Regions of the herpes simplex virus type 1 latency-associated transcript that protect cells from apoptosis in vitro and protect neuronal cells in vivo

Abstract

Recent studies have suggested that the latency-associated transcript (LAT) region of herpes simplex virus type 1 (HSV-1) is effective at blocking virus-induced apoptosis both in vitro and in the trigeminal ganglia of acutely infected rabbits (Inman et al., J. Virol. 75:3636-3646, 2001; Perng et al., Science 287:1500-1503, 2000). By transfecting cells with a construct expressing the Pst-Mlu segment of the LAT, encompassing the LAT exon 1, the stable 2.0-kb intron, and 5' part of exon 2, we confirmed that this region was able to diminish the onset of programmed cell death initiated by anti-Fas and camptothecin treatment. In addition, caspase 8-induced apoptosis was specifically inhibited in cells expressing the Pst-Mlu LAT fragment. To further delineate the minimal region of LAT that is necessary for this antiapoptotic function, LAT mutants were used in our cotransfection assays. In HeLa cells, the plasmids lacking exon sequences were the least effective at blocking apoptosis. However, similar to previous work (Inman et al., op. cit.), our data also indicated that the 5' end of the stable 2.0-kb LAT intron appeared to contribute to the promotion of cell survival. Furthermore, cells productively infected with the 17N/H LAT mutant virus, a virus deleted in the LAT promoter, exon 1, and about half of the intron, exhibited a greater degree of DNA fragmentation than cells infected with wild-type HSV-1. These data support the finding that the exon 1 and 2.0-kb intron region of the LAT transcription unit display an antiapoptotic function both in transfected cells and in the context of the virus infection in vitro. In trigeminal ganglia of mice acutely infected with the wild-type virus, 17, and 17DeltaSty, a virus lacking most of exon 1, apoptosis was not detected in cells that were positive for virus particles. However, dual staining was observed in cells from mice infected with 17N/H virus, indicating that the LAT antiapoptotic function demonstrated in cells transfected by LAT-expressing constructs may also play a role in protecting cells from virus-induced apoptosis during acute viral infection in vivo.

FIG. 1.

HSV-1 LATs. (A) Linear map of the HSV-1 genome with its unique long (UL) and unique short (US) regions flanked by inverted repeat (IR) elements. (B) LAT region of the HSV-1 genome. The LAT region is enlarged to show the different LAT transcripts that map to this area, as well as the other RNAs (L/ST’s, ICP0, ICP4, ICP34.5, UL54, UL55, and UL56). The minor LAT (mLAT), the putative 8.5-kb primary transcript, and the potential spliced exons are shown (including 2.0-kb LAT intron). In addition, a linear diagram of the pcDNA3.Pst-Mlu plasmid expressing the 2.0-kb LAT intron and the exon 1 and 2 regions is shown. (C) LAT deletion mutants in the pcDNA3.Pst-Mlu background.

FIG. 2.

In vitro inhibition of apoptosis by LAT in HeLa cells (A) and in neuron-like SY5Y cells (B). Cells were transfected with 1 μg of pEGFP-C1 (GFP-expressing construct) and 3 μg of pcDNA3 vector, pcDNA3.Pst-Mlu, or pCIp35 expressing the baculovirus antiapoptotic protein. At 48 h posttransfection, anti-Fas antibody was added to HeLa cells, while camptothecin was added to SY5Y cultures. At various times after treatment, GFP-positive cells were identified under a fluorescent microscope. ✻, positive control. The number of GFP-positive cells in control untreated cells represents 100% survival. Data are averages from five separate experiments.

FIG. 3.

Inhibition of caspase 8-induced apoptosis by LAT in vitro. HeLa cells were transfected with 3 μg of pcDNA3, pcDNA3.Pst-Mlu, or pICp35 together with 1 μg of pEGFP-C1 and 1 μg of the plasmid expressing caspase 8 (pC8). The pC8 plasmid was not transfected into control cells. (A) At 24 h after transfection, GFP-positive cells were visualized under a fluorescent scope and photographed. (B) GFP-positive cells were counted at 24, 48, and 72 h posttransfection, and data are expressed as the percentage of surviving GFP-positive cells over the control cells.

FIG. 3.

Inhibition of caspase 8-induced apoptosis by LAT in vitro. HeLa cells were transfected with 3 μg of pcDNA3, pcDNA3.Pst-Mlu, or pICp35 together with 1 μg of pEGFP-C1 and 1 μg of the plasmid expressing caspase 8 (pC8). The pC8 plasmid was not transfected into control cells. (A) At 24 h after transfection, GFP-positive cells were visualized under a fluorescent scope and photographed. (B) GFP-positive cells were counted at 24, 48, and 72 h posttransfection, and data are expressed as the percentage of surviving GFP-positive cells over the control cells.

FIG. 4.

FACS analysis to confirm the ability of LAT to protect cells from caspase 8-induced apoptosis. HeLa cells were transfected with 6 μg of pcDNA3, pcDNA3.Pst-Mlu, or pICp35 together with 2 μg of pCG239.GFP (membrane-bound GFP) and 1 μg of pC8. Control cells were transfected without pC8. At 24 and 48 h posttransfection, cells were harvested and processed for propidium iodide (PI) staining. Analysis of sub-G₁ peaks of GFP-positive cells after induction of apoptosis was carried out by the flow cytometry facility at the Wistar Institute. (A) Representative FACS data at 24 h after transfection. (B) Percent protection of GFP-positive cells at 24 and 48 h posttransfection.

FIG. 5.

Inhibition of apoptosis by LAT mutants expressed from the pcDNA3 vector. (A) Northern blot analysis measuring the expression of LAT mutants. HeLa cells were transfected with 3 μg of pcDNA3, pcDNA3.Pst-Mlu, or the LAT mutant plasmids pΔXcm, pΔBstE, pΔSty, pΔCMV(Pst-Mlu), and pCons (described in Table 1). In cells cotransfected with both pCons and pΔSty, 3 mg of each DNA was used. Total RNA was extracted at 48 h posttransfection, and Northern blot analysis was carried out as previously described (Materials and Methods). The filters were hybridized with ³²P-labeled probes specific for the 2-kb LAT and exposed for autoradiography for visualization. (B) Inhibition of apoptosis by LAT mutants. HeLa cells were transfected with 3 μg of each of the indicated plasmids together with 1 μg of pEGFP-C1 and 1 μg of the plasmid expressing caspase 8 (pC8). In the experiment where pΔSty and pCons were cotransfected into cells, 3 μg of each plasmid was added. Control cells were transfected without pC8. GFP-positive cells were counted at 24, 48, and 72 h posttransfection. Data are expressed as a percentage of GFP-positive cells in control dishes. Results represent an average of four independent experiments.

FIG. 6.

DNA fragmentation in HeLa cells productively infected with LAT deletion viruses. Mock-infected cells or cells infected with strain 17, 17N/H, or ΔSty virus (10 PFU/cell) were lysed in buffer containing 0.5% Triton X-100 and incubated for 1 h at 37°C with RNase A (0.1 mg/ml). The lysates were centrifuged to separate chromosomal DNA from the low-molecular-weight DNA in the supernatant. Proteinase K (1 mg/ml) was added to the supernatant and incubated at 50°C for 1 h in the presence of 1% SDS. DNA was extracted with phenol-chloroform, precipitated in ethanol, and subjected to electrophoresis on a 1.5% agarose gel. The presence of low-molecular-weight DNA in the gel represents DNA fragmentation by apoptosis.

FIG. 7.

Detection of apoptosis in mice trigeminal ganglia. BALB/c mice were ocularly infected in both eyes with HSV 17, 17N/H, or ΔSty viruses at 5 × 10⁴ PFU/eye. At days 3 (A) and 6 (B) postinfection, five mice were sacrificed per group, and trigeminal ganglia were obtained. Trigeminal ganglia were fixed and serial sections were cut and processed for immunohistochemistry to detect replicating virus (pink; described in Materials and Methods). Apoptotic cells were distinguished by the DeadEnd colorimetric apoptosis detection system (brown; Promega). Sections were visualized by light microscopy and photographed. Virus strain: i, 17; ii, none (mock infected); iii, ΔSty; iv, 17N/H.

FIG. 7.

Detection of apoptosis in mice trigeminal ganglia. BALB/c mice were ocularly infected in both eyes with HSV 17, 17N/H, or ΔSty viruses at 5 × 10⁴ PFU/eye. At days 3 (A) and 6 (B) postinfection, five mice were sacrificed per group, and trigeminal ganglia were obtained. Trigeminal ganglia were fixed and serial sections were cut and processed for immunohistochemistry to detect replicating virus (pink; described in Materials and Methods). Apoptotic cells were distinguished by the DeadEnd colorimetric apoptosis detection system (brown; Promega). Sections were visualized by light microscopy and photographed. Virus strain: i, 17; ii, none (mock infected); iii, ΔSty; iv, 17N/H.