Oligomerization paths of the nucleoprotein of influenza A virus.

The influenza viruses contain a segmented, negative strand RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP) and is associated with the polymerase complex into ribonucleoprotein (RNP) particles. Despite its importance in the virus life cycle, the interactions between the NP and the genome are not well understood. Here, we studied the assembly process of NP-RNA oligomers and analyzed how the oligomeric/monomeric status of RNA-free NP affects RNA binding and oligomerization. Recombinant wild-type NP purified in low salt concentrations and a derived mutant engineered for oligomerization deficiency (R416A) were mainly monomeric in RNA-free solutions as shown by biochemical and electron microscopy techniques. NP monomer formed with RNA a fast 1/1 complex characterized by surface plasmon resonance. In a subsequent and slow process that depended on the RNA length, oligomerization of NP was mediated by RNA binding. In contrast, preparations of wild-type NP purified in high salt concentrations as well as mutant Y148A engineered for deficiency in nucleic acid binding were partly or totally oligomeric in RNA-free solutions. These trimer/tetramer NP oligomers bind directly as oligomers to RNA with a higher affinity than that of the monomers. Both oligomerization routes we characterized could be exploited by cellular or viral factors to modulate or control viral RNA encapsidation by NP.

Description of two new plasmids isolated from Francisella philomiragia strains and construction of shuttle vectors for the study of Francisella tularensis.

Francisella tularensis is the causative agent of tularemia, a zoonotic disease often transmitted to humans by infected animals. The lack of useful specific genetic tools has long hampered the study of F. tularensis subspecies. We identified and characterized two new plasmids, pF242 and pF243, isolated from Francisella philomiragia strains ATCC 25016 and ATCC 25017, respectively. Sequence analysis revealed that pF242 and pF243 are closely related to pC194 and pFNL10 plasmids, respectively. Two generations of pF242- and pF243-based shuttle vectors, harboring several antibiotic resistance markers, were developed. We used the first generation to compare transformation efficiencies in two virulent F. tularensis subspecies. We found that electroporation was more efficient than cryotransformation: almost all vectors tested were successfully introduced by electroporation into Francisella strains with a high level of efficiency. The second generation of shuttle vectors, containing a multiple cloning site and/or gfp gene downstream of Francisella groES promotor, was used for GFP production in F. tularensis. The development of new shuttle vectors offers new perspectives in the genetic manipulation of F. tularensis, helping to elucidate the mechanisms underlying its virulence.