A point mutation within a distinct conserved region of the herpes simplex virus DNA polymerase gene confers drug resistance

C B Hwang, K L Ruffner, D M Coen
1992 Journal of Virology  
We have shown that a drug-resistant mutant from a clinical isolate of herpes simplex virus contains a single point mutation in the DNA polymerase gene that confers resistance to both acyclovir and foscarnet. The mutated amino acid is located within a distinct conserved region shared among of-like DNA polymerases which we designate region VII. We infer that these conserved sequences are directly or indirectly involved in the recognition and binding of nucleotide and PP1 substrates. Herpes
more » ... virus (HSV) DNA polymerase (Pol) is both an important target for antiviral therapy and an excellent model for the a-like DNA polymerases (reviewed in reference 6). HSV DNA Pol contains several regions, designated I through VI, conserved among a-like DNA polymerases (20). Sequence analysis of several HSVpol mutants revealed that mutations that confer resistance to antiviral drugs that mimic and/or compete with deoxynucleoside triphosphate (dNTP) or PPi are distributed within regions I, II, III, and V and in region A, which is shared by only certain a-like DNA polymerases (Fig. 1B ). This suggests that these conserved regions are directly or indirectly involved in recognition and binding of nucleotides and PPi (reviewed in reference 6). Here we report the sequence of a pol mutant derived from a clinical isolate that implicates a distinct conserved region, which we term region VII, in these functions. Sacks et al. (17) previously reported a heterogeneous HSV type 1 isolate from an immunocompromised patient who had suffered severe progressive herpetic disease despite acyclovir (ACV) therapy. This isolate included a mutant, termed 615.8. A marker transfer experiment in which a 3.3-kbp BamHI fragment from the 615.8pol gene transferred foscarnet (PFA) and ACV resistance to genetically related pretreatment isolate 294.1 or laboratory strain KOS established that the 615.8 pol gene contains a mutation(s) that confers resistance to both PFA and ACV (17) . Finer mapping of the 615.8 drug resistance mutations. To localize the PFA resistance marker of 615.8 more finely, a marker transfer experiment was performed as described previously (5) . Three ICnI subfragments from plasmid p615.8BQ (referred to as 615.8 3.3-kbp pol in reference 17) containing the 3.3-kbp BamHI pol DNA of 615.8 were used (Fig. 1A) . Two of the subfragments were similar in size (1.1 kbp) and extended from the RpnI site of the pUC19 vector to the second KpnI site within the 3.3-kbppol DNA insert and were isolated together from an agarose gel. The third fragment (3.7 kbp) included the remaining sequences of the pol insert, extending from the second Kpnl site, and also included sequences of the cloning vector (Fig. 1A) . This 3.7-kbp KpnI fragment transferred PFA resistance to infectious KOS DNA at a frequency (0.12%) substantially higher * Corresponding author. than that obtained when no fragment (0.0047%) or the 1.1-kbp KpnI fragments (0.002%) were added. PFA-resistant recombinant virus (615.8 P'K1) from the marker transfer experiment using the 3.7-kbp KpnI fragment was then plaque purified and tested for sensitivity to ACV. Both the original resistant isolate, 615.8, and the recombinant virus, 615.8 PrK1, were similarly resistant to ACV with a 50% effective dose of 40 ,uM, while KOS exhibited a 50% effective dose of 6 ,uM (Fig. 2) . Since the corresponding DNA fragments derived from pretreatment isolate 294.1 failed to transfer the PFA resistance marker to KOS (17) and those fragments from 615.8 did transfer the resistance marker to both 294.1 and KOS (17; this report), these results localized the PFA and ACV resistance markers of the 615.8 isolate to the 1,125 bp between the KpnI and BamHI sites of the 615.8 DNA pol gene. Identification of the mutated amino acid that confers PFA and ACV resistance. The 1,125-bp IKpnI-BamHI regions of both 615.8 and pretreatment isolate 294.1 were then sequenced on double-stranded plasmid DNA by using the M13 universal primer (Promega Biotec) and pol-specific oligonucleotide primers (10) and Sequenase (U.S. Biochemicals) as recommended by the manufacturers. The region of the 615.8 pol gene contained a single change of C to T at nucleotide 2821, relative to that of 294.1. This transition mutation converts tyrosine to histidine at amino acid residue 941. We conclude that the change at amino acid 941 is the mutation responsible for the resistance of 615.8 to both PFA and ACV. In addition, both 294.1 and 615.8 contained two nucleotides different from those of the wild-type strain KOS sequence. One of these, an A-to-G change, generated a methionine-to-valine change at position 905. However, both nucleotides are identical to those of the HSV strain 17 sequence (16). The 615.8 mutation alters a highly conserved tyrosine within region VII of sequence similarity. Amino acid 941 is not located within any of the five conserved regions (I, II, III, V, and A) previously shown to mutate to confer drug resistance (reviewed in reference 6) nor in the other conserved regions identified by Wong et al. (20) . However, amino acid 941 is part of a motif recognized by several investigators and termed variously region 4, 9, or IX (3, 13, 18) . This motif is illustrated in Fig. 1B and contains a highly conserved sequence, Lys-Lys-X-Tyr, with the second Lys and the Tyr conserved in all of the polymerases examined. Remarkably, it is this Tyr that is altered in 615.8. The first Lys is
doi:10.1128/jvi.66.3.1774-1776.1992 fatcat:ijl4nqc5wvbt5pchfojlcu7c2y