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JM Hill, R Tran, S Higaki, JM Loutsch, ME Myles, K Maruyama, DC Bloom; Analysis of Spacing and Configuration Requirements within the 5' End of the Latently Associated Transcript (LAT ) Related to Epinephrine-Induced Reactivation in the Rabbit Eye Model of HSV Latency . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4321.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine, by the use of recombinant HSV-1, if the spacing and/or essential elements in the 5' end of LAT are necessary for HSV reactivation. Methods: The following recombinant viruses were constructed from the parental HSV-1 strain 17Syn+: 17Δ;348 (a 348 base pair deletion in the 5' end of LAT); 17Δ;110, 17Δ;91, 17Δ;116 (these make up most of the 348 bp deletion in 17Δ;d 348); a 17Δ;348 stuffer (a sequence from the E. coli Lac Z gene replacing the 348 bp); 17Δ;201 and 17Δ;207 (covering together the entire 348 base pair region); and 17Δ;348 rescue. Scarified rabbit corneas received 1 x 105 PFU per eye of viral suspension. Acute infection of the corneal epithelium was assessed by slit lamp examination (SLE). At 4-5 weeks postinoculation, rabbits underwent transcorneal iontophoresis of 0.015% epinephrine once daily for 3 consecutive days; ocular swabs were done for 7 consecutive days beginning on the day after the first iontophoresis. Quantitative PCR of rabbit trigeminal ganglia was done to assess HSV copy number. Results: All viruses showed similar SLE scores from PI day 3 through 12. Two recombinant viruses -- 17Δ;348 and 17Δ;348 stuffer -- showed significantly lower induced reactivation following epinephrine iontophoresis. The other five recombinant viruses with smaller deletions spanning the 348 region (17Δ;110, 17Δ;91, 17Δ;116, 17Δ;207, 17Δ;201) had high ocular reactivation, similar to that of the parent, 17Syn+. Semi-quantitative PCR showed no significant differences in HSV genome copy number in the trigeminal ganglia for all viruses tested. Conclusion: The 348 base pair deletion and substitution with nonsense in the LAT 5' exon results in a significant decrease in reactivation following epinephrine iontophoresis. This demonstrates that this entire region contains an essential element or elements for reactivation. Since the smaller deletions retain the parental phenotype, we suggest that spacing and configuration, in addition to the sequence, are essential components for HSV-induced reactivation from elements in this region.
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