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Computer design and chemical synthesis generated viable variants of poliovirus type 1 (PV1), whose ORF (6,189 nucleotides) carried up to 1,297 "Max" mutations (excess of overrepresented synonymous codon pairs) or up to 2,104 "SD" mutations (randomly scrambled synonymous codons). "Min" variants (excess of underrepresented synonymous codon pairs) are nonviable except for P2Min, a variant temperature-sensitive at 33 and 39.5 °C. Compared with WT PV1, P2Min displayed a vastly reduced specific infectivity (si) (WT, 1 PFU/118 particles vs. P2Min, 1 PFU/35,000 particles), a phenotype that will be discussed broadly. Si of haploid PV presents cellular infectivity of a single genotype. We performed a comprehensive analysis of sequence and structures of the PV genome to determine if evolutionary conserved cis-acting packaging signal(s) were preserved after recoding. We showed that conserved synonymous sites and/or local secondary structures that might play a role in determining packaging specificity do not survive codon pair recoding. This makes it unlikely that numerous "cryptic, sequence-degenerate, dispersed RNA packaging signals mapping along the entire viral genome" [Patel N, et al. (2017) Nat Microbiol 2:17098] play the critical role in poliovirus packaging specificity. Considering all available evidence, we propose a two-step assembly strategy for +ssRNA viruses: step I, acquisition of packaging specificity, either (a) by specific recognition between capsid protein(s) and replication proteins (poliovirus), or (b) by the high affinity interaction of a single RNA packaging signal (PS) with capsid protein(s) (most +ssRNA viruses so far studied); step II, cocondensation of genome/capsid precursors in which an array of hairpin structures plays a role in virion formation. [ABSTRACT FROM AUTHOR]

We have developed algorithms that measure the relative historical significance of the over 800,000 people appearing in Wikipedia, producing significance rankings that correlate well with a wide variety of validation criteria. In our bookWho's bigger?: where historical figures really rank(Skiena and Ward 2013), we use our significance scores to provide insight into a variety of historical and cultural questions. Here we apply our significance measure to study several issues related to the history and stature of computing. Who are the most historically significant computer scientists? The most significant Turing award winners? The Turing award purports to be the ‘Nobel Prize of Computing’, but does the stature of these awards really compare? Our answers to all these questions prove revealing. To lend credence to our findings, we describe our ranking methodology and validation criteria. Our complete rankings for all historical figures in Wikipedia are available for inspection athttp://www.whoisbigger.com. [ABSTRACT FROM PUBLISHER]

Genomes of RNA viruses contain multiple functional RNA elements required for translation or RNA replication. We use unique approaches to identify functional RNA elements in the coding sequence of poliovirus (PV), a plus strand RNA virus. The general method is to recode large segments of the genome using synonymous codons, such that protein sequences, codon use, and codon pair bias are conserved but the nucleic acid sequence is changed. Such recoding does not affect the growth of PV unless it destroys the sequence/structure of a functional RNA element. Using genetic analyses and a method called "signal location search," we detected two unique functionally redundant RNA elements (α and β), each about 75 nt long and separated by 150 nt, in the 3'-ter-minal coding sequence of RNA polymerase, 3Dpol. The presence of wild type (WT) α or β was sufficient for the optimal growth of PV, but the alteration of both segments in the same virus yielded very low titers and tiny plaques. The nucleotide sequences and predicted RNA structures of α and β have no apparent resemblance to each other. In a, we narrowed down the functional domain to a 48-nt-long, highly conserved segment. The primary determinant of function in β is a stable and highly conserved hairpin. Reporter constructs showed that the α- and β-segments are required for RNA replication. Recoding offers a unique and effective method to search for unknown functional RNA elements in coding sequences of RNA viruses, particularly if the signals are redundant in function. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV) is a single-stranded parvovirus retaining the unique capacity for site-specific integration into a transcriptionally silent region of the human genome, a characteristic requiring the functional properties of the Rep 78/68 polypeptide in conjunction with AAV terminal repeat integrating elements. Previous strategies designed to assemble these genetic elements into adenoviral (Ad) backbones have been limited by the general intolerability of AAV Rep sequences, prompting us to computationally reengineer the Rep gene by using synonymous codon pair recoding. Rep mutants generated by using de novo genome synthesis maintained the polypeptide sequence and endonuclease properties of Rep 78, while dramatically enhancing Ad replication and viral titer yields, characteristics indistinguishable from adenovirus lacking coexpressed Rep. Parallel approaches using domain swaps encompassing WT and recoded genomic segments, coupled with iterative computational algorithms, collectively established that 3' cis-acting Rep genetic elements (and not the Rep 78 polypeptide) retain dominant-acting sequences inhibiting Ad replication. These data provide insights into the molecular relationships of AAV Rep and Ad replication, while expanding the applicability of synonymous codon pair reengineering as a strategy to effect phenotypic endpoints. [ABSTRACT FROM AUTHOR]

Despite existing vaccines and enormous efforts in biomedical research, influenza annually claims 250,000–500,000 lives worldwide, motivating the search for new, more effective vaccines that can be rapidly designed and easily produced. We applied the previously described synthetic attenuated virus engineering (SAVE) approach to influenza virus strain A/PR/8/34 to rationally design live attenuated influenza virus vaccine candidates through genome-scale changes in codon-pair bias. As attenuation is based on many hundreds of nucleotide changes across the viral genome, reversion of the attenuated variant to a virulent form is unlikely. Immunization of mice by a single intranasal exposure to codon pair–deoptimized virus conferred protection against subsequent challenge with wild-type (WT) influenza virus. The method can be applied rapidly to any emerging influenza virus in its entirety, an advantage that is especially relevant when dealing with seasonal epidemics and pandemic threats, such as H5N1- or 2009-H1N1 influenza. [ABSTRACT FROM AUTHOR]