Recombinant herpes simplex virus type 1 strains with targeted mutations relevant for aciclovir susceptibility

Abstract

Here, we describe a novel reliable method to assess the significance of individual mutations within the thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1) to nucleoside analogue resistance. Eleven defined single nucleotide polymorphisms that occur in the TK gene of clinical HSV-1 isolates and a fluorescence reporter were introduced into the HSV-1 strain 17(+) that had been cloned into a bacterial artificial chromosome. The susceptibility of these different strains to aciclovir, penciclovir, brivudin, and foscarnet was determined with a modified cytopathic effect reduction assay. The strains were also tested for their aciclovir susceptibility by measuring the relative fluorescence intensity as an indicator for HSV-1 replication and by quantifying the virus yield. Our data indicate that the amino acid substitutions R41H, R106H, A118V, L139V, K219T, S276R, L298R, S345P, and V348I represent natural polymorphisms of the TK protein, whereas G61A and P84L mediate broad cross-resistance against aciclovir, penciclovir, brivudin, and susceptibility to foscarnet. This method allows the definition of the resistance genotype of otherwise unclear mutations in the TK gene of HSV-1. Thus, it provides a scientific basis for antiviral testing in clinical isolates of patients suffering from serious diseases and will facilitate testing of new antivirals against HSV-1.

Figure 1. One-step replication kinetics of HSV-1 recombinants.

Vero cells were infected with an MOI of 5 and the supernatant was harvested at several time points to show viral replication behaviour as function of time. The TK-mutated strains R41H, G61A, P84L, R106H, A118V, L139V, K219T, S276R, L298R, S345P, and V348I (dotted lines) were compared with their EGFP-tagged counterparts (solid lines). The growth kinetic of the HSV1(17⁺)Lox strain is depicted separately (dashed line). The data were plotted as a mean and SD of triplicates.

Figure 2. TK gene expression after transfection of Vero cells.

(a) RT-PCR was performed to confirm UL23 gene transcription. The analysis of GAPDH served as cellular control. The PCR was conducted in presence (+) and absence (−) of reverse transcriptase to exclude DNA contamination. (b) Immunoblotting was performed to confirm TK protein expression. The analysis of gD protein served as viral-load control and beta-actin as cellular control. (c) Immunofluorescence microscopy revealed TK protein expression in infected Vero cells. Plaques by EGFP-tagged virus strains (first column) were stained with anti-TK antibody (second column). The third column displays the merged images.

Figure 3. Quantitative PCR for the determination of viral load under various ACV concentrations.

A constant number of 2 × 10⁵ Vero cells were infected with a MOI of 0.01 and 3.3% of the eluate volume from whole cell DNA extracts obtained one day p.i. was used as template for qPCR. (a) The HSV1(17⁺)Lox strain was tested with ACV concentrations ranging from 35.2 to 0 μM. HSV1(17⁺)Lox was graded as susceptible reference strain. (b) The TK-modified strains were tested with a high concentration (35.2 μM, filled bars) or in absence (0 μM, empty bars) of ACV. Data are plotted as the mean and SD of three measurements from one representative experiment.