Interaction of Chandipura Virus N and P Proteins: Identification of Two Mutually Exclusive Domains of N Involved in Interaction with P

The nucleocapsid protein (N) and the phosphoprotein (P) of nonsegmented negative-strand (NNS) RNA viruses interact with each other to accomplish two crucial events necessary for the viral replication cycle. First, the P protein binds to the aggregation prone nascent N molecules maintaining them in a soluble monomeric (N0) form (N0-P complex). It is this form that is competent for specific encapsidation of the viral genome. Second, the P protein binds to oligomeric N in the nucleoprotein complex (N-RNA-P complex), and thereby facilitates the recruitment of the viral polymerase (L) onto its template. All previous attempts to study these complexes relied on co-expression of the two proteins in diverse systems. In this study, we have characterised these different modes of N-P interaction in detail and for the first time have been able to reconstitute these complexes individually in vitro in the chandipura virus (CHPV), a human pathogenic NNS RNA virus. Using a battery of truncated mutants of the N protein, we have been able to identify two mutually exclusive domains of N involved in differential interaction with the P protein. An unique N-terminal binding site, comprising of amino acids (aa) 1–180 form the N0-P interacting region, whereas, C-terminal residues spanning aa 320–390 is instrumental in N-RNA-P interactions. Significantly, the ex-vivo data also supports these observations. Based on these results, we suggest that the P protein acts as N-specific chaperone and thereby partially masking the N-N self-association region, which leads to the specific recognition of viral genome RNA by N0

Exploration of nucleoprotein α-MoRE and XD interactions of Nipah and Hendra viruses

International audienceHenipavirus, including Hendra virus (HeV) and Nipah virus (NiV), is a newly discovered human pathogen genus. The nucleoprotein of Henipavirus contains an α-helical molecular recognition element (α-MoRE) that folds upon binding to the X domain (XD) of the phosphoprotein (P). In order to explore the conformational dynamics of free α-MoREs and the underlying binding-folding mechanism with XD, atomic force field-based and hybrid structure-based MD simulations were carried out. In our empirical force field-based simulations, characteristic structures and helicities of α-MoREs reveal the co-existence of partially structured and disordered conformations, as in the case of the well characterized cognate measles virus (MeV) α-MoRE. In spite of their overall similarity, the two α-MoREs display subtle helicity differences in their C-terminal region, but much different from that of MeV. For the α-MoRE/XD complexes, the results of our hybrid structure-based simulations provide the coupled binding-folding landscapes, and unveil a wide conformational selection mechanism at early binding stages, followed by a final induce-fit mechanism selection process. However, the HeV and NiV complexes have a lower binding barrier compared to that of MeV. Moreover, the HeV α-MoRE/XD complex shows much less coupling effects between binding and folding compared to that from both NiV and MeV. Our analysis revealed that contrary to NiV and MeV, the N- and C-terminal regions of the HeV α-MoRE maintains a low helicity also in the bound form