Herpes simplex virus latency-associated transcript sequence downstream of the promoter influences type-specific reactivation and viral neurotropism

Abstract

Herpes simplex virus (HSV) establishes latency in sensory nerve ganglia during acute infection and may later periodically reactivate to cause recurrent disease. HSV type 1 (HSV-1) reactivates more efficiently than HSV-2 from trigeminal ganglia while HSV-2 reactivates more efficiently than HSV-1 from lumbosacral dorsal root ganglia (DRG) to cause recurrent orofacial and genital herpes, respectively. In a previous study, a chimeric HSV-2 that expressed the latency-associated transcript (LAT) from HSV-1 reactivated similarly to wild-type HSV-1, suggesting that the LAT influences the type-specific reactivation phenotype of HSV-2. To further define the LAT region essential for type-specific reactivation, we constructed additional chimeric HSV-2 viruses by replacing the HSV-2 LAT promoter (HSV2-LAT-P1) or 2.5 kb of the HSV-2 LAT sequence (HSV2-LAT-S1) with the corresponding regions from HSV-1. HSV2-LAT-S1 was impaired for reactivation in the guinea pig genital model, while its rescuant and HSV2-LAT-P1 reactivated with a wild-type HSV-2 phenotype. Moreover, recurrences of HSV-2-LAT-S1 were frequently fatal, in contrast to the relatively mild recurrences of the other viruses. During recurrences, HSV2-LAT-S1 DNA increased more in the sacral cord compared to its rescuant or HSV-2. Thus, the LAT sequence region, not the LAT promoter region, provides essential elements for type-specific reactivation of HSV-2 and also plays a role in viral neurotropism. HSV-1 DNA, as quantified by real-time PCR, was more abundant in the lumbar spinal cord, while HSV-2 DNA was more abundant in the sacral spinal cord, which may provide insights into the mechanism for type-specific reactivation and different patterns of central nervous system infection of HSV-1 and HSV-2.

FIG. 1.

Virus construction. Genomic and endonuclease restriction sites are shown relevant to HSV-2 strain 333 and HSV-1 strain 17+. The HSV genome consists of unique long and short (U_L and U_S) regions flanked by internal and terminal repeats (IR_L/IR_S and TR_L/TR_S). The primary LAT is transcribed from the repeat regions in the antisense direction from ICP0 and ICP34.5 (transcripts are shown here as lines, with arrowheads indicating the direction of transcription). The primary LAT is spliced into a single stable LAT intron in HSV-2 and two differentially spliced introns in HSV-1. Boxes on the lines denote regions that are spliced out of transcripts and not easily detectable after splicing. Within the SphI-BamHI fragment, the black region from NotI to PvuI sites was the replacement region for HSV2-LAT-P1 and the shaded region from PvuI to XhoI sites was the replacement region for HSV2-LAT-S1. Together, they represent the replacement region in the previously described HSV-2 333/LAT1.

FIG. 2.

Acute lesion severity in acutely infected female guinea pigs. Lesion severity is graphed as the mean for each group of guinea pigs on each day of observation from days 1 to 14 p.i., with 0 being no symptoms and 4 being the most severe. HSV-2, n = 21; HSV-1, n = 6; HSV2-LAT-P1, n = 12; HSV2-LAT-S1, n = 15; HSV2-LAT-S1-R, n = 8.

FIG. 3.

Percentages of guinea pigs from each group experiencing urinary tract dysfunction (UTD) on days 1 to 14 p.i., indicative of autonomic nervous system involvement HSV-2, n = 21; HSV-1, n = 6; HSV2-LAT-P1, n = 12; HSV2-LAT-S1, n = 15; HSV2-LAT-S1-R, n = 8.

FIG. 4.

Cumulative recurrences per guinea pig for each group, adjusted for number of days of observation. HSV-2, n = 19; HSV-1, n = 5; HSV2-LAT-P1, n = 12; HSV2-LAT-S1, n = 13; HSV2-LAT-S1-R, n = 8. Guinea pigs that did not survive acute disease were excluded. Significant P values are as follows: HSV-2 versus HSV-1, 0.023; HSV-2 versus HSV2-LAT-S1, 0.005; HSV-1 versus HSV2-LAT-P1, 0.011; HSV2-LAT-S1 versus HSV2-LAT-P1, 0.006.

FIG. 5.

Survival of infected guinea pigs. HSV-2, n = 21; HSV-1, n = 6; HSV2-LAT-P1, n = 12; HSV2-LAT-S1, n = 15; HSV2-LAT-S1-R, n = 8.

FIG. 6.

Quantities of viral DNA in the DRG, sacral spinal cord, and lumbar spinal cord, quantified by Taqman PCR assay and normalized to the gene for the 18S rRNA. (A) Viral DNA copy numbers quantified in guinea pig tissues obtained during latent infection with no observable symptoms. HSV-1 differs significantly from all HSV-2-based viruses in the lumbar cord (P < 0.0005). HSV2-LAT-P1, n = 12; HSV-2, n = 13; HSV2-LAT-S1, n = 7; HSV2-LAT-S1-R, n = 2; HSV-1, n = 3. (B) Viral DNA copy numbers quantified in guinea pig tissues obtained during symptomatic recurrences. HSV-1 differs significantly from HSV-2 viruses in the sacral cord (P < 0.022) and lumbar cord (P < 0.0005). HSV2-LAT-S1 differs significantly in the DRG from HSV-2 (P = 0.021) and in the sacral cord from HSV-2 (P = 0.007), HSV2-LAT-S1-R (P = 0.007), and HSV-1 (P < 0.0005). HSV2-LAT-P1, no tissues extracted during symptomatic recurrences; HSV-2, n = 5; HSV2-LAT-S1, n = 5; HSV2-LAT-S1-R, n = 4; HSV-1, n = 2.