Frustration and Direct-Coupling Analyses to Predict Formation and Function of Adeno-Associated Virus.

Adeno-associated virus (AAV) is a promising gene therapy vector because of its efficient gene delivery and relatively mild immunogenicity. To improve delivery target specificity, researchers use combinatorial and rational library design strategies to generate novel AAV capsid variants. These approaches frequently propose high proportions of nonforming or noninfective capsid protein sequences that reduce the effective depth of synthesized vector DNA libraries, thereby raising the discovery cost of novel vectors. We evaluated two computational techniques for their ability to estimate the impact of residue mutations on AAV capsid protein-protein interactions and thus predict changes in vector fitness, reasoning that these approaches might inform the design of functionally enriched AAV libraries and accelerate therapeutic candidate identification. The Frustratometer computes an energy function derived from the energy landscape theory of protein folding. Direct-coupling analysis (DCA) is a statistical framework that captures residue coevolution within proteins. We applied the Frustratometer to select candidate protein residues predicted to favor assembled or disassembled capsid states, then predicted mutation effects at these sites using the Frustratometer and DCA. Capsid mutants were experimentally assessed for changes in virus formation, stability, and transduction ability. The Frustratometer-based metric showed a counterintuitive correlation with viral stability, whereas a DCA-derived metric was highly correlated with virus transduction ability in the small population of residues studied. Our results suggest that coevolutionary models may be able to elucidate complex capsid residue-residue interaction networks essential for viral function, but further study is needed to understand the relationship between protein energy simulations and viral capsid metastability.

Control of synthetic microbial consortia in time, space, and composition.

While synthetic microbial systems are becoming increasingly complicated, single-strain systems cannot match the complexity of their multicellular counterparts. Such complexity, however, is much more difficult to control. Recent advances have increased our ability to control temporal, spatial, and community compositional organization, including modular adhesive systems, strain growth relationships, and asymmetric cell division. While these systems generally work independently, combining them into unified systems has proven difficult. Once such unification is proven successful we will unlock a new frontier of synthetic biology and open the door to the creation of synthetic biological systems with true multicellularity.

An essential N-terminal serine-rich motif in the AAV VP1 and VP2 subunits that may play a role in viral transcription.

Adeno-associated virus (AAV) is one of the most researched, clinically utilized gene therapy vectors. Though clinical success has been achieved, transgene delivery and expression may be hindered by cellular and tissue barriers. Understanding the role of receptor binding, entry, endosomal escape, cytoplasmic and nuclear trafficking, capsid uncoating, and viral transcription in therapeutic efficacy is paramount. Previous studies have shown that N-terminal regions of the AAV capsid proteins are responsible for endosomal escape and nuclear trafficking, however the mechanisms remain unknown. We identified a highly-conserved three-residue serine/threonine (S/T) motif in the capsid N-terminus, previously uncharacterized in its role in intracellular trafficking and transduction. Using alanine scanning mutagenesis, we found S155 and the flanking residues, D154 and G158, are essential for AAV2 transduction efficiency. Remarkably, specific capsid mutants show a 5 to 9-fold decrease in viral mRNA transcripts, highlighting a potential role of the S/T motif in transcription of the viral genome.