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. IMPORTANCEBy 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.DOI: http://dx.doi.org/10.7554/eLife.03753.001
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 .T he genomes of RNA viruses, such as poliovirus (PV), often contain complex RNA secondary and tertiary structures. These structures are crucial for translation and replication of the viral genome and may play additional roles in other processes, such as genome packaging and modulation of the host antiviral response.Poliovirus, the prototypical picornavirus and causative agent of poliomyelitis, is a nonenveloped virus with a single-stranded RNA genome of positive polarity. The virion consists of an icosahedral protein shell, composed of four capsid proteins (VP1, VP2, VP3, and VP4), which encapsidates the RNA genome (1). Poliovirus has a rapid replication cycle, with approximately 8 h elapsing between infection and release of progeny virions upon host cell lysis. During an infection, high yields of both viral proteins and genomes are produced. These yields ensure synthesis of up to 10,000 virions per cell (2), which can be widely disseminated to neighboring cells and/or new hosts. The compact nature of the viral genome (less than 7,500 nucleotides [nt] long) facilitates this rapid exponential growth. The viral genome acts as an mRNA and can be divided into a highly conserved 742-nt 5= untranslated region (5= UTR), a single long open reading frame encoding the viral polyprotein, a 68-nt 3= untranslated region (3= UTR), and a polyadenosine tract of a variable length (see Fig. 2A). A small viral protein of 22 amino acids, VPg (3B), is covalently attached to the 5= end of the RNA. The 5= UTR contains a structure critical for viral translation (the internal ribosome entry site or IRES) (3, 4), as well the 5=-cloverleaf (5=-CL) structure, which i...
The flavonoid pigment pathway in plants has been used as a model system for studying gene regulatory mechanisms. C2-Idf is a stable dominant mutation of the chalcone synthase gene, c2, which encodes the first dedicated enzyme in this biosynthetic pathway of maize. Homozygous C2-Idf plants show no pigmentation. This allele also inhibits expression of functional C2 alleles in heterozygotes, producing a less pigmented condition instead of the normal deeply pigmented phenotype. To explore the nature of this effect, the C2-Idf allele was cloned. The gene structure of the C2-Idf haplotype differs substantially from that of the normal c2 gene in that three copies are present. Two of these are located in close proximity to each other in a head-to-head orientation and the third is closely linked. Previous experiments showed that the lower level of pigmentation in heterozygotes is correlated with reduced enzyme activity and low steady-state mRNA levels. We found that c2 transcription occurs in nuclei of C2-Idf/C2 heterozygotes, but mRNA does not accumulate, suggesting that the inhibition is mediated by RNA silencing. Infection of C2-Idf/C2 heterozygotes with viruses that carry suppressors of RNA silencing relieved the phenotypic inhibition, restoring pigment production and mRNA levels. Finally, we detected small interfering RNAs (siRNAs) in plants carrying C2-Idf, but not in plants homozygous for the wild-type C2 allele. Together, our results indicate that the inhibitory effect of C2-Idf occurs through RNA silencing.
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. Curr. Protoc. Microbiol. 29:15H.2.1‐15H.2.32. © 2013 by John Wiley & Sons, Inc.
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