Investigating the specificity and stoichiometry of RNA binding by the nucleocapsid protein of Bunyamwera virus.
Bunyamwera virus (BUNV) is the prototypic member of both the Orthobunyavirus genus and the Bunyaviridae family of negative stranded RNA viruses. In common with all negative stranded RNA viruses, the BUNV genomic and anti-genomic strands are not naked RNAs, but instead are encapsidated along their entire lengths with the virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex. This association is critical for the negative strand RNA virus life cycle because only RNPs are active for productive RNA synthesis and RNA packaging. We are interested in understanding the molecular details of how N and RNA components associate within the bunyavirus RNP, and what governs the apparently selective encapsidation of viral replication products. Toward this goal, we recently devised a protocol that allowed generation of native BUNV N protein that maintained solubility under physiological conditions and allowed formation of crystals that yielded high-resolution x-ray diffraction data. Here we extend this work to show that this soluble N protein is able to oligomerize and bind RNA to form a highly uniform RNP complex, which exhibits characteristics in common with the viral RNP. By extracting and sequencing RNAs bound to these model RNPs, we determined the stoichiometry of N-RNA association to be approximately 12 nucleotides per N monomer. In addition, we defined the minimal sequence requirement for BUNV RNA replication. By comparing this minimal sequence to those bound to our model RNP, we conclude that N protein does not obligatorily require a sequence or structure for RNA encapsidation.
Mohl
Bjorn-Patrick
BP
Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS29JT, West Yorkshire, United Kingdom.
Barr
John N
JN
D002042:Bunyamwera virus / Q000378:metabolism*; D002850:Chromatography, Gel; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D014461:Ultracentrifugation; D014961:X-Ray Diffraction
D019590:Nucleocapsid Proteins; D012367:RNA, Viral