Abstract
In spite of decades-long studies, the mechanism of morphogenesis of plus-stranded RNA viruses belonging to the genus Enterovirus of Picornaviridae, including poliovirus (PV), is not understood. Numerous attempts to identify an RNA encapsidation signal have failed. Genetic studies, however, have implicated a role of the non-structural protein 2CATPase in the formation of poliovirus particles. Here we report a novel mechanism in which protein-protein interaction is sufficient to explain the specificity in PV encapsidation. Making use of a novel “reporter virus”, we show that a quasi-infectious chimera consisting of the capsid precursor of C-cluster coxsackie virus 20 (C-CAV20) and the nonstructural proteins of the closely related PV translated and replicated its genome with wild type kinetics, whereas encapsidation was blocked. On blind passages, encapsidation of the chimera was rescued by a single mutation either in capsid protein VP3 of CAV20 or in 2CATPase of PV. Whereas each of the single-mutation variants expressed severe proliferation phenotypes, engineering both mutations into the chimera yielded a virus encapsidating with wild type kinetics. Biochemical analyses provided strong evidence for a direct interaction between 2CATPase and VP3 of PV and CAV20. Chimeras of other C-CAVs (CAV20/CAV21 or CAV18/CAV20) were blocked in encapsidation (no virus after blind passages) but could be rescued if the capsid and 2CATPase coding regions originated from the same virus. Our novel mechanism explains the specificity of encapsidation without apparent involvement of an RNA signal by considering that (i) genome replication is known to be stringently linked to translation, (ii) morphogenesis is known to be stringently linked to genome replication, (iii) newly synthesized 2CATPase is an essential component of the replication complex, and (iv) 2CATPase has specific affinity to capsid protein(s). These conditions lead to morphogenesis at the site where newly synthesized genomes emerge from the replication complex.
Highlights
Morphogenesis is a crucial step at the end of the virus’ life cycle that provides newly synthesized genomes with a protective shell to survive in the extracellular environment yet assures attachment to and penetration into subsequent host cells
We describe our studies of the morphogenesis of a group of single, plus-stranded RNA viruses that belong to the genus Enterovirus of Picornaviridae, a family of viruses containing a large number of human and animal pathogens
A chimera, with the coxsackie A viruses (CAVs) capsid domain and the poliovirus two nonstructural domains of the polyprotein, synthesized its genome with wt kinetics yet was blocked in morphogenesis
Summary
Morphogenesis is a crucial step at the end of the virus’ life cycle that provides newly synthesized genomes with a protective shell to survive in the extracellular environment yet assures attachment to and penetration into subsequent host cells. Morphogenesis of viral genomes must be specific because encapsidation of non-progeny nucleic acid is wasteful for the virus, for which reason elaborate mechanisms have evolved to discriminate against nucleic acids other than its own genome. We describe our studies of the morphogenesis of a group of single, plus-stranded RNA viruses that belong to the genus Enterovirus of Picornaviridae, a family of viruses containing a large number of human and animal pathogens. Poliovirus (PV), the prototype enterovirus, has been extensively studied for a century and much is known about its virion structure, uptake into host cell, genome structure and macromolecular events of replication, the mechanism of particle assembly is only poorly understood [1]. The key requirement of morphogenesis, namely the specific selection of viral genomes, has remained obscure. It should be noted that this mechanism is different from the one used by some other RNA viruses such as hepatitis B virus and alphaviruses [2,3]
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