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ANIMAL VIRUSES

Human Cytomegalovirus oriLyt Sequence Requirements

Yuao Zhu, Lili Huang, David G. Anders
Yuao Zhu
The David Axelrod Institute, Wadsworth Center for Laboratories and Research, New York State Department of Health, and
Department of Biomedical Sciences, State University of New York at Albany School of Public Health, Albany, New York 12201-2002
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Lili Huang
The David Axelrod Institute, Wadsworth Center for Laboratories and Research, New York State Department of Health, and
Department of Biomedical Sciences, State University of New York at Albany School of Public Health, Albany, New York 12201-2002
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David G. Anders
The David Axelrod Institute, Wadsworth Center for Laboratories and Research, New York State Department of Health, and
Department of Biomedical Sciences, State University of New York at Albany School of Public Health, Albany, New York 12201-2002
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DOI: 10.1128/JVI.72.6.4989-4996.1998
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  • Fig. 1.
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    Fig. 1.

    Kanr cassette insertion mutants. (A) Plasmid constructs. Positions of the Kanr cassette insertions are plotted in relation to landmark features of oriLyt. The name, insertion coordinate, and relative activity for each mutant are noted at the right. Features located on the oriLyt line above the mutants include the UL59 open reading frame and transcript (16), the 29-bp repeats (triangle), the Y block (hollow box), and the two large, imperfect dyad symmetries A and B (3). The core region is shaded and bound by dotted lines, and the flanking regions within which partially inactivated insertions are represented by a gradient. (B) Transient transfection assay of the Kanr cassette insertion constructs. The abilities of the constructs described in panel A to mediate DNA replication were tested as described in Materials and Methods (lanes 1 to 14).DpnI-resistant products of replication were detected by Southern blotting; a replica of the resulting autoradiogram is shown. The tested plasmids are indicated at the top of the panel. The marker (lane 16) contains 0.1 ng each of EcoRI-treated plasmids SP54, SP50, and pGEM7Zf(−). Plasmid SP50 (lane 15), the parent to most of the insertions, and the vector pGEM7Zf(−) (lane 17) were transfected in parallel as wild-type and negative standards, respectively, for comparison. (C) Transient assay of the Kanr cassette insertion constructs from which the insertion was deleted by PstI treatment, leaving a residualPstI linker insertion. Each plasmid was tested in duplicate. The autoradiogram is reproduced here. The deletion constructs, which correspond to the Kanr insertions described in panel A, are indicated at the top of each lane.

  • Fig. 2.
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    Fig. 2.

    Overlapping deletions across HCMV oriLyt. (A) A schematic of the PvuII (nt 89796)-to-KpnI (nt 94860) fragment encompassing oriLyt and deletion constructs. The deleted region of each plasmid is indicated by nucleotide coordinates and by a gap in the line corresponding to the position in the oriLyt core. The open box in the line of pYZ20+1r represents the reiterated dyad sequence (2). Relative replication efficiencies are given at the right; ND, none detected. (B) Quantitative replication assay. The indicated test plasmids were cotransfected with pSP50 or pSP54 and pGEM-7Zf(−) as described in Materials and Methods. An autoradiogram of the resulting Southern blot is reproduced here. Deletion mutants pSP90, pSP72-24, pLH13Δ, pLH34Δ, and pLH50Δ were cotransfected with pSP54 as the positive internal standard. The other deletion mutants were pSP54 derived, and SP50 was used for the internal wild-type comparison. The pSP68 sample was treated with DpnI plus EcoRI andHindIII because pSP68 lacks an EcoRI site, and thus the pSP50 internal standard migrated to the position of pGEM-7Zf(−). (C) Qualitative assay. The indicated test plasmids were assayed for replication competence by transient transfection as described in Materials and Methods. A representative autoradiogram is reproduced here; only the region of the autoradiogram containing replicated (rep’d) signals is shown.

  • Fig. 3.
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    Fig. 3.

    Replicator activities of insertion and deletion mutants relative to those of the wild type. Exterior deletions that impinge upon the oriLyt region (thick grey bar) progressively reduce activity (3, 22). The positions and relative replicator activities of insertions (closed triangles) and deletions (thin grey bars) are plotted. The trough in the activity plot defines the core region (cross-hatched rectangle). Essential regions I and II, defined by deletions that completely abrogated replicator activity (black rectangles I and II), and the intervening deletable segment (open rectangle D) are indicated.

  • Fig. 4.
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    Fig. 4.

    Mutations in essential region I. (A) A schematic of the mutations in essential region I. Essential region I is enlarged directly below a schematic of the oriLyt region highlighting landmark features. For each plasmid, the deleted sequence is indicated by nucleotide coordinates and by a gap in the line. Relative replication efficiencies estimated in the quantitative replication assay are noted at the right; ND, none detected. (B) Quantitative replication assay of the small deletions. For each test plasmid, pSP50 and pGEM-7Zf(−) were used as wild-type and negative internal standards, respectively. The relative replication efficiencies of each plasmid were measured as described in Materials and Methods and are indicated at the right of in panel A. Samples for lanes 14 to 17 were from a transfection experiment and blot separate from those for lanes 1 to 13.

Tables

  • Figures
  • Table 1.

    Deletion plasmids

    PlasmidCentral primer(s)Flanking primer(s)Site/vectorCoordinates (nt)Reference or source
    pSP54 XbaI-NheI/pSP5190504–94860 3
    pSP50 KpnI/pSP38, pGEM89795–94860 3
    pYZ1YUAO3 and -4YUAO1 and -2 EcoRI-NotI/pSP54Δ92227–92255This paper
    pYZ3a YUAO7 and -8YUAO1 and 2 EcoRI-NotI/pSP54Δ92294–92322This paper
    pYZ3′YUAO7 and -8YUAO1 and -2 NsiI-XcmI/pSP54Δ92294–92322This paper
    pYZ4YUAO9 and -10YUAO1 and -2 EcoRI-NotI/pSP54Δ92338–92360This paper
    pYZ5YUAO11 and -12YUAO1 and -2 EcoRI-NotI/pSP54Δ92361–92390This paper
    pYZ6YUAO13 and -14YUAO1 and -2 EcoRI-NotI/pSP54Δ92227–92322This paper
    pYZ7YUAO15 and -16YUAO1 and -2 EcoRI-NotI/pSP54Δ92400–92420This paper
    pYZ8YUAO17 and -18YUAO1 and -2 EcoRI-NotI/pSP54Δ92439–92453This paper
    pYZ9YUAO19 and -20YUAO1 and -2 NsiI-XcmI/pSP54Δ92471–92492 17
    pYZ9RRP1 and RP2 NsiI-XcmI/pSP54Δ92471–92492 17
    pYZ9R29RP1 and 9RP2 NsiI-XcmI/pSP54Δ92471–92492 17
    pYZ10YUAO21 and -22YUAO1 and -2 EcoRI-NotI/pSP54Δ92506–92516This paper
    pYZ11YUAO23 and -24YUAO1 and -2 EcoRI-NotI/pSP54Δ92517–92533This paper
    pYZ12YUAO25 and -26YUAO1 and -2 EcoRI-NotI/pSP54Δ92542–92573This paper
    pYZ13YUAO31 and -32YUAO29 and -30 BsmI-EcoRI/pSP54Δ91498–91697This paper
    pYZ14YUAO33 and -34YUAO29 and -30 BsmI-EcoRI/pSP54Δ91598–91797This paper
    pYZ15YUAO35 and -36YUAO29 and -30 BsmI-EcoRI/pSP54Δ91698–91897This paper
    pYZ15L PstI-XcmI/pYZ14/pYZ15Δ91598–91897This paper
    pYZ15R PstI-XcmI/pYZ15/pYZ16Δ91698–91997This paper
    pYZ15LR PstI-XcmI/pYZ14/pYZ16Δ91598–91997This paper
    pYZ16YUAO37 and -38YUAO29 and -30 BsmI-EcoRI/pSP54Δ91798–91997This paper
    pYZ17YUAO39 and -40YUAO29 and -30 BsmI-EcoRI/pSP54Δ91898–92097This paper
    pYZ18YUAO41YUAO29 BsmI-EcoRI/pSP54Δ91998–92219This paper
    pYZ18L PstI-XcmI/pYZ17/pYZ18Δ91898–92219This paper
    pYZ18LL PstI-XcmI/pYZ16/pYZ18Δ91798–92219This paper
    pSP68 EcoRI-ExoIII/pSP54Δ92111–92391 3
    pYZ19YUAO43 and -44YUAO1 and 92654 EcoRI-XcmI/pSP54Δ92400–92573This paper
    pSP90 NotI,ExoIII/pSP61Δ92454–92979This paper
    pSP72-24 SphI,ExoIII/pSP62Δ92574–92979This paper
    pYZ20 NotI-BstXI/pSP54Δ92887–93145This paper
    pYZ22 BstXI-BamHI/pSP54Δ93142–93361This paper
    pLH13Δ PstI/pSP50Kan13Δ93163–93561This paper
    pLH34Δ PstI/pSP50Kan34Δ93701–94370This paper
    pLH50Δ PstI/pSP50Kan50Δ93561–94370This paper
    • ↵a pYZ3 contains a single base substitution in the remaining 29-bp repeat as indicated at the bottom of Fig. 4A.

  • Table 2.

    Oligonucleotides used in mutagenesis

    Oligo- nucleo- tideSequenceCoordinates (nt)
    YUAO15′-GGCTTCTCCGTCTACCGG-3′91963–91970
    YUAO25′-CTCGCGCTCCCTAGGTGC-3′92892–92909
    YUAO35′-CACGCATACGCCGTATGTCCGGAATTCC-3′92209–92265
    YUAO45′-GGACATACGGCGTATGCGTGCGTCATCT-3′92217–92273
    YUAO75′-AACGGACTGATCAGAGATGATTTCCGCC-3′92277–92332
    YUAO85′-TCATCTCTGATCAGTCCGTTTTACGTAT-3′92284–92340
    YUAO95′-GCCCCTACGGCGTAAAACGGACTGATGA-3′92320–92370
    YUAO105′-CCGTTTTACGCCGTAGGGGCGGAGCCTA-3′92328–92378
    YUAO115′-TATGCATCCTGGCGTCGCCTAGCATCCG-3′92343–92400
    YUAO125′-AGGCGACGCCAGGATGCATACCCTATAT-3′92351–92408
    YUAO135′-AACGGACTGACCGTATGTCCGGAATTCC-3′92209–92332
    YUAO145′-GGACATACGGTCAGTCCGTTTTACGTAT-3′92217–92340
    YUAO155′-GTTTCGTTAGCTGCAGATGCATCCTAGGGGTGG-3′92383–92430
    YUAO165′-TAGGATGCATCTGCAGCTAACGAAACGTTCTAC-3′92390–92437
    YUAO175′-GGTTCCCGTTCTGCAGCGTAGAACGTTTCGTTA-3′92422–92463
    YUAO185′-ACGTTCTACGCTGCAGAACGGGAACCACCGTAA-3′92429–92470
    YUAO195′-CGGAGGAGAACTGCAGTTACGGTGGTTCCCGTT-3′92454–92502
    YUAO205′-ACCACCGTAACTGCAGTTCTCCTCCGGAACCGG-3′92461–92509
    YUAO215′-GTAAAAATTTCTGCAGTTCCGGAGGAGAAGGGG-3′92489–92526
    YUAO225′-TCCTCCGGAACTGCAGAAATTTTTACCAAATTT-3′92496–92533
    YUAO235′-ATGGTTGCCCCTGCAGGCCCCCCCCGGTTCCGG-3′92500–92543
    YUAO245′-CGGGGGGGGCCTGCAGGGGCAACCATGATTTCC-3′92507–92550
    YUAO255′-GACTGCGCATCTGCAGGGTTGCCCAAATTTGGT-3′92525–92583
    YUAO265′-TTGGGCAACCCTGCAGATGCGCAGTCGGGGCGA-3′92532–92590
    YUAO295′-TGTAACCTGAAACCGCCGTG-3′91381–91400
    YUAO305′-CCGTATGTCCGGAATTCCAC-3′92207–92226
    YUAO315′-GACGTTGGCACTGCAGCGATCGCCACATTCGAT-3′91481–91707
    YUAO325′-GTGGCGATCGCTGCAGTGCCAACGTCATAATCA-3′91488–91714
    YUAO335′-ATATGGCTACCTGCAGTGCCTGTTCTTATGCCG-3′91581–91807
    YUAO345′-AGAACAGGCACTGCAGGTAGCCATATCCGCTTA-3′91588–91814
    YUAO355′-CGTACAAGGGCTGCAGTCTGGCACCGCCTCTTG-3′91681–91907
    YUAO365′-CGGTGCCAGACTGCAGCCCTTGTACGGAAATTT-3′91688–91914
    YUAO375′-AGCGTCTACGCTGCAGACGTAATGGGTGTGGCT-3′91781–92007
    YUAO385′-CCCATTACGTCTGCAGCGTAGACGCTACTCCCG-3′91788–92014
    YUAO395′-ACAGAGGAAGCTGCAGGTGGAGTCTAGGGAGGG-3′91881–92107
    YUAO405′-TAGACTCCACCTGCAGCTTCCTCTGTTTTGGCC-3′91888–92114
    YUAO415′-TCCGGAATTCCTGCAGTCAGCAGGTGTATATTT-3′91981–92219
    YUAO425′-CACCTGCTGACTGCAGGAATTCCGGACATACGG-3′91988–92226
    YUAO435′-GACTGCGCATCTGCAGATGCATCCTAGGGGTGG-3′92383–92583
    YUAO445′-TAGGATGCATCTGCAGATGCGCAGTCGGGGCGA-3′92390–92590
    926545′-CGGCGCATGCGCACTCGAGT-3′92635–92654
    RP15′-TTCTAGAACCGCTGGATGCA-3′
    RP25′-TCCAGCGGTTCTAGAATGCA-3′
    9RP15′-TTCTAGACGCGATGCA-3′
    9RP25′-TCGCGTCTAGAATGCA-3′
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Human Cytomegalovirus oriLyt Sequence Requirements
Yuao Zhu, Lili Huang, David G. Anders
Journal of Virology Jun 1998, 72 (6) 4989-4996; DOI: 10.1128/JVI.72.6.4989-4996.1998

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Human Cytomegalovirus oriLyt Sequence Requirements
Yuao Zhu, Lili Huang, David G. Anders
Journal of Virology Jun 1998, 72 (6) 4989-4996; DOI: 10.1128/JVI.72.6.4989-4996.1998
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