RNA viruses exist as genetically diverse populations1. It is thought that diversity and genetic structure of viral populations determine the rapid adaptation observed in RNA viruses2 and hence their pathogenesis3. However, our understanding of the mechanisms underlying virus evolution has been limited by the inability to accurately describe the genetic structure of virus populations. Next-generation sequencing technologies generate data of sufficient depth to characterize virus populations, but are limited in their utility because most variants are present at very low frequencies and are thus indistinguishable from next-generation sequencing errors. Here we present an approach that reduces next-generation sequencing errors and allows the description of virus populations with unprecedented accuracy. Using this approach, we define the mutation rates of poliovirus and uncover the mutation landscape of the population. Furthermore, by monitoring changes in variant frequencies on serially passaged populations, we determined fitness values for thousands of mutations across the viral genome. Mapping of these fitness values onto three-dimensional structures of viral proteins offers a powerful approach for exploring structure–function relationships and potentially uncovering new functions. To our knowledge, our study provides the first single-nucleotide fitness landscape of an evolving RNA virus and establishes a general experimental platform for studying the genetic changes underlying the evolution of virus populations.

Insect viruses have evolved strategies to control the host RNAi antiviral defense mechanism. In nature Drosophila C Virus (DCV) infection causes low mortality and persistent infection, whereas the closely related Cricket Paralysis Virus (CrPV) causes a lethal infection. We show these viruses use different strategies to modulate the host RNAi defense machinery. The DCV RNAi suppressor (DCV-1A) binds to long double-stranded RNA (dsRNA) and prevents processing by Dicer2. In contrast, the CrPV suppressor (CrPV-1A) interacts with the endonuclease Ago2 and inhibits its activity, without affecting the miRNA-Ago1 mediated silencing. The link between viral RNAi suppressors and the outcome of infection was examined using recombinant Sindbis viruses encoding either CrPV-1A or DCV-1A. Flies infected with Sindbis virus expressing CrPV-1A showed a dramatic increase in virus production, spread and mortality. In contrast, Sindbis pathogenesis was only modestly increased by expression of DCV- 1A. We conclude that RNAi suppressors function as virulence factors.

A major limitation of high-throughput DNA sequencing is the high rate of erroneous base calls produced. For instance, Illumina sequencing machines produce errors at a rate of ∼0.1-1 × 10 −2 per base sequenced. These technologies typically produce billions of base calls per experiment, translating to millions of errors. We have developed a unique library preparation strategy, "circle sequencing," which allows for robust downstream computational correction of these errors. In this strategy, DNA templates are circularized, copied multiple times in tandem with a rolling circle polymerase, and then sequenced on any high-throughput sequencing machine. Each read produced is computationally processed to obtain a consensus sequence of all linked copies of the original molecule. Physically linking the copies ensures that each copy is independently derived from the original molecule and allows for efficient formation of consensus sequences. The circlesequencing protocol precedes standard library preparations and is therefore suitable for a broad range of sequencing applications. We tested our method using the Illumina MiSeq platform and obtained errors in our processed sequencing reads at a rate as low as 7.6 × 10 −6 per base sequenced, dramatically improving the error rate of Illumina sequencing and putting error on par with low-throughput, but highly accurate, Sanger sequencing. Circle sequencing also had substantially higher efficiency and lower cost than existing barcode-based schemes for correcting sequencing errors.next-generation sequencing | barcoding | rare variants

Evenly spaced nucleosomes directly correlate with condensed chromatin and gene silencing. The ATP-dependent chromatin assembly factor (ACF) forms such structures in vitro and is required for silencing in vivo. ACF generates and maintains nucleosome spacing by constantly moving a nucleosome towards the longer flanking DNA faster than the shorter flanking DNA. But how the enzyme rapidly moves back and forth between both sides of a nucleosome to accomplish bidirectional movement is unknown. We show that nucleosome movement depends cooperatively on two ACF molecules, suggesting that ACF functions as a dimer of ATPases. Further, the nucleotide state determines whether the dimer closely engages one vs. both sides of the nucleosome. Three-dimensional reconstruction by single particle electron microscopy of the ATPase-nucleosome complex in an activated ATP state reveals a dimer architecture in which the two ATPases face each other. Our results suggest a model in which the two ATPases work in a coordinated manner, taking turns to engage either side of a nucleosome, thereby allowing processive bidirectional movement. This novel dimeric motor mechanism differs from that of dimeric motors such as kinesin and dimeric helicases that processively translocate unidirectionally and reflects the unique challenges faced by motors that move nucleosomes.Chromatin-remodeling motors play essential roles in organizing the chromatin state for regulating eukaryotic genomes, yet how they carry out their myriad activities is poorly understood. Their substrate, the nucleosome, contains 147 bp of DNA wrapped in ~1.5 turns Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms around an octamer of histone proteins. Even the smallest movement of the histone octamer relative to the DNA presumably requires a coordinated process of breaking and reforming the many histone-DNA contacts. The ACF chromatin-remodeling complex exemplifies the task, as it is able to move nucleosomes to create evenly spaced nucleosomal arrays that contain equal DNA on either side of each nucleosome1-10. These evenly spaced arrays are important for packaging the underlying DNA into silent chromatin structures in vivo1-10. HHS Public AccessACF is part of the ISWI family of remodeling complexes. The ATPase subunits of ISWI complexes can move nucleosomes by themselves while the accessory subunits modulate this basic activity11-15. The human ACF complex consists of one ATPase subunit, SNF2h and one accessory subunit, Acf16,7. SNF2h is part of the SF2 family of DExx box proteins that includes helicases and nucleic acid translocases16. The ATPase domain of SNF2h has two RecA-like domains, which are thought to form a cleft within which ATP binds. SNF2h also has an alpha-helical extension comprised of three additional domains, HAND, SANT and SLIDE which are thought to play a role in bi...

Summary RNA viruses exist as dynamic and diverse populations shaped by constant mutation and selection. Yet little is known about how the mutant spectrum contributes to virus evolvability and pathogenesis. Because several codon choices are available for a given amino acid, a central question concerns whether viral sequences have evolved to optimize not only the protein coding consensus, but also the DNA/RNA sequences accessible through mutation. Here we directly test this hypothesis by comparing wild type poliovirus to synthetic viruses carrying reengineered capsid sequences with hundreds of synonymous mutations. Strikingly, such rewiring of the population's mutant network reduced its robustness and attenuated the virus in an animal model of infection. We conclude that the position of a virus in sequence space defines its mutant spectrum, evolutionary trajectory, and pathogenicity. This organizing principle for RNA virus populations confers tolerance to mutations and facilitates replication and spread within the dynamic host environment.