Drug resistance patterns of recombinant herpes simplex virus DNA polymerase mutants generated with a set of overlapping cosmids and plasmids

Abstract

Herpes simplex virus (HSV) DNA polymerase (Pol) mutations can confer resistance to all currently available antiherpetic drugs. However, discrimination between mutations responsible for drug resistance and those that are part of viral polymorphism can be difficult with current methodologies. A new system is reported for rapid generation of recombinant HSV type 1 (HSV-1) DNA Pol mutants based on transfection of a set of overlapping viral cosmids and plasmids. With this approach, twenty HSV-1 recombinants with single or dual mutations within the DNA pol gene were successfully generated and subsequently evaluated for their susceptibilities to acyclovir (ACV), foscarnet (FOS), cidofovir (CDV), and adefovir (ADV). Mutations within DNA Pol conserved regions II (A719T and S724N), VI (L778M, D780N, and L782I), and I (F891C) were shown to induce cross-resistance to ACV, FOS, and ADV, with two of these mutations (S724N and L778M) also conferring significant reduction in CDV susceptibility. Mutant F891C was associated with the highest levels of resistance towards ACV and FOS and was strongly impaired in its replication capacity. One mutation (D907V) lying outside of the conserved regions was also associated with this ACV-, FOS-, and ADV-resistant phenotype. Some mutations (K522E and Y577H) within the delta-region C were lethal, whereas others (P561S and V573M) induced no resistance to any of the drugs tested. Recombinants harboring mutations within conserved regions V (N961K) and VII (Y941H) were resistant to ACV but susceptible to FOS. Finally, mutations within conserved region III were associated with various susceptibility profiles. This new system allows a rapid and accurate evaluation of the functional role of various DNA Pol mutations, which should translate into improved management of drug-resistant HSV infections.

FIG. 1.

Schematic representation of viral cosmids and plasmids used to generate HSV-1 recombinants. (A) Inserts of the five cosmids (24, 32, 48, 51, and 71) covering the complete genome of the HSV-1 strain 17 (16), along with the three plasmids (pNEB23, pPol6, and pNEB10) designed to replace cos71. Tr, terminal repeat; U_L, unique long; U_S, unique short; Ir, internal repeat. (B) Construction of the pNEB23, pPol6, and pNEB10 plasmids. The nucleotide positions of inserts in the HSV strain 17 genome are indicated.

FIG. 2.

Southern blot analysis of some recombinant HSV-1 wt and mutant viruses along with the parental laboratory strain HSV 17 by using some of the viral cosmids (cos 24, 51, and 71) as probes. Numbers on the left indicate molecular sizes in kilobase pairs.

FIG. 3.

Plaque morphologies of some recombinant HSV-1 wt and mutant viruses in 24-well plates containing Vero cells. At 96 h postinfection, cells were fixed and stained. Note that viral inoculums were not necessarily the same for each recombinant virus.

FIG. 4.

Influence of DNA Pol mutations on HSV replication capacities in single-step growth kinetics. On the day of infection, confluent Vero cells in 24-well plates were infected with either wt virus or different recombinant mutants (V573M, N961K, V715M, and L778M) at a MOI of 5. Extracellular viruses were collected at different times (up to 144 h postinfection) and titrated. Curves are representative of results from two independent experiments.