Abstract
Human metapneumovirus (HMPV) is a major cause of respiratory illness in young children. The polymerase complex of HMPV consists of two obligate components, the L polymerase and its cofactor, the phosphoprotein P. During replication and transcription, the L/P complex traverses the viral RNA genome, which is encapsidated within multimerized N nucleoproteins. An essential interaction between N and a C-terminal region of P is required for tethering of the L/P polymerase to the RNA template. This N-P interaction is also involved in the formation of cytoplasmic viral factories in infected cells, called inclusion bodies. To define how L/P recognizes N-encapsidated RNA (N-RNA) we employed cryogenic electron microscopy (cryo-EM) and molecular dynamics simulations, coupled to polymerase activity assays and imaging of inclusion bodies in transfected cells. We report a 2.9 Å resolution structure of a triplecomplex between multimeric N, bound to both RNA and the C-terminal region of P. Furthermore, we also present cryo-EM structures of assembled N in different oligomeric states, highlighting the plasticity of N. Combined with our functional assays, these structural data delineate in molecular detail how P attaches to N-RNA whilst retaining substantial conformational dynamics. Moreover, the N-RNA-P triple complex structure provides a molecular blueprint for the design of therapeutics to potentially disrupt the attachment of L/P to its template.