FLU VIRUS RNP COMPLEX

Baylor College Of Medicine, Houston TX

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The crystal structure of influenza A virus NP has recently been determined in our laboratory. Organized as trimers in the crystal, NP folds into a two-domain structure with a topology completely different from that of the rhabdovirus NP. A short tail loop, consisting of residues 402 to 428, is likely to play an important role in NP oligomerization, as single-residue mutation in this region causes a total loss of oligomerization. A large positively charged groove was identified at the exterior of the NP trimer at the interface between the two domains. An external RNA binding site indicates that RNA is likely to be exposed in the influenza virus RNPs, different from the situation in non-segmented RNA viruses. This structural difference explains previous results that showed fundamental differences between influenza RNPs and the RNPs of non-segmented (-)-strand RNA viruses like VSV and rabies virus. For example, the viral RNA in influenza virus RNPs is digested by RNase treatment, whereas the viral RNA in the RNPs of parainfluenza viruses and rhabdoviruses is completely resistant to RNase digestion. In addition, polyvinylsufate (PVS), a negatively charged polymer, is able to completely displace RNA from influenza virus RNP, whereas it has no effect on RNPs from VSV. To provide a definite evidence for our external RNA binding model and to elucidate RNP structure and assembly, we propose to determine the structure of the NP:RNA complex. Unfortunately we have not been able to obtain any crystals of NP-RNA complex even after extensive trials, and that leaves cryo-EM reconstruction as our next choice. The size of the NP trimer is ~220kD with a three-fold rotation symmetry. By comparing structural differences of NP and NP:RNA complexes, we will be able to determine not only the RNA binding site, but also the conformational changes induced by binding of RNA. Therefore, this study will provide critical information for the structural organization of RNP and its function during virus infection.