Structural Investigation Of Thogoto Virus Matrix Protein And Bunyavirus Nucleoprotein

Objectives:(1) The matrix proteins (M) play multiple roles in every stages of viral life cycle in Orthomyxovirus, especially the process of assembly and uncoating. Based on structural and functional research results about influenza virus, a model of pH-dependent conformational transitions of M1 was proposed for the pH-dependent assembly/disassembly transition of matrix layer. In order to investigate the mechanism of viral assembly and uncoating in Thogoto virus (THOV), this study attempted to obtain the crystal of THOV matrix protein and detemine the crystal structure using structural study approaches. (2) The nucleoprotein (NP) of bunyavirus has the ability to bind the naked genomic RNA for protection and assembly into ribonucleoprotiens (RNPs) as the templates of replication and transcription. Several crystal structures of NP proteins from Bunyaviridae family have been reported in recent years, except for the NP in this study. To verify the molecular mechniasm of RNPs assembly, this study attempted to process the structural investigation of a NP from the Bunyaviridae family and identificate the key sites of oligomer formation and RNA binding.Methods:(1) The genes encoding THOV matrix proteins were cloned into pGEX-6p-1 expression vector; The GST fusion proteins were expressed using the E. coli strain and purificated with proper method; Matrix proteins with high concentration and high purity were used to crystallization and optimization; X-ray diffraction data of matrix protein crystals were collected and processed; The structures of matrix protein crystal were determined and analyzed. (2) The genes encoding NP proteins were cloned into pRSFDuet-1 expression vector; The HIS fusion proteins were expressed using the E. coli strain and purificated with proper method; NP proteins with high concentration and high purity were used to crystallization and optimization; X-ray diffraction data of NP crystals were collected and processed; The structures of NP were determined and analyzed. The hydrophobic surface and electrostatic potential surface of NP were characterized.Results:(1) In this study, the crystal structures of the N-terminal domain of matrix protein (MN) at both neutral and acidic pH were determined. THOV MN is structurally homologous to influenza virus M1N. Both them comprise 9 helices and could bedivided into two 4-helix bundles. THOV matrix protein and MN tends to oligomerize in vitro and exist as dimer or higher oligomers in solution. In both situations, the two molecules in an asymmetric unit could pack into a dimer. In the neutral crystal, two THOV MN monomers could form a dimer mediated by interactions between two loops. While in acidic crystal, two monomers could form a dimer mediated by interactions between four loops and two helices. These interactions were the structural basis of oligomer formation. By comparing the structures obtained in two different pH conditions, the conformational changes in monomer and dimer formation were identified, mainly caused by conformational differences of two flexible loops. In Se-MN structures, monomer structure presents transition conformation between neutral and acidic structures and dimer structure presents a neutral-likely packing model. (2) In this study, the crystal structure of NP was determined. During the purification process, the recombinant NP forms oligomers and binds host celluar RNA. The monomeric NP structure could be divided into four domains: N-arm, N-lobe, C-lobe and C-tail, that was similar to the NP from Orthobunyavirus. One trimeric structure of NP was observed by analyzing the crystal packing and formed a ring-like assembly, which was suggested as the basic unit of RNPs. The N-arm and C-tail protruding out of the protein to the left and right, respectively, carry out oligomerization. The hydrophobic regions locating in N-arm, C-tail, N-lobe and C-lobe domains were involved in oligomerization of NP. The continuous positively charged cleft in the monomeric and trimeric structure were observed by analyzing the electrostatic potential surface of NP, confirming the RNA binds sites. Compared to other NP structures, conservation and diversity of oligomer formation and RNA binding were observed within Bunyavirus.Conclusions:(1) By analyzing the THOV MN structures at neutral, acidic and transition conditions, conformational changes of monomers and dimmers were identified, mainly caused by two flexible loops, L3 and L5. This study suggested the characteristic of pH-dependent conformational transition would reflect the basis of viral capsid assembly or disassembly in virus. (2) By analyzing the monomeric and trimeric NP structures, this study identified that hydrophobic contacts are the structural basis of oligomerization and the positively charged cleft locating between the N-lobe and C-lobe is the structural basis of RNA binding. These key sites could explain the molecular mechanism of RNPs assembly.