Investigating the specificity and stoichiometry of RNA binding by the nucleocapsid protein of Bunyamwera virus

Abstract

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 ;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.

Structure of the Leanyer orthobunyavirus nucleoprotein–RNA complex reveals unique architecture for RNA encapsidation

“…Each protomer contains residues 6-232 and one tetramer of N encapsidates 44 nt of ssRNA. Each protomer contains 11 nt of ssRNA; this number is one nucleotide less than the number previously reported for BUNV N proteins based on biochemical studies (23,24). Incidentally, the RNA extracted from the bacterially expressed tetrameric LEAV NssRNA complex exhibited a 40-to 50-nt size during electrophoresis, which is consistent with previous reports (23,24) (Fig.…”

“…S2D), suggesting that the oligomeric state of the LEAV N could be maintained in the absence of RNA. Such a feature is also exhibited by BUNV N (23,24) and other negative-strand RNA viruses, such as VSV (25). RNA-free N could bind ssRNA (K d = 0.166 μM) or ssDNA (K d = 0.505 μM) with high affinity, as suggested by isothermal titration calorimetry (ITC) assays ( Fig.…”

Expression, purification and crystallization of the Crimean–Congo haemorrhagic fever virus nucleocapsid protein

“…4) or = 60 (data not shown). Tetrameric or hexameric complexes similar to those seen for other bunyaviruses do not appear to be present, unless a combination of asymmetric units results in, for instance, a tetramer (Mohl & Barr, 2009) Diffraction image of a CCHFV N crystal. The oscillation was 0.2 and reflections were observed to beyond 2.1 Å .…”

Crimean–Congo hemorrhagic fever virus nucleoprotein reveals endonuclease activity in bunyaviruses

“…The binding affinities of cap analogs of wildtype CCHFV NP, i.e., m 7 G, m 7 Gpp, and m 7 Gppp, were measured using isothermal titration calorimetry (ITC). A 0.1-mM CCHFV NP protein solution was titrated against 0.3 mM cap-analog solutions (Sigma-Aldrich) using a VP-ITC microcalorimeter (MicroCal).…”

“…The bunyavirus genomes consist of small (S), medium (M), and large (L) RNA segments, which encode a viral nucleocapsid protein (NP), glycoprotein precursor, and polymerase proteins, respectively (3). According to previous studies on the function of nucleoproteins in bunyaviruses, some of these NPs may be able to recognize specific viral RNA sequences (4-6), but mostly bind to single-stranded RNA (ssRNA) in a nonspecific way (7,8). Because the Bunyaviridae family includes hundreds of different genuses and the nucleoproteins of each genus show little homology or other features in common, the addressing of the exact function and mechanism of each group of nucleoproteins case by case is necessary for our understanding on bunyaviruses replication and assembly.…”