As a result of the redundancy of the genetic code, adjacent pairs of amino acids can be encoded by as many as 36 different pairs of synonymous codons. A species-specific "codon pair bias" provides that some synonymous codon pairs are used more or less frequently than statistically predicted. We synthesized de novo large DNA molecules using hundreds of over-or underrepresented synonymous codon pairs to encode the poliovirus capsid protein. Underrepresented codon pairs caused decreased rates of protein translation, and polioviruses containing such amino acid-independent changes were attenuated in mice. Polioviruses thus customized were used to immunize mice and provided protective immunity after challenge. This "death by a thousand cuts" strategy could be generally applicable to attenuating many kinds of viruses.

Exploring the utility of de novo gene synthesis with the aim of designing stably attenuated polioviruses (PV), we followed two strategies to construct PV variants containing synthetic replacements of the capsid coding sequences either by deoptimizing synonymous codon usage (PV-AB) or by maximizing synonymous codon position changes of the existing wild-type (wt) poliovirus codons (PV-SD). Despite 934 nucleotide changes in the capsid coding region, PV-SD RNA produced virus with wild-type characteristics. In contrast, no viable virus was recovered from PV-AB RNA carrying 680 silent mutations, due to a reduction of genome translation and replication below a critical level. After subcloning of smaller portions of the AB capsid coding sequence into the wt background, several viable viruses were obtained with a wide range of phenotypes corresponding to their efficiency of directing genome translation. Surprisingly, when inoculated with equal infectious doses (PFU), even the most replication-deficient viruses appeared to be as pathogenic in PV-sensitive CD155tg (transgenic) mice as the PV(M) wild type. However, infection with equal amounts of virus particles revealed a neuroattenuated phenotype over 100-fold. Direct analysis indicated a striking reduction of the specific infectivity of PV-AB-type virus particles. Due to the distribution effect of many silent mutations over large genome segments, codon-deoptimized viruses should have genetically stable phenotypes, and they may prove suitable as attenuated substrates for the production of poliovirus vaccines.

Influenza claims 250,000 - 500,000 lives annually worldwide. Despite existing vaccines and enormous efforts in biomedical research, these staggering numbers have not changed significantly over the last two decades1, motivating the search for new, more effective, vaccines that can be rapidly designed and easily produced. Using influenza virus strain A/PR/8/34, we describe a systematic, rational approach, termed Synthetic Attenuated Virus Engineering (SAVE), to develop new, efficacious live attenuated influenza virus vaccine candidates through genome-scale changes in codon pair bias. Attenuation is based on many hundreds of nucleotide changes across the viral genome, offering high genetic stability and a wide margin of safety. The method can be applied rapidly to any emerging influenza virus in its entirety, an advantage that is significant for dealing with seasonal epidemics and pandemic threats, such as H5N1- or 2009-H1N1 influenza.

Actin-based cell-cell adherens junctions (AJs) are crucial not only for mechanical adhesion but also for cell morphogenesis and differentiation. While organization of homotypic AJs is attributed mostly to classic cadherins, the adhesive mechanism of heterotypic AJs in more complex tissues remains to be clarified. Nectin, a member of a family of immunoglobulin-like adhesion molecules at various AJs, is a possible organizer of heterotypic AJs because of its unique heterophilic trans-interaction property. Recently, nectin-2 (-/-) mice have been shown to exhibit the defective sperm morphogenesis and the male-specific infertility, but the role of nectin in testicular AJs has not been investigated. We show here the heterotypic trans-interaction between nectin-2 in Sertoli cells and nectin-3 in spermatids at Sertoli-spermatid junctions (SspJs), heterotypic AJs in testes. Moreover, each nectin-based adhesive membrane domain exhibits one-to-one colocalization with each actin bundle underlying SspJs. Inactivation of the mouse nectin-2 gene causes not only impaired adhesion but also loss of the junctional actin scaffold at SspJs, resulting in aberrant morphogenesis and positioning of spermatids. Localization of afadin, an adaptor protein of nectin with the actin cytoskeleton, is also nectin-2 dependent at SspJs. These results indicate that the nectin-afadin system plays essential roles in coupling cell-cell adhesion and the cortical actin scaffold at SspJs and in subsequent sperm morphogenesis.