Viral precursor protein P3 and its processed products perform discrete and essential functions in the PV RNA replication complex
Poliovirus (PV) is one of the best characterized RNA viruses known, yet a host of questions remain regarding the molecular mechanisms that drive viral replication. In the search for new antiviral drug targets, the characterization of the protein-protein and protein-RNA interactions central to viral replication is critical. Our results showed that viral precursor proteins and their processed products play distinct and essential roles in the assembly of the RNA replication complex. Importantly, we showed that viral protein, P3, is the preferred precursor that binds the 5’ cloverleaf (5’ CL) in the viral genome to form a functional 5’ CL-RNP complex. In addition, we discovered that both the viral polymerase (3Dpol) and VPg protein primer are recruited to this complex in the form of their precursors, which are then processed to release active 3Dpol and VPg.
Work performed by Lucia Eisner Smerage during her tenure as a graduate student in the Flanegan laboratory laid the foundation for this study. She showed that a poliovirus polymerase mutant could not be rescued by providing wild-type polymerase (3Dpol) in genetic complementation assays. Surprisingly, she found that the immediate precursor to the polymerase, 3CD, could efficiently restore replication even though 3CD itself is inactive as a polymerase. This observation led a new student, Allyn Spear, to ask the simple question: Why? What properties did 3CD possess as a precursor that made it more suitable to do the task?
Viruses, particularly ones with small RNA genomes like poliovirus, must make efficient use of their limited coding capacity. Known functions of 3CD and its processed products included: protease activity, polymerase activity, RNA binding activity, multimerization, etc…… Where to start? Thankfully, we were able to take advantage of a wide-range of previously characterized poliovirus mutations and our expertise in using cell-free replication reactions to conduct genetic complementation assays.
After testing several mutations, it became clear that the viral protein composition of the replication complex was more “complex” than originally thought. The 3CD story quickly became a P3 story when we found that the larger precursor, P3, was actually preferred for certain functions. P3 bound with high affinity to the 5’ CL, a key RNA element in the genome that serves as a platform for replication complex assembly. Our data suggests that at least two independent molecules of P3 are recruited to the replication complex via a network of protein-protein and protein-RNA interactions, and that each molecule has a discrete function and a unique destiny within the replication complex. These findings support a new model (see figure) in which the first molecule of P3 binds to the 5’ CL to form the 5’ CL-RNP complex. The bound P3 does not serve as the precursor for either 3Dpol or VPg. Instead, these functions are provided by a second molecule of P3 which is recruited to the complex via protein-protein interactions with the P3 bound to the 5’ CL. It is proposed that the bound P3 provides the protease activity required to cleave the second molecule of P3. This allows for the release of active 3Dpol and VPg at the optimal time and location in the replication complex and provides an efficient mechanism to replicate the viral genome.
Model showing the role of P3 precursor protein in the multi-step process of poliovirus replication complex assembly and the initiation of RNA synthesis. The 5’ CL-RNP complex containing PCBP and P3 serves as a platform to recruit additional molecules of P3 to the complex via protein-protein interactions. Pathway on the right: A second molecule of P3 is recruited to the 5’CL-RNP complex and is then cleaved to release active 3D polymerase. Pathway on the left: A second molecule of P3 is recruited to the 5’CL-RNP complex and is cleaved to release VPg. The 3D polymerase is then used to uridylylate VPg and initiate VPgpUpU-primed RNA synthesis.
About the authors
Pictured from left to right are Allyn Spear, Sushma A. Ogram, B. Joan Morasco, Lucia Eisner Smerage and James B. Flanegan. All of the work was carried out in the Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida.