The latency-associated transcript gene enhances establishment of herpes simplex virus type 1 latency in rabbits

Abstract

The latency-associated transcript (LAT) gene the only herpes simplex virus type 1 (HSV-1) gene abundantly transcribed during neuronal latency, is essential for efficient in vivo reactivation. Whether LAT increases reactivation by a direct effect on the reactivation process or whether it does so by increasing the establishment of latency, thereby making more latently infected neurons available for reactivation, is unclear. In mice, LAT-negative mutants appear to establish latency in fewer neurons than does wild-type HSV-1. However, this has not been confirmed in the rabbit, and the role of LAT in the establishment of latency remains controversial. To pursue this question, we inserted the gene for the enhanced green fluorescent protein (EGFP) under control of the LAT promoter in a LAT-negative virus (DeltaLAT-EGFP) and in a LAT-positive virus (LAT-EGFP). Sixty days after ocular infection, trigeminal ganglia (TG) were removed from the latently infected rabbits, sectioned, and examined by fluorescence microscopy. EGFP was detected in significantly more LAT-EGFP-infected neurons than DeltaLAT-EGFP-infected neurons (4.9% versus 2%, P < 0.0001). The percentages of EGFP-positive neurons per TG ranged from 0 to 4.6 for DeltaLAT-EGFP and from 2.5 to 11.1 for LAT-EGFP (P = 0.003). Thus, LAT appeared to increase neuronal latency in rabbit TG by an average of two- to threefold. These results suggest that LAT enhances the establishment of latency in rabbits and that this may be one of the mechanisms by which LAT enhances spontaneous reactivation. These results do not rule out additional LAT functions that may be involved in maintenance of latency and/or reactivation from latency.

FIG. 1

Structure of LAT-EGFP and ΔLAT-EGFP viruses. (A) Structure of the HSV-1 McKrae genome in the prototypic orientation. The open rectangles represent the repeat regions of the virus (TRL, terminal repeat long; IRL, internal repeat long; IRS, internal repeat short; TRS, terminal repeat short) that bound the unique long (UL) and unique short (US) regions. The long repeats are expanded to show more detailed structure of the LAT region (one in each long repeat). The largest arrow represents the location of the primary LAT. Locations of the ICP0 and ICP34.5 transcripts are shown for reference. The solid rectangle represents the very stable 2-kb LAT. The start of LAT transcription is indicated by the arrow at +1. (B) dLAT2903 has a deletion from LAT nucleotides −161 to +1667 in both copies of LAT, makes no LAT RNA, and reactivates poorly. We have previously described the construction and properties of dLAT2903 (16). (C) ΔLAT-EGFP was constructed from dLAT2903 by homologous recombination between dLAT2903 DNA and a plasmid containing the complete LAT promoter and the entire structural EGFP gene [including a 3′ poly(A) signal] flanked by regions of LAT contained in dLAT2903 (LAT nucleotides −798 to +76 and 1667 to 1850) as described in Materials and Methods. The resulting virus contains two copies of EGFP, one in each long repeat, under transcriptional control of the LAT promoter. (D) LAT-EGFP was derived from ΔLAT-EGFP (vertical arrow) by insertion by homologous recombination of a 3.3-kb restriction fragment comprising the LAT promoter and the first 1.5 kb of LAT in an ectopic location between UL37 and UL38. We previously showed that insertion of this 3.3-kb LAT fragment into this location completely restored wt levels of spontaneous reactivation to the LAT null mutant dLAT2903 (19). (E) LAT3.3A (previously designated LAT1.5a) (19) is deleted for LAT in both long repeats, contains the 3.3-kb LAT ectopic insert described in panel D, and has wt levels of spontaneous reactivation. (F) LAT-EGFP-2 was constructed from LAT3.3A (vertical arrow) by insertion of the LAT promoter and the entire structural EGFP gene [including a 3′ poly(A) signal] as described for panel C. The final structures of LAT-EGFP and LAT-EGFP-2 should be identical. The structures of all the viruses were confirmed by restriction enzyme digestion and Southern analyses.

FIG. 2

Detection of EGFP in TG of rabbits latently infected with LAT-EGFP and ΔLAT-EGFP. Rabbits were bilaterally ocularly infected with the HSV-1 McKrae strain-derived mutant LAT-EGFP or ΔLAT-EGFP (2 × 10⁵ PFU/eye) without corneal scarification as described in Materials and Methods. Sixty days postinfection, the rabbits were euthanized and the TG were removed. As described in Materials and Methods, frozen TG sections were prepared and the presence of EGFP was determined, without additional manipulation, by immunofluorescence microscopy. Representative results are shown. (A) TG from a rabbit latently infected with LAT-EGFP; (B) TG from a rabbit latently infected with ΔLAT-EGFP; (C) TG from an uninfected rabbit. Panel C is slightly overexposed compared to panels A and B to confirm the lack of EGFP fluorescence. The arrows indicate neurons that were scored positive for EGFP.

FIG. 3

Quantitation of EGFP-positive neurons in TG of rabbits latently infected with LAT-EGFP or ΔLAT-EGFP. (A) Total EGFP-positive neurons. TG from rabbits latently infected with LAT-EGFP (one TG from each of 8 rabbits) or ΔLAT-EGFP (one TG from each of 11 rabbits) were sectioned and examined for EGFP-positive neurons as described in Materials and Methods. In the ΔLAT-EGFP group, 20,858 neurons were examined for EGFP on 68 sections from 11 TG (average of 6.2 sections/TG). In the LAT-EGFP group, 12,070 neurons were examined for EGFP on 49 sections from 8 TG (average of 6.1 sections/TG). P for the number of EGFP-positive neurons in TG from LAT-EGFP versus ΔLAT-EGFP latently infected rabbits was determined by the Student t test. (B) Percentage of EGFP-positive neurons per TG. Each point represents the percentage of EGFP-positive neurons in a single TG. The horizontal bars indicate the means for each group. P was determined by the Mann-Whitney rank sum test.

FIG. 4

Distribution of EGFP-positive neurons in sections from individual TG. The percentage of EGFP-positive neurons in each of the sections from the 19 TG in Fig. 3 is shown. Each point represents the percentage of EGFP-positive neurons on a single section. Each vertical collection of points represents a single TG. The horizontal bars indicate the mean for each TG. For ease of comparison, the TG are arranged in order from the lowest to the highest percentage of EGFP-positive neurons.

FIG. 5

Replication of LAT-EGFP and ΔLAT-EGFP in rabbit eyes. Rabbits were ocularly infected, and tear films were collected at the times shown as described in Materials and Methods. The amount of virus recovered from individual eyes was determined by plaque assays on RS cells. Each point is the mean ± standard deviation of five eyes (one per rabbit). P.I., postinfection.

FIG. 6

Serum neutralizing antibody in rabbits 60 days postinfection. Rabbits were infected as described for Fig. 5. Serum was collected 60 days postinfection (>30 after latency had been established). HSV-1 neutralizing antibody titers were determined on individual serum samples as described in Materials and Methods. Each point represents the reciprocal of the neutralizing antibody titer for one serum sample. The horizontal bars and numbers show the mean neutralizing antibody titer. P values were determined by the Student t test.