Production of Pseudoinfectious Yellow Fever Virus with a Two-Component Genome

Abstract

Application of genetically modified, deficient-in-replication flaviviruses that are incapable of developing productive, spreading infection is a promising means of designing safe and effective vaccines. Here we describe a two-component genome yellow fever virus (YFV) replication system in which each of the genomes encodes complete sets of nonstructural proteins that form the replication complex but expresses either only capsid or prM/E instead of the entire structural polyprotein. Upon delivery to the same cell, these genomes produce together all of the viral structural proteins, and cells release a combination of virions with both types of genomes packaged into separate particles. In tissue culture, this modified YFV can be further passaged at an escalating scale by using a high multiplicity of infection (MOI). However, at a low MOI, only one of the genomes is delivered into the cells, and infection cannot spread. The replicating prM/E-encoding genome produces extracellular E protein in the form of secreted subviral particles that are known to be an effective immunogen. The presented strategy of developing viruses defective in replication might be applied to other flaviviruses, and these two-component genome viruses can be useful for diagnostic or vaccine applications, including the delivery and expression of heterologous genes. In addition, the achieved separation of the capsid-coding sequence and the cyclization signal in the YFV genome provides a new means for studying the mechanism of the flavivirus packaging process.The Flavivirus genus of the family Flaviviridae contains a variety of important human and animal pathogens. In nature, flaviviruses circulate between vertebrate hosts and arthropod vectors mainly represented by a large number of mosquito and tick species. Almost 40 members of this genus, classified into four distinct antigenic complexes, are capable of causing human disease.The flavivirus genome is a single-stranded RNA of positive polarity of almost 12 kb. It encodes a single polypeptide that is co-and posttranslationally processed by cellular and viral proteases into the viral structural proteins C, prM/M, and E that form infectious viral particles and the nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 that form the enzyme complex required for replication of the viral genome (27). The flavivirus genome mimics the structure of cellular messenger RNAs by having a 5Ј methylguanylate cap but differs from the cellular RNA templates due to the absence of a 3Ј-terminal poly(A) sequence.In flavivirus virions, a single copy of viral genomic RNA is packaged by the C (capsid) protein into a nucleocapsid surrounded by a lipid envelope with embedded dimers of E and M proteins (23). The mechanism of interaction between the nucleocapsid and the envelope is not completely understood yet, but it appears to be less specific than, for instance, the alphavirus nucleocapsid-envelope interaction, and the flavivirus virions can be efficiently formed by capsid and envelope proteins derived from the...

Construction of yellow fever virus subgenomic replicons by yeast-based homologous recombination cloning technique

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Inhibition of Dengue Virus through Suppression of Host Pyrimidine Biosynthesis

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Identification of Mutated Cyclization Sequences That Permit Efficient Replication of West Nile Virus Genomes: Use in Safer Propagation of a Novel Vaccine Candidate

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