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Pathogenesis and Immunity

Identical 371-Base-Pair Deletion Mutations in the LAT Genes of Herpes Simplex Virus Type 1 McKrae and 17syn+ Result in Different In Vivo Reactivation Phenotypes

Jeannette M. Loutsch, Guey-Chuen Perng, James M. Hill, Xiaodong Zheng, Mary E. Marquart, Timothy M. Block, Homayon Ghiasi, Anthony B. Nesburn, Steven L. Wechsler
Jeannette M. Loutsch
LSU Eye Center, Department of Ophthalmology, Microbiology and Immunology, and Department of Pharmacology, Louisiana State University Medical Center School of Medicine, New Orleans, Louisiana 70112-2234;
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Guey-Chuen Perng
Ophthalmology Research Laboratories, Cedars-Sinai Medical Center Burns and Allen Research Institute, Los Angeles, California 90048;
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James M. Hill
LSU Eye Center, Department of Ophthalmology, Microbiology and Immunology, and Department of Pharmacology, Louisiana State University Medical Center School of Medicine, New Orleans, Louisiana 70112-2234;
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Xiaodong Zheng
LSU Eye Center, Department of Ophthalmology, Microbiology and Immunology, and Department of Pharmacology, Louisiana State University Medical Center School of Medicine, New Orleans, Louisiana 70112-2234;
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Mary E. Marquart
LSU Eye Center, Department of Ophthalmology, Microbiology and Immunology, and Department of Pharmacology, Louisiana State University Medical Center School of Medicine, New Orleans, Louisiana 70112-2234;
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Timothy M. Block
Jefferson Center for Biomedical Research, Thomas Jefferson College, Doylestown, Pennsylvania 18901; and
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Homayon Ghiasi
Ophthalmology Research Laboratories, Cedars-Sinai Medical Center Burns and Allen Research Institute, Los Angeles, California 90048;
Department of Ophthalmology, UCLA School of Medicine, Los Angeles, California 90024
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Anthony B. Nesburn
Ophthalmology Research Laboratories, Cedars-Sinai Medical Center Burns and Allen Research Institute, Los Angeles, California 90048;
Department of Ophthalmology, UCLA School of Medicine, Los Angeles, California 90024
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Steven L. Wechsler
Ophthalmology Research Laboratories, Cedars-Sinai Medical Center Burns and Allen Research Institute, Los Angeles, California 90048;
Department of Ophthalmology, UCLA School of Medicine, Los Angeles, California 90024
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DOI: 10.1128/JVI.73.1.767-771.1999
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    Fig. 1.

    Structures of dLAT371 and 17ΔSty. The top of the figure shows a schematic representation of the prototypic orientation of HSV-1 (strains McKrae and 17syn+). HSV-1 contains a unique long region and a unique short region, each of which is flanked by inverted repeats. The unique regions are shown as solid lines. The repeats are shown as open rectangles. UL, unique long; US, unique short; TRL, terminal repeat, long; IRL, internal repeat, long; TRS, terminal repeat, short; IRS, internal repeat, short. The long-repeat regions are expanded to show details of the LAT domain and are indicated by the dashed lines. The locations of the genes for ICP0 and ICP34.5 are shown for reference. (A) Detailed enlargement of the internal-repeat region of parental and marker-rescued viruses. This region contains the 8.3-kb primary LAT transcript. The direction of transcription is indicated by the arrowhead. TATA indicates the location (in the genomic DNA) of the LAT promoter TATA box. The start of LAT transcription is indicated as +1, corresponding to nt 118801 of the genome (15, 20). The filled rectangle within the primary 8.3-kb LAT transcript indicates the location of the stable 2-kb LAT intron starting at LAT nt 662. The locations and directions of the ICP34.5 and ICP0 transcripts are shown for reference. In all viruses shown here, the copy of LAT in the terminal long repeat is identical to the internal long repeat (enlargement shown). (B) dLAT371, a McKrae mutant. The 371-nt deletion is indicated by the break in the line and is bounded by the StyI restriction sites at LAT nt 76 and 447. This deletion is present in both copies of LAT (one in each long repeat). (C) 17ΔSty, a 17syn+ mutant. TheStyI-StyI deletion is identical to that shown in panel B. (D) dLAT2903, a McKrae LAT null mutant. The deletion from nt −161 to +1667 encompasses part of the LAT promoter containing the TATA box. The region of LAT downstream of theHpaI site is drawn with a dashed line to indicate thatdLAT2903 makes no LAT RNA.

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  • Table 1.

    In vivo spontaneous reactivationa

    Virus or P value comparisonNo. of virus-positive rabbits/total no. of rabbits (%) or P valueNo. of virus-positive eyes/total no. of eyes (%) or P valueNo. of virus-positive eye swabs/total no. of eye swabsb (%) or P value
    Viruses
     17syn+6/8 (75)11/15 (73)35/297 (11.8)
     17ΔSty3/13 (23)4/25 (16)11/492 (2.2)
     17ΔSty-Res7/10 (70)13/19 (64)38/377 (10.1)
    P value comparisonc
     17ΔSty vs 17syn+0.030.0005<0.0001
     17ΔSty vs 17ΔSty-Res0.040.0006<0.0001
     17syn+ vs 17ΔSty-Res1.0 1.0   0.53  
    Virusesd
     McKrae25/30 (83)44/60 (73)183/1,570 (11.7)
     dLAT3715/6 (83)9/12 (75)38/290 (13.1)
     dLAT290313/28 (46)15/56 (27)37/1,516 (2.4)
    P value comparisonc
     17syn+ vs McKrae0.621.0     0.42  
     17ΔSty vs dLAT3710.040.0008<0.0001
     17ΔSty vsdLAT29030.190.40    0.93  
    • ↵a Rabbits were infected with 2 × 105 PFU of the indicated virus per eye. Tears (eye swabs) were collected daily from latently infected rabbits from day 20 to 39 p.i. Spontaneous reactivation was monitored by culturing tears for the presence of infectious HSV-1 as described previously (7).

    • ↵b The total numbers of cultures are slightly less than the numbers of eyes times 20 days, because occasional cultures were lost due to contamination.

    • ↵c Two-sided Fisher exact test or chi-square test if the numbers were too large for the Fisher exact test. Analyses were done with the personal computer program Instat. A P of <0.05 was considered significant.

    • ↵d Data reproduced from a previous publication (19).

  • Table 2.

    In vivo epinephrine-induced ocular reactivation in latently infected rabbitsa

    Virus or P value comparisonNo. of virus-positive rabbitsb/total no. of rabbits (%) or P valueNo. of virus-positive eyes/total no. of eyes (%) or P valueNo. of virus-positive eye swabs/total no. of eye swabs (%) or P value
    Viruses
     McKrae4/7 (57)7/14 (50)23/98 (24)
     dLAT3717/7 (100)9/14 (64)20/98 (20)
     dLAT29034/16 (25)4/32 (13)8/192 (4)
    P value comparisonsc
     dLAT371 vs McKrae0.19 0.70    0.73  
     dLAT371 vsdLAT29030.0010.0008<0.0001
     dLAT2903 vs McKrae0.18 0.01  <0.0001
    Virusesc
     17syn+7/11 (64)12/22 (55)48/154 (31)
     17ΔSty2/9 (22)2/18 (11)4/126 (3)
     17ΔSty-Res5/8 (63)10/16 (63)35/112 (31)
    P value comparisons
     McKrae vs 17syn+1.0  1.0     0.2   
     dLAT371 vs 17ΔSty0.0030.003 <0.0001
     dLAT2903 vs 17ΔSty1.0  1.0     0.77  
     17ΔSty-Res vs 17syn+1.0  0.74   1.0   
    • ↵a Rabbits were infected with 2 × 105 PFU of the indicated virus per eye and subjected to epinephrine iontophoresis approximately 30 days p.i., and in vivo-induced reactivation was monitored by culturing tears daily for 7 consecutive days, as described previously (7). Data presentation and statistical analyses are as described for Table 1.

    • ↵b Number of rabbits in each group that had at least one virus-positive tear culture within 7 days of induction.

    • ↵c Data reproduced from a previous publication (7).

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Identical 371-Base-Pair Deletion Mutations in the LAT Genes of Herpes Simplex Virus Type 1 McKrae and 17syn+ Result in Different In Vivo Reactivation Phenotypes
Jeannette M. Loutsch, Guey-Chuen Perng, James M. Hill, Xiaodong Zheng, Mary E. Marquart, Timothy M. Block, Homayon Ghiasi, Anthony B. Nesburn, Steven L. Wechsler
Journal of Virology Jan 1999, 73 (1) 767-771; DOI: 10.1128/JVI.73.1.767-771.1999

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Identical 371-Base-Pair Deletion Mutations in the LAT Genes of Herpes Simplex Virus Type 1 McKrae and 17syn+ Result in Different In Vivo Reactivation Phenotypes
Jeannette M. Loutsch, Guey-Chuen Perng, James M. Hill, Xiaodong Zheng, Mary E. Marquart, Timothy M. Block, Homayon Ghiasi, Anthony B. Nesburn, Steven L. Wechsler
Journal of Virology Jan 1999, 73 (1) 767-771; DOI: 10.1128/JVI.73.1.767-771.1999
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KEYWORDS

Herpesvirus 1, Human
Virus Activation
Virus Latency

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