A Point Mutation in a Herpesvirus Polymerase Determines Neuropathogenicity
Figure 7
Differential Sensitivity of the Pol D/N752 Virus and Protein Variants to Aphidicolin and Structural Modeling of the Pol Region of Interest
(A) Cells were infected with the Pol D752 revertant (•) or N752 mutant (○) virus, treated with aphidicolin, incubated 3 d, then lysed; final virus yield was titrated on new cells.
(B) DNA was also extracted after the lysing step and qPCR performed to quantify normalized viral genome copies.
(C) The DNA polymerase activity of Pol D752 and Pol N752 proteins, in the absence and in the presence of pORF18 (Pol accessory subunit), was analyzed by measuring the incorporation of [3H]dTTP into a poly(dA)-oligo(dT) template. (▪) Pol D752; (□) Pol N752; (•) Pol D752 + pORF18; (○) Pol N752 + pORF18.
(D) The effect of aphidicolin on polymerase activity of Pol D752 (•) and of Pol N752 (○) was assayed by measuring the incorporation of [3H]dTTP into a poly(dA)-oligo(dT) template in the presence of pORF18. Graphs show the average of three experiments with standard deviations (error bars). Asterisk * indicates p < 0.05.
(E) Ribbon diagram of EHV-1 Pol N752 is based on HSV-1 Pol crystal structure [23].
(F) Space-filling diagram highlights the region between HSV-1 Pol secondary structure elements P3 and PB on the outside surface of the palm domain.
(G) Prediction of structural changes caused by the residue variation in Pol N752 as opposed to Pol D752.