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Pathogenesis and Immunity

Diverse Herpes Simplex Virus Type 1 Thymidine Kinase Mutants in Individual Human Neurons and Ganglia

Kening Wang, Gowtham Mahalingam, Susan E. Hoover, Erik K. Mont, Steven M. Holland, Jeffrey I. Cohen, Stephen E. Straus
Kening Wang
1Medical Virology Section
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  • For correspondence: kwang@niaid.nih.gov
Gowtham Mahalingam
1Medical Virology Section
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Susan E. Hoover
1Medical Virology Section
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Erik K. Mont
1Medical Virology Section
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Steven M. Holland
2Immunopathogenesis Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
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Jeffrey I. Cohen
1Medical Virology Section
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Stephen E. Straus
1Medical Virology Section
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DOI: 10.1128/JVI.00166-07
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  • FIG. 1.
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    FIG. 1.

    Schematic representation of HSV-1 tk gene and mutations in the seven-G homopolymer region. The top panel represents the tk gene, with the locations of the seven-G homopolymer region and the PCR primers shown. The bottom panel represents the wild-type TK protein and the mutated polypeptides predicted to correspond to mutations in the seven-G region. Gray boxes, ATP binding domains; striped boxes, nucleoside binding domains; bold lines, amino acid sequences resulting from frameshifts; aa, amino acids; dl, deletion; ins, insertion. The oval on the bottom polypeptide diagram indicates an insertion of one amino acid.

  • FIG. 2.
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    FIG. 2.

    Diffuse skin lesions on patient 708. This photograph of patient 708 was taken when the patient was 13 years old and shows diffuse skin lesions on the face and neck. A swab sample collected from the face on the day of the photograph grew HSV-1 that was sensitive to ACV (IC50 = 0.22 μg/ml).

  • FIG. 3.
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    FIG. 3.

    Extremely high HSV-1 DNA loads in TG and DRG of patient 708. Total DNA isolated from ganglia was quantified by real-time PCR with primers and a probe specific for the HSV-1 gG gene. (A) DNA samples isolated from TG of patient 708 and seven other patients were each used as templates in PCRs (500 ng/reaction). *, median; **, maximum. (B) DNA samples isolated from the left TG and left DRG of patient 708 were each used as templates in PCRs (50 ng/reaction).

  • FIG. 4.
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    FIG. 4.

    PCR subcloning and sequencing of HSV-1 tk DNA samples from ganglia of patient 708. Total DNA samples isolated from ganglia were amplified by PCR with primers specific for HSV-1 tk so that the product encompassed the entire ORF of tk. The PCR products were then subcloned into plasmid vectors, and DNA samples prepared from single bacterial colonies were sequenced. (A) Representative gel showing the 1.2-kb PCR products from the LT2, LT9, and LL1 DRG, indicated by an arrow. MW, molecular weight standard. (B to F) Chromatograms showing the seven-G homopolymer regions of tk clones from patient 708 with a variety of mutations: one-G deletion (B), seven-G WT sequence (C), one-G insertion (D), two-G insertion (E), and three-G insertion (F).

  • FIG. 5.
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    FIG. 5.

    Comparison of the frequencies of nucleotide mutations in clones obtained by PCR amplification from ganglia of patient 708 and from a plasmid with WT tk. The nucleotide mutation rates (calculated as the number of nucleotides mutated divided by the total number of nucleotides sequenced from tk clones derived from individual ganglia) were determined for clones from patient 708 ganglion DNA and for clones from a plasmid containing WT tk. The nucleotide mutation rates for clones from 11 of 12 ganglia were lower than that for the WT tk control plasmid.

Tables

  • Figures
  • TABLE 1.

    Oligonucleotides used for PCR and sequencing

    NameSequence (5′ to 3′)
    tkF1b CGCCAAGCTTAAGCCACCATGGCTTCGTACCCCG
    tkR1 CCGTTCTAGAGTTAGCCTCCCCCATCT
    tkF51 GAAACTCCCGCACCTCTTCGG
    tkR61 GGTTCCTTCCGGTATTGTCTCC
    tkF82 AAGCGCCCAGATAACAATGG
    tkR21 AACACAGGAGGGCGGCGATG
    tkF5 CAAGAAGCCACGGAAGTC
    tkR2 GCTGTCCCCAATCCTCCCG
    M13 Forward (−20) GTAAAACGACGGCCAG
    M13 Reverse CAGGAAACAGCTATGAC
    pc3.1F AGAGAACCCACTGCTTACTGGC
    pc3.1R CTAGAAGGCACAGTCGAGGCTG
  • TABLE 2.

    Results of drug sensitivity testing of HSV-1 clinical isolates from patient 708

    Patient agea (yrs)Site of swabIC50 (μg/ml)b of:Last recorded therapy
    ACVFoscarnet
    18.84Skin1322ACV and foscarnet
    18.79Left inguinal area0.514Foscarnet
    14.39Labium3.219ACV
    14.39Abdomen1.916ACV
    13.67Chin0.127Foscarnet
    13.62Face0.248Foscarnet
    13.10Vagina3.9NRACV
    13.10Vagina1.8NRACV
    • ↵ a Age at which the corresponding swab sample was taken.

    • ↵ b Resistance is defined as an IC50 of ACV of ≥2 μg/ml and an IC50 of foscarnet of ≥150 μg/ml. NR, not recorded.

  • TABLE 3.

    Diverse mutations in the HSV-1 tk seven-G homopolymer region detected in individual ganglia from patient 708

    Source of DNANo. of clones sampledNo. of clonesa with:% of mutant clones
    7-G (WT) sequence1-G del1-G ins2-G ins3-G ins
    LTG484620004.2
    LT2 ganglion2316006130
    LT6 ganglion75590212221
    LT7 ganglion2320120013
    LT8 ganglion2312026348
    LT9 ganglion23100110257
    LL1 ganglion2315070135
    LL3 ganglion2150112376
    LS1 ganglion2419005021
    LS2 ganglion2418122125
    RT6 ganglion232120008.7
    RS1 ganglion242400000.0
    Total354265617531325
    • ↵ a del, deletion; ins, insertion.

  • TABLE 4.

    Determining the PCR subcloning fidelity of the seven-G homopolymer region in WT HSV-1 tk

    PCR templateNo. of clones sampledNo. of clonesa with:% of mutant clones
    7-G (WT) sequence1-G del1-G ins2-G ins3-G ins
    LT6 DRG DNA from patient 7084638025117
    Plasmid with WT tk434300000.0b
    TG DNA from patient 725242400000.0c
    • ↵ a del, deletion; ins, insertion.

    • ↵ b The difference between the results for the WT tk control template and the template from patient 708 were statistically significant (P = 0.006).

    • ↵ c The difference between the results for the template from patient 725 and that from patient 708 were statistically significant (P = 0.04).

  • TABLE 5.

    Determining the mutation rate during PCR subcloning of HSV-1 tk plasmid clones from patient 708 with nine-G sequences in the seven-G homopolymer region

    PCR templateNo. of clones sampledNo. of clones with:% of mutant clones
    9-G sequence9- and 8-G sequences8-G sequence7-G sequence
    Topo143-2.7322811213
    Topo143-4.1112901225
    Total443712416
  • TABLE 6.

    Polymorphisms in HSV-1 tk clones from patient 708

    Nucleotide positionNucleotide at indicated position in:
    HSV-1 17+332 HSV-1 tk clones from ganglia21 tk clones from RT6 and 1 clone from RS1
    68AGG
    102AAG
    106AGG
    171CTT
    261CTC
    266GAA
    271TCC
    513CTC
    717TCC
    719GAG
    793GAA
    915TTC
    933TCC
    969TCC
    1065ACC
  • TABLE 7.

    HSV-1 WT and tk mutants detected in individual neurons from a ganglion of patient 708

    Composition of virus population in single neurons (% of total no. of neurons)Single neurona identificationNo. of clones sampledNo. of clonesb with:% of mutant clones
    7-G (WT) sequence1-G del1-G ins2-G ins3-G ins
    Both WT and mutant clones insn-11612425
        each neuron (42.9)sn-2171616
    sn-31612425
    sn-42171467
    sn-5245116279
    sn-6171615.9
    WT only in each neuron (28.6)sn-713130.0
    sn-8660.0
    sn-923230.0
    sn-1010100.0
    Mutants only in each neuron (28.6)sn-111313100
    sn-1221183100
    sn-1322220100
    sn-141019100
    • ↵ a Single neurons were retrieved from LT9 DRG tissue sections by LCM.

    • ↵ b del, deletion; ins, insertion.

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Diverse Herpes Simplex Virus Type 1 Thymidine Kinase Mutants in Individual Human Neurons and Ganglia
Kening Wang, Gowtham Mahalingam, Susan E. Hoover, Erik K. Mont, Steven M. Holland, Jeffrey I. Cohen, Stephen E. Straus
Journal of Virology Jun 2007, 81 (13) 6817-6826; DOI: 10.1128/JVI.00166-07

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Diverse Herpes Simplex Virus Type 1 Thymidine Kinase Mutants in Individual Human Neurons and Ganglia
Kening Wang, Gowtham Mahalingam, Susan E. Hoover, Erik K. Mont, Steven M. Holland, Jeffrey I. Cohen, Stephen E. Straus
Journal of Virology Jun 2007, 81 (13) 6817-6826; DOI: 10.1128/JVI.00166-07
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KEYWORDS

Drug Resistance, Viral
Ganglia, Spinal
Herpesvirus 1, Human
Point Mutation
thymidine kinase
Trigeminal Nuclei
Viral Proteins

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