Unlabelled

Viral infections often begin with a very small number of initiating particles. Accordingly, the outcome of an infection is likely to be affected by variability in the initial molecular interactions between virus and host. In this study, we investigated the range of outcomes upon infection of single cells. We isolated individual cells infected with poliovirus at low or high multiplicities of infection (MOI) and measured viral genomic replication and infectious viral progeny in each cell. We first determined that at 7 h postinfection, the ratio of positive to negative strands in individual cells varies from 5:1 to more than 190:1, with and average of 20:1, suggesting a significant variability in RNA synthesis. We further found that while virus genome production is higher in cells infected at a high multiplicity, the production of infectious particles is largely independent of the number of viruses infecting each cell. Strikingly, by correlating RNA and particle production within individual infections, we uncovered a significant contribution of stochastic noise to the outcome of infection. At low MOI, stochastic influences appear as kinetic effects which are most critical at the initial steps in infection. At high MOI, stochastic influences appear to dictate the virus's ability to harness cellular resources. We conclude that biological noise is a critical determinant of the overall productivity of viral infections. The distinct nature of stochasticity in the outcome of infection by low and high numbers of viral particles may have important implications for our understanding of the determinants of successful viral infections.

Importance

By correlating genome and particle production in single-cell infections, we elucidated sources of noise in viral infections. When a cell was infected by only a single infectious particle, variation in the kinetics of the initial steps of replication contributed significantly to the overall productivity of the infection. Additionally, variation in the distribution of subcellular resources impacted infections initiated by one or many infectious particles. We also observed that when a cell was infected with multiple particles, more genomes were produced, while particle production was hindered by an apparent cellular resource limit. Understanding variations in viral infections may illuminate the dynamics of infection and pathogenesis and has implications for virus adaptation and evolution.

Life history theory posits that the sequence and timing of events in an organism's lifespan are fine-tuned by evolution to maximize the production of viable offspring. In a virus, a life history strategy is largely manifested in its replication mode. Here, we develop a stochastic mathematical model to infer the replication mode shaping the structure and mutation distribution of a poliovirus population in an intact single infected cell. We measure production of RNA and poliovirus particles through the infection cycle, and use these data to infer the parameters of our model. We find that on average the viral progeny produced from each cell are approximately five generations removed from the infecting virus. Multiple generations within a single cell infection provide opportunities for significant accumulation of mutations per viral genome and for intracellular selection.

Poliovirus (PV) is the prototypical picornavirus. It is a non-enveloped RNA virus with a small (~7.5-kb) genome of positive polarity. cDNA clones of several strains are available, and infectious virus can be produced by the transfection of in vitro-transcribed viral genomes into an appropriate host cell. The ease of genetic studies in poliovirus is a primary reason that it has long served as a model to study RNA virus biology, pathogenesis, and evolution. Protocols for the generation and characterization of PV mutants are presented.

The genomes of RNA viruses often contain RNA structures that are crucial for translation and RNA replication and may play additional, uncharacterized roles during the viral replication cycle. For the picornavirus family member poliovirus, a number of functional RNA structures have been identified, but much of its genome, especially the open reading frame, has remained uncharacterized. We have now generated a global RNA structure map of the poliovirus genome using a chemical probing approach that interrogates RNA structure with single-nucleotide resolution. In combination with orthogonal evolutionary analyses, we uncover several conserved RNA structures in the open reading frame of the viral genome. To validate the ability of our global analyses to identify functionally important RNA structures, we further characterized one of the newly identified structures, located in the region encoding the RNA-dependent RNA polymerase, 3D(pol), by site-directed mutagenesis. Our results reveal that the structure is required for viral replication and infectivity, since synonymous mutants are defective in these processes. Furthermore, these defects can be partially suppressed by mutations in the viral protein 3C(pro), which suggests the existence of a novel functional interaction between an RNA structure in the 3D(pol)-coding region and the viral protein(s) 3C(pro) and/or its precursor 3CD(pro).