Cellular FLIP can substitute for the herpes simplex virus type 1 latency-associated transcript gene to support a wild-type virus reactivation phenotype in mice

Abstract

Latency-associated transcript (LAT) deletion mutants of herpes simplex virus type 1 (HSV-1) have reduced reactivation phenotypes. Thus, LAT plays an essential role in the latency-reactivation cycle of HSV-1. We have shown that LAT has antiapoptosis activity and demonstrated that the chimeric virus, dLAT-cpIAP, resulting from replacing LAT with the baculovirus antiapoptosis gene cpIAP, has a wild-type HSV-1 reactivation phenotype in mice and rabbits. Thus, LAT can be replaced by an alternative antiapoptosis gene, confirming that LAT's antiapoptosis activity plays an important role in the mechanism by which LAT enhances the virus' reactivation phenotype. However, because cpIAP interferes with both of the major apoptosis pathways, these studies did not address whether LAT's proreactivation phenotype function was due to blocking the extrinsic (Fas-ligand-, caspase-8-, or caspase-10-dependent pathway) or the intrinsic (mitochondria-, caspase-9-dependent pathway) pathway, or whether both pathways must be blocked. Here we constructed an HSV-1 LAT(-) mutant that expresses cellular FLIP (cellular FLICE-like inhibitory protein) under control of the LAT promoter and in place of LAT nucleotides 76 to 1667. Mice were ocularly infected with this mutant, designated dLAT-FLIP, and the reactivation phenotype was determined using the trigeminal ganglia explant model. dLAT-FLIP had a reactivation phenotype similar to wild-type virus and significantly higher than the LAT(-) mutant dLAT2903. Thus, the LAT function responsible for enhancing the reactivation phenotype could be replaced with an antiapoptosis gene that primarily blocks the extrinsic signaling apoptosis pathway.

Figure 1

Structure of the LAT region of dLAT-FLIP. (A) HSV-1 genomic structure. TRL and IRL indicate the viral long repeats (terminal and internal). IRS and TRS indicate the viral short repeats. UL and US indicate the long and short unique regions. The dashed lines indicate that the region of the TRL and IRL are expanded below with the TRL inverted relative to the IRL so that both identical regions can be represented by a single image in the subsequent schematics. (B) Wild-type (wt) McKrae. The LAT promoter is indicated by open rectangle. The solid black rectangle indicates the stable 2-kb LAT. The large arrow indicates the extent of the primary 8.3-kb LAT RNA. The relative locations of ICP0 and ICP34.5 are shown for reference, as are various restriction enzyme sites. LAT transcription starts at LAT nucleotide (nt) +1 (genomic nt 118,801 for the copy of LAT in the ILR). All nt numbers are shown relative LAT nt +1. The location of the LAT TATA box is shown at nt −28. (C) dLAT2903. dLAT2903 is deleted from LAT nt −161 to +1667 (XXXXXX). dLAT2903 is a true LAT-null mutant that is missing primary LAT promoter elements between −161 and +1. dLAT2903 is also deleted for a putative secondary LAT promoter, LAP2, located within the 5’ end of the primary LAT transcript prior to the start of the 2-kb LAT. This mutant therefore is not capable of expressing any LAT RNA (dashed lines) (Perng et al, 1994). (D) dLAT-FLIP contains the antiapoptosis gene FLIP followed by a poly-A signal inserted in place of LAT nts 76 to 1667. The entire LAT promoter is present. No LAT RNA is transcribed past the poly-A site.

Figure 2

Southern hybridization analysis of dLAT-FLIP genomic structure. dLAT-FLIP (F), wt McKrae (wt), and dLAT2903 (dL) viral DNAs were purified, digested with SalI, separated using a 0.8% agarose gel, transferred to a membrane, and hybridized to a ³²P-labeled SmaI-EcoRV (LAT nts −864 to −161) restriction fragment as described in the text. The numbers indicate the approximate size in base pairs of the restriction fragments.

Figure 3

Replication of dLAT-FLIP in RS cells. Cell monolayers were infected with virus at a low MOI of 0.01. At the indicated times, infected cell monolayers with the culture medium were harvested, freeze-thawed to release virus, and total infectious virus was determined using standard plaque assays.

Figure 4

Replication of dLAT-FLIP in mouse eyes. Mice were ocularly infected with 2 × 10⁵ PFU/eye. Tear swabs were collected on the indicated days. Each symbol represents the average titer for each group (n = 20). The results show the combined data from two independent experiments.

Figure 5

Western blot detection of FLIP protein expressed by dLAT-FLIP. RS cells were infected at an MOI of 0.1 with wild-type McKrae (wt) or dLAT-FLIP (F). Total cell extracts were harvested at the indicated times post infection, separated by SDS-PAGE, transferred to a membrane, and probed using rabbit FLIP-specific antibody (Anaspec). The antibody bound to the blots was visualized with anti-rabbit IgG conjugated to horseradish peroxidase (Chemicom). The arrows indicate the sizes of the two main forms of FLIP. Lane M = molecular weight markers in kDa.

Figure 6

Explant reactivation of dLAT-FLIP from mouse TG. Eight- to 10-week-old female Swiss Webster mice were infected with 2 × 10⁵ PFU/eye of virus. On day 30 p.i. individual TG were explanted into tissue culture medium. Aliquots were removed daily and plated on indicator cells (RS cells) to look for the presence and time of first appearance of reactivated virus. The numbers next to each plot are the number of TG that reactivated (i.e., produced infectious virus)/the total number of TG in that group. F = dLAT-FLIP; wt = wild-type McKrae. P value calculated by Kaplan-Meier survival analysis. The combined results of three experiments are shown.

Figure 7

Survival of mice from the experiment shown in Figure 6. Numbers above each bar represent the number of mice the survived/total number of mice. P values are compared to dLAT-FLIP and were determined by the Fisher’s exact test.