Briefly, T400, T404 and S408 were mutated to Ala or Glu (single or multiple mutations) with the Change-IT™ Multiple Mutation Site Directed Mutagenesis Kit (USB).
HCV NS3 or NS5A variants identified either during in vitro selection experiments in cell culture, or in patients who experienced virologic failure in the PEARL-I study, were introduced into the wild-type NS3 or NS5A GT4a or 4d replicon constructs using the Change-IT multiple mutation site-directed mutagenesis kit (Affymetrix, Santa Clara, CA).
Thus, while certain protein sites permit multiple mutations, sites critical to viral structure-function are evolutionarily robust and highly conserved.
Variants in NS3, NS5A, or NS5B were each introduced into the GT1a strain H77 replicon using a Change-IT multiple-mutation site-directed mutagenesis kit (Affymetrix, Santa Clara, CA).
A pair of sequences with saturation is expected to have more single mutations per site than multiple mutations per site.
The mtDNA divergence between the C.c. recticrista populations across the Jezreel Valley included multiple mutations in sites with potential functionality (Bar-Yaacov, Arbel-Thau, et al. 2012).
That is, despite the conservative nature of this coding region, when looking very broadly there are likely to be multiple mutations at sites throughout the gene causing either less accurate multiple sequence alignments or causing clustering methods to break up the matrix into smaller sections.
We assumed uniform prior distributions and applied an HKY model of mutation [ 60] to allow for the possibility of multiple mutations per site.
We therefore tried to use manual review of mutation sites and multiple online databases as well as literature review to consolidate our findings.
This permits us to avoid the "corrections" used in many approaches to capture the possibility of multiple mutations at individual sites.
