Mechanism of parainfluenza virus genome replication

SUMMARY The non-segmented negative strand RNA viruses (nsNSVs) include pathogens that infect humans, mammals, birds, reptiles, fish, insects, and plants. The nsNSVs encompasses 11 virus families, including the paramyxoviruses (e.g., measles, mumps and the parainfluenza viruses), filoviruses (e.g., Ebola virus), pneumoviruses (e.g., respiratory syncytial virus and human metapneumovirus), and rhabdoviruses (e.g., rabies virus). The nsNSV polymerase complex, which is comprised of the large polymerase subunit (L) and the phosphoprotein (P), is the major target of antiviral development for these pathogens. The L-P complex performs the key processes of transcription, to produce viral mRNAs, and RNA replication, to produce antigenomes and new genomes. The template for both processes is the viral genome encapsidated by the nucleocapsid (N) protein. Proper balance of transcription and replication is key to viral infection, but mechanisms by which these processes are regulated are poorly understood. Recent work by the Fearns group suggests a novel model for transcription-replication regulation. We found that the L-P polymerase of human parainfluenza virus type 3 (HPIV-3) can adopt a dimeric form, and that dimer formation is required for RNA replication. In Aim 1 of this project, this exciting discovery will serve as the foundation to direct structure-guided analysis of HPIV-3 L, utilizing the wealth of assays and expertise at our disposal. These studies will identify important molecular features of the polymerase. We will test the novel hypothesis that monomeric L-P complexes perform transcription, whereas dimeric L-P complexes perform RNA replication by coupling RNA synthesis and encapsidation. In Aim 2, we will dissect how viral inclusion bodies influence L dimerization and the balance between transcription and replication. We hypothesize that accumulation of N protein triggers inclusion body formation, which in turn promotes L dimerization and RNA replication. We will determine if RNA replication occurs exclusively within inclusion bodies and whether disruption of inclusion bodies downregulates RNA replication but not transcription. In addition, we will determine the effect of inclusion body formation on L dimerization and analyze polymerase complexes at the molecular level. The combined expertise of the Fearns, Dutch and Bullitt groups will be utilized for this work, and innovative approaches including AmpFISH, for highly specific detection of RNA species in cells, and electron tomography to characterize polymerase complexes within inclusions and in the cytoplasm will be employed. These exciting studies will provide novel insight into the mechanisms of HPIV-3 transcription-replication regulation that can likely be applied across the nsNSVs. This information could aid antiviral drug development and our understanding of nsNSV replication and pathogenesis.