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JAC Advance Access published online on February 25, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn057
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

A single drug-resistance mutation in HSV-1 UL52 primase points to a difference between two helicase–primase inhibitors in their mode of interaction with the antiviral target

Subhajit Biswas1, Gerald Kleymann2, Mihaiela Swift1, Laurence S. Tiley1, Jonathan Lyall1, Jesús Aguirre-Hernández1 and Hugh J. Field1,*

1 Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK 2 Leopoldshöherstraβe 7, D-32107 Bad-Salzuflen, Germany

* Corresponding author. Tel: +44-1223-330810; Fax: +44-1223-337610; E-mail: hjf10{at}cam.ac.uk

Received 28 November 2007; returned 23 January 2008; revised 17 January 2008; accepted 24 January 2008


   Abstract
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Objectives: To investigate the mechanism of action of the helicase–primase inhibitors (HPIs) BAY 57-1293 and BILS 22 BS by selection and characterization of drug-resistant herpes simplex virus (HSV)-1 mutants.

Methods: HSV-1 mutants were selected using BAY 57-1293 in Vero cells. Resistance mutations identified in the UL5 helicase or UL52 primase genes were validated by marker transfer. Cross-resistance to the structurally distinct BILS 22 BS was measured by ID50 determinations.

Results: (i) A single mutation (UL52: A899T) confers 43-fold resistance to BAY 57-1293, but does not confer any resistance to BILS 22 BS. (ii) A double mutant (UL52: A899T and UL5: K356T) is 2500-fold resistant to BAY 57-1293, which is more than 17 times the sum of fold-resistance due to the individual mutations, UL52: A899T (43-fold) and UL5: K356T (100-fold). (iii) Virus containing the single helicase mutation and the double mutant with mutations in both helicase and primase showed equal resistance to BILS 22 BS (70-fold).

Conclusions: By measuring the relative inhibitory concentrations required to overcome particular mutations in the helicase and primase proteins, evidence was obtained that BAY 57-1293 interacts with both components of the helicase–primase complex to achieve maximum potency, whereas for BILS 22BS, this may not be the case. Furthermore, our observations suggest that BAY 57-1293 interacts simultaneously with UL5 and UL52. Overall, the results suggest that these two potent HPIs interact differently with the helicase–primase complex.

Key Words: herpes simplex virus , BAY 57-1293 , BILS 22 BS , mutation


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DNA replication is an essential step in the multiplication cycle of herpes simplex virus (HSV), and several virus-coded enzymes are involved. The UL5 helicase is a 5'–3' helicase that unwinds the DNA double helix at the replication fork and UL52 primes the lagging strand.1 The latter is then extended by HSV DNA polymerase, which has been the most important target, to date, for nucleoside analogue antivirals such as aciclovir.

BAY 57-1293 and BILS 22 BS are representatives of a new class of HSV antiviral compound that target the HSV helicase–primase complex, which comprises the products of HSV UL5, UL8 and UL52 genes.1 Helicase–primase inhibitors (HPIs) form a heterogeneous group including thiazole, thiazoleamide (e.g. BAY 57-1293), thiazoleurea or thiazolylphenyl (e.g. BILS 22 BS) derivatives.

The drug–enzyme interaction possibly involves concomitant inhibition of helicase, primase and DNA-dependent ATPase activities.2,3 These published data mostly point to interaction with the HSV-1 helicase (UL5) involving several critical residues (Gly352, Met355 and Lys356) at a site immediately downstream of the proposed helicase motif IV.

Previously, a mutation (A897T) in HSV-1 UL52 primase was reported to confer resistance to two other thiazoleamide derivatives.3 Here, we report on viruses that contain the same resistance mutation selected by BAY 57-1293 and an additional HPI-resistance mutation in UL5. The pattern of cross-resistance shown by the mutants provides a novel insight into the mode of interaction between these interesting inhibitors and the enzyme complex.


   Materials and methods
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HPIs and antiviral assays

BAY 57-1293 (molecular mass: 402.5 Da) and BILS 22 BS (molecular mass: 449 Da) powders were kindly provided by Arrow Therapeutics (London, UK). The ID50 values were determined by plaque reduction assays (PRAs) in Vero cells using a standard method.4

Selection of HPI-resistant viruses

Parental viruses were the laboratory working-stocks of HSV-1 SC16 and PDK [derived from the laboratory strain Cl (101)] and their three-times plaque-purified substrains, namely, ‘HSV-1 SC16 cl-2’ and ‘PDK cl-1’.4,5 HPI-resistant mutants were selected in Vero cells using inhibitory concentrations of BAY 57-1293.5 Resistant plaques were picked and three-times plaque-purified. The resistance mutations and fold-resistance of the mutants are summarized (Table 1).


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  		Table 1. Sensitivity of HSV-1 wild-type strains and HPI-resistant mutants/variants selected from them to the two different HPIs (BAY 57-1293 and BILS 22 BS)

 
DNA sequencing and marker transfer

Total DNA from virus-infected Vero cells was used to amplify and bidirectionally sequence the HSV-1 UL5 (full) or UL52 (partial) genes,6 using multiple pairs of overlapping virus-specific primers. The role of UL52 or UL5 mutations in HPI resistance was confirmed by marker transfer using methods described previously.6

Putative drug-resistant recombinants normally occurred at ≥10–4, which was more than 100-fold over the background of ≤10–6 pfu.6 See Supplementary data at JAC Online (http://jac.oxfordjournals.org/) for further information.


   Results and discussion
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Two mutants were selected from the parental strain PDK cl-1 for resistance to BAY 57-1293 following a single passage in 0.8 µM BAY 57-1293 (BAY-Pr1) or two passages in 0.8 and 25 µM, respectively (BAY-Pr2).5 The two mutants, BAY-Pr1 and BAY-Pr2, showed 43- and 2500-fold resistance, respectively, to BAY 57-1293. However, BAY-Pr1 was fully sensitive to the alternative HPI, BILS 22 BS, and BAY-Pr2 was just 70-fold resistant (Table 1).

DNA sequence alignments revealed that BAY-Pr1 had no nucleotide substitutions relative to PDK cl-1 in the entire UL5 gene (2649 nt coding for 882 amino acids); BAY-Pr2 showed a single substitution, resulting in K356T (Table 1).

A 294 bp fragment of the UL52 (primase) gene was sequenced, and a point mutation g2695cg-> acg in the primase, leading to A899T, was located in both BAY-Pr1 and BAY-Pr2. This fragment was chosen for sequencing because it had previously been reported to contain a resistance mutation to BAY 54-6322, a different HPI.3 The role of this mutation was confirmed by marker transfer of a 1.1 kb primase fragment from BAY-Pr2 to SC16 cl-2 where it conferred 100-fold resistance (e.g. Pr2-cl-2-Rec). The fragment used for marker transfer was shown to have an identical sequence alignment to wild-type PDK cl-1, except for the single resistance mutation.

The role of the helicase mutation (UL5: K356T) in HPI resistance was confirmed by marker transfer of a 2.1 kb helicase fragment, containing the helicase mutation to BAY-Pr1 (originally sensitive to BILS 22 BS). The recombinants (e.g. Pr2-Pr1-Rec) displayed a resistance profile to each HPI identical to the double-mutant BAY-Pr2 and, therefore, conferred 70-fold resistance to BILS 22 BS. The same point mutation aa1067g->acg was also mapped to UL5 of BAY 57-1293-resistant variants (A-cl-4 and C-cl-2), selected from the SC16 working-stock, showing approximately 100-fold resistance to BAY 57-1293 and 70-fold co-resistance to BILS 22 BS (Table 1). Neither of these variants revealed any amino acid change in the previously mentioned 294 bp fragment of UL52 primase when compared with strain 17 or PDK. It might be argued that the K356T mutation could confer a different degree of resistance in different backgrounds (PDK or SC16). However, the influence of strain variation is likely to be relatively small as wild-type PDK is only slightly less sensitive to BAY 57-1293 than wild-type SC16. This is further supported by the observation that Lys356Gln in an HSV-1 F background confers approximately 150-fold resistance to BAY 57-1293;3 the same mutation confers a similar level of resistance (100-fold) to the same drug in the SC16 cl-2 background.6 Furthermore, K356T in SC16 or PDK background confers the same degree of resistance (70-fold) to BILS 22 BS.

The main findings from this study are:

  1. An HSV-1 PDK drug-resistant mutant with a single amino acid substitution in the primase protein (A899T) but with no change in UL5 helicase conferred moderate resistance (43-fold) to BAY 57-1293.
  2. The same mutation (A899T) did not confer resistance to BILS 22 BS. Neither the primase mutant (PDK-BAY-Pr1) nor the recombinant primase mutant (Pr2-cl-2-Rec) was co-resistant to BILS 22 BS. This is to the best of our knowledge the first evidence of HSV resistance to one HPI but sensitivity to another.
  3. A single UL5 helicase mutation (K356T) in HSV SC16 conferred co-resistance to both drugs: approximately 100-fold resistance to BAY 57-1293 and 70-fold resistance to BILS 22 BS. This supports previous observations6 that single helicase mutations (Lys356Gln in BAYr1 or Gly352Arg in BAYr2) confer co-resistance (Table 1).
From (i) and (iii), we conclude that most likely BAY 57-1293 interacts with both UL5 helicase and UL52 primase to exert its antiviral activity, inferring that interaction with helicase or primase alone is not sufficient to achieve maximum potency. Alternatively, the substitution of threonine (which has a bulky side chain compared with alanine) at position 899 might impose steric hindrance to BAY 57-1293-interaction with UL5 helicase. A third possible explanation for our results is that the substitution of A899T in UL52 generates a predictive phosphorylation site on the primase subunit of the helicase–primase complex, which could then result in allosteric hindrance to drug interaction with UL5 helicase, increasing the threshold of inhibition by BAY 57-1293. This idea is supported by close interdependence between the UL5 and UL52 for helicase and primase activities.7

The surprising sensitivity of the primase mutant to BILS 22 BS suggests that the primase mutation (A899T) may not prevent BILS 22 BS from inhibiting primase activity. Furthermore, if the primase mutation (A899T) imposes steric hindrance or allosteric inhibition to BAY 57-1293-interaction with UL5 helicase, this is not the case for BILS 22 BS.

Alternatively, BILS 22 BS may not need to interact with UL52 in order to achieve full potency. Unlike BAY 57-1293, BILS 22 BS does not have the sulphonamide moiety, which is thought to be involved in interaction with primase.8 Although previous reports9 indicate that BILS 22 BS inhibits DNA-helicase, RNA-primase and ATPase activities of the HSV helicase–primase holoenzyme, it is also known that these activities are interdependent,7 inhibiting one will inhibit the other(s).

A third possibility is that BILS 22 BS and BAY 57-1293 interact with UL52 primase at different sites. However, there is no evidence, to date, to support this proposition because, so far, we know of no other mutations in UL52 (other than A899T or its equivalent A897T in strain HSV-1 F3) that have been reported to confer resistance to any HPI. The position of the primase mutation in HSV-1 F3 as A897T (not A899T) arises because of an apparent deletion of two N-terminal amino acids N695 and D696 in UL52 (data not shown).

Returning to the proposition that BAY 57 1293 interacts with both components of the complex (helicase and primase), this raises the question of whether single molecules of BAY 57-1293 interact simultaneously with both proteins or different molecules interact with the two components independently. If the binding were independent, we would expect BAY 57-1293, at concentrations >100 times the ID50 for wild-type, to overcome both the 100-fold resistance conferred by the single helicase mutation and the lesser 43-fold resistance due to the primase mutation. However, we observed this not to be the case as the double mutant was 2500-fold resistant to BAY 57-1293, which is >17 times the sum of the two individual resistance components (43- and 100-fold). Furthermore, if the sites were independent, then neither of the single mutations could confer resistance. We believe these are evidences that the molecules of BAY 57-1293 interact simultaneously with both UL5 helicase and UL52 primase (Figure 1).


Figure 1
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  		Figure 1. Schematic of resistance profile of HSV-1 helicase–primase single or double mutants to BAY 57-1293 or BILS 22 BS. (a) Wild-type HSV-1 UL5 and UL52; (b) UL5 helicase single mutant; (c) UL52 primase single mutant; (d) UL5 and UL52 double mutant. S, sensitive; R, resistant (a virus is defined as resistant to an HPI when its ID50 value is >ID90 for the respective wild-type; this is the same as >4-fold IC50 of BAY 57-1293 and >2-fold IC50 of BILS 22 BS for wild-type HSV-1). Star symbol denotes single amino acid mutation in HSV-1 UL5/UL52 protein.

 
From the above results and published data, it appears that an {alpha}-helical region8 of the UL5 helicase (Gly352-Asn-Leu-Met-Lys356), close to helicase motif IV, and the region of UL52 primase, containing the residue Ala899, form an important site of interaction between these two enzymes in the complex. In a recent report,10 the site of interaction between UL5 and UL52 has been suggested as an interface for the binding of the helicase–primase complex with the viral DNA. So, it appears that BAY 57-1293 possibly interacts with the helicase–primase complex at its DNA-binding site and stabilizes the interaction between the HSV helicase–primase complex and nucleic acid, as suggested previously2 for other HPIs. Our study should stimulate in-depth three-dimensional study of drug–enzyme interactions to resolve these issues.

Although more work is required to resolve these questions, nonetheless, we conclude that BAY 57-1293 and BILS 22 BS differ in their mode of interaction with the virus UL5–UL52 complex.


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Arrow Therapeutics (London, UK) provided materials (including several PCR and sequencing primers) and a small grant-in-aid. S. B. is supported by the Cambridge Commonwealth Trust by means of a Cambridge Nehru Scholarship.


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H. J. F. held a research collaboration with AiCuris GmbH & Co. KG, Wuppertal, Germany during the period of the study. Other authors: none to declare.


   Supplementary data
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Further information on DNA sequencing and marker transfer is available at JAC Online (http://jac.oxfordjournals.org/).


   Acknowledgements
 
S. B. gratefully acknowledges support from the Cambridge Commonwealth Trust by means of a Cambridge Nehru Scholarship and we gratefully acknowledge the gift of materials (including several PCR and sequencing primers) and a small grant-in-aid from Arrow Therapeutics, London, UK. We thank Mrs Liz Lay for expert technical assistance. We are also grateful to Drs Ken Powell and Dagmar Alber, Arrow Therapeutics, London, UK; Dr Helga Rübsamen-Waigmann and colleagues at AiCuris GmbH & Co. KG, Germany; and Dr Stacey Efstathiou, University of Cambridge, for helpful advice and discussion of the data.


   References
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1 . Crute JJ, Tsurumi L, Zhu L, et al. Herpes simplex virus 1 helicase–primase: a complex of three herpes encoded gene products. Proc Natl Acad Sci USA (1989) 86:2186–9.[Abstract/Free Full Text]

2 . Crute JJ, Grygon CA, Hargrave KD, et al. Herpes simplex virus helicase–primase inhibitors are active in animal models of human disease. Nat Med (2002) 8:386–91.[CrossRef][Web of Science][Medline]

3 . Kleymann G, Fischer R, Betz UA, et al. New helicase–primase inhibitors as drug candidates for the treatment of herpes simplex disease. Nat Med (2002) 8:392–8.[CrossRef][Web of Science][Medline]

4 . Biswas S, Smith C, Field HJ. Detection of HSV-1 variants highly resistant to the helicase–primase inhibitor BAY 57-1293 at high frequency in 2 of 10 recent clinical isolates of HSV-1. J Antimicrob Chemother (2007) 60:274–9.[Abstract/Free Full Text]

5 . Biswas S, Swift M, Field HJ. High frequency of spontaneous helicase primase inhibitor (BAY 57-1293) drug-resistant variants in certain laboratory isolates of HSV-1. Antivir Chem Chemother (2007) 18:13–23.[Medline]

6 . Biswas S, Jennens L, Field HJ. Single amino acid substitutions in the HSV-1 helicase protein that confer resistance to the helicase–primase inhibitor BAY 57-1293 are associated with increased or decreased virus growth characteristics in tissue culture. Arch Virol (2007) 152:1489–500.[CrossRef][Medline]

7 . Chen Y, Carrington-Lawrence SD, Bai P, et al. Mutations in the putative zinc-binding motif of UL52 demonstrate a complex interdependence between the UL5 and UL52 subunits of the human herpes simplex virus type 1 helicase/primase complex. J Virol (2005) 79:9088–96.[Abstract/Free Full Text]

8 . Kleymann G. Agents and strategies in development for improved management of herpes simplex virus infection and disease. Expert Opin Investig Drugs (2005) 14:135–61.[CrossRef][Medline]

9 . Liuzzi M, Kibler P, Bousquet C, et al. Isolation and characterization of herpes simplex virus type 1 resistant to aminothiazolylphenyl-based inhibitors of the viral helicase–primase. Antiviral Res (2004) 64:161–70.[CrossRef][Web of Science][Medline]

10 . Chen Y, Livingston CM, Carrington-Lawrence SD, et al. A mutation in the human herpes simplex virus type I UL52 zinc finger motif results in defective primase activity but can recruit viral polymerase and support viral replication efficiently. J Virol (2007) 81:8742–51.[Abstract/Free Full Text]


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A mutation in helicase motif IV of herpes simplex virus type 1 UL5 that results in reduced growth in vitro and lower virulence in a murine infection model is related to the predicted helicase structure
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