Characterized attenuated alphavirus mutants contain changes to the genomic 5′ UTR and mutations that result in altered non-structural protein cleavage timing leading to altered levels of antigen expression and interferon (IFN) induction.
The chosen 4 R-selective mutant contained four changes in addition to the E192N substitution: A93V, F109I, N153K, and S208G.
In place of L7Ae, we substituted L7Ae-K37A (referred to here as L7Ae-K), L7Ae-D54A-K79A (L7Ae-K37A-K79AL7Ae-K37A-K79A (L7Ae-KK), mutants containing amino acid changes predicted to affect RNA binding from analysis of the cocrystal structure and from high degrees of conservation in a sequence alignment (Supplementary Figure S1).
Some of the mutants contained multiple mutations, as shown in Fig. 1.
Doubly truncated mutants contained BamHI and XhoI restriction sites.
In contrast all of the H5 HA proteins bound to fewer cells and of the cells bound the majority were ciliated although the mutants containing the G228S change bound to some cells that were non-ciliated (dark blue).
We sequenced the mutant protein-coding sequence for each allele of Ago1, Dicer-1, Drosha, and Pasha, and except for one Ago1 and Pasha allele, each mutant contained a single change in the coding sequence that altered the polypeptide product (Table 1 and Figure S2).
This cefotaxime resistant mutant contained six amino acid changes (including E104K, G238S and M182T) and an engineered promoter mutation located between the -35 and -10 sites of the β-lactamase P3 promoter that increased β-lactamase expression levels by 2 3-fold.
This mutant contained a single base change in the regulatory region upstream of the riboflavin biosynthetic genes.
In MD simulations of the P39G mutant (and other mutants containing the P39G mutation), we observe a conformational change in the backbone of residues 74 76, whereas the average structure of residues 74 76 is well maintained in MD simulations of WT KSI and mutants not containing the P39G mutation.
