The control and prevention of communicable disease is directly impacted by the genetic mutability of the underlying etiological agents. In the case of RNA viruses, genetic recombination may impact public health by facilitating the generation of new viral strains with altered phenotypes and by compromising the genetic stability of live attenuated vaccines. The landscape of homologous recombination within a given RNA viral genome is thought to be influenced by several factors; however, a complete understanding of the genetic determinants of recombination is lacking. Here, we utilize gene synthesis and deep sequencing to create a detailed recombination map of the poliovirus 1 coding region. We identified over 50 thousand breakpoints throughout the genome, and we show the majority of breakpoints to be concentrated in a small number of specific “hotspots,” including those associated with known or predicted RNA secondary structures. Nucleotide base composition was also found to be associated with recombination frequency, suggesting that recombination is modulated across the genome by predictable and alterable motifs. We tested the predictive utility of the nucleotide base composition association by generating an artificial hotspot in the poliovirus genome. Our results imply that modification of these motifs could be extended to whole genome re-designs for the development of recombination-deficient, genetically stable live vaccine strains.

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...

JBrowse is a web-based genome browser, allowing many sources of data to be visualized, interpreted and navigated in a coherent visual framework. JBrowse uses efficient data structures, pre-generation of image tiles and client-side rendering to provide a fast, interactive browsing experience. Many of JBrowse's design features make it well suited for visualizing high-volume data, such as aligned next-generation sequencing reads.

The Multiple Sequence Alignment (MSA) is a computational abstraction that represents a partial summary either of indel history, or of structural similarity. Taking the former view (indel history), it is possible to use formal automata theory to generalize the phylogenetic likelihood framework for finite substitution models (Dayhoff's probability matrices and Felsenstein's pruning algorithm) to arbitrary-length sequences. In this paper, we report results of a simulation-based benchmark of several methods for reconstruction of indel history. The methods tested include a relatively new algorithm for statistical marginalization of MSAs that sums over a stochastically-sampled ensemble of the most probable evolutionary histories. For mammalian evolutionary parameters on several different trees, the single most likely history sampled by our algorithm appears less biased than histories reconstructed by other MSA methods. The algorithm can also be used for alignment-free inference, where the MSA is explicitly summed out of the analysis. As an illustration of our method, we discuss reconstruction of the evolutionary histories of human protein-coding genes.