Analysis of Serine Codon Conservation Reveals Diverse Phenotypic Constraints on Hepatitis C Virus Glycoprotein Evolution

Serine is encoded by two divergent codon types, UCN and AGY, which are not interchangeable by a single nucleotide substitution. Switching between codon types therefore occurs via intermediates (threonine or cysteine) or via simultaneous tandem substitutions. Hepatitis C virus (HCV) chronically infects 2 to 3% of the global population. The highly variable glycoproteins E1 and E2 decorate the surface of the viral envelope, facilitate cellular entry, and are targets for host immunity. Comparative sequence analysis of globally sampled E1E2 genes, coupled with phylogenetic analysis, reveals the signatures of multiple archaic codonswitching events at seven highly conserved serine residues. Limited detection of intermediate phenotypes indicates that associated fitness costs restrict their fixation in divergent HCV lineages. Mutational pathways underlying codon switching were probed via reverse genetics, assessing glycoprotein functionality using multiple in vitro systems. These data demonstrate selection against intermediate phenotypes can act at the structural/functional level, with some intermediates displaying impaired virion assembly and/or decreased capacity for target cell entry. These effects act in residue/isolate-specific manner. Selection against intermediates is also provided by humoral targeting, with some intermediates exhibiting increased epitope exposure and enhanced neutralization sensitivity, despite maintaining a capacity for target cell entry. Thus, purifying selection against intermediates limits their frequencies in globally sampled strains, with divergent functional constraints at the protein level restricting the fixation of deleterious mutations. Overall our study provides an experimental framework for identification of barriers limiting viral substitutional evolution and indicates that serine codon-switching represents a genomic "fossil record" of historical purifying selection against E1E2 intermediate phenotypes

Formation of Stable Nanocarriers by in Situ

We present an in situ hydrophobic salt forming technique for the encapsulation of weakly hydrophobic, ionizable active pharmaceutical ingredients (API) into stable nanocarriers (NCs) formed via a rapid precipitation process. Traditionally, NC formation via rapid precipitation has been difficult with APIs in this class because their intermediate solubility makes achieving high supersaturation difficult during the precipitation process and the intermediate solubility causes rapid Ostwald ripening or recrystallization after precipitation. By forming a hydrophobic salt in situ, the API solubility and crystallinity can be tuned to allow for NC formation. Unlike covalent API modification, the hydrophobic salt formation modifies properties via ionic interactions, thus circumventing the need for full FDA re-approval. This technique greatly expands the types of APIs that can be successfully encapsulated in NC form. Three model API’s were investigated and successfully incorporated into NCs by forming salts with hydrophobic counter ions: cinnarizine, an antihistamine, clozapine, an antipsychotic and α-lipoic acid, a common food supplement. We focus on cinnarizine to develop the rules for the in situ nanoprecipitation of salt NCs. These rules include the pK(a)’s and solubilities of the API and counter ion, the effect of the salt former-to-API ratio on particle stability and encapsulation efficiency, and the control of NC size. Finally, we present results on the release rates of these ion pair APIs from the NCs

Analysis of Serine Codon Conservation Reveals Diverse Phenotypic Constraints on Hepatitis C Virus Glycoprotein Evolution

Serine is encoded by two divergent codon types, UCN and AGY, which are not interchangeable by a single nucleotide substitution. Switching between codon types therefore occurs via intermediates (threonine or cysteine) or via simultaneous tandem substitutions. Hepatitis C virus (HCV) chronically infects 2 to 3% of the global population. The highly variable glycoproteins E1 and E2 decorate the surface of the viral envelope, facilitate cellular entry, and are targets for host immunity. Comparative sequence analysis of globally sampled E1E2 genes, coupled with phylogenetic analysis, reveals the signatures of multiple archaic codon-switching events at seven highly conserved serine residues. Limited detection of intermediate phenotypes indicates that associated fitness costs restrict their fixation in divergent HCV lineages. Mutational pathways underlying codon switching were probed via reverse genetics, assessing glycoprotein functionality using multiple in vitro systems. These data demonstrate selection against intermediate phenotypes can act at the structural/functional level, with some intermediates displaying impaired virion assembly and/or decreased capacity for target cell entry. These effects act in residue/isolate-specific manner. Selection against intermediates is also provided by humoral targeting, with some intermediates exhibiting increased epitope exposure and enhanced neutralization sensitivity, despite maintaining a capacity for target cell entry. Thus, purifying selection against intermediates limits their frequencies in globally sampled strains, with divergent functional constraints at the protein level restricting the fixation of deleterious mutations. Overall our study provides an experimental framework for identification of barriers limiting viral substitutional evolution and indicates that serine codon-switching represents a genomic “fossil record” of historical purifying selection against E1E2 intermediate phenotypes