MHV Leader RNA Secondary Structure Affects Binding to the Nucleocapsid Protein
Leader RNA is found at the 5′ end of the mouse hepatitis virus (MHV) genomic RNA, at the 5′ end of the seven viral mRNAs, and free in the cell. Leader RNA is synthesized by a transcriptional activity separate from the activities that synthesize both (-) sense genomic length RNA and the virus mRNAs1. It is believed to function as a primer, binding to complementary intergenic sites, on (-) sense template, situated 5′ of each of the initiation sites for viral mRNAs2. Using monoclonal antibodies specific for the nucleocapsid (N) protein, immunoprecipitations of RNA/protein complexes from infected cells indicate that the N protein is complexed to: 1) genomic RNA; Z) viral mRNAs; and 3) even free leader containing RNA fragments as small as 60 nucleotides in length3. Northwestern blot analysis showed that the viral N protein exhibits RNA binding activity that is specific for viral leader containing RNA when expressed in the (+) sense4,5. However, this system has several limitations. First, in denaturing conditions, RNA/protein interactions which require the interaction of multiple protein subunits cannot be studied, and second, it is not possible to quantify relative affinities and binding characteristics.
KeywordsLeader Sequence Viral mRNAs Mouse Hepatitis Virus Infected Cell Lysate Nonspecific Competitor
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