 102 and 282; 2) lack of competition by nonspecific RNAs; 3) a 3-fold increase in RNA binding activity during hCG-induced receptor down-regulation; and 4) limited tissue expression. This report describes the first evidence of an LH/hCG receptor mRNA binding protein, which we term LRBP-1, for luteinizing hormone receptor RNA binding protein-1. This protein is a candidate for a trans-acting factor involved in the hormonal regulation of LH/hCG receptor mRNA stability in rat ovary.