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Comparative Study
. 2005 May;79(10):6162-71.
doi: 10.1128/JVI.79.10.6162-6171.2005.

The locus encompassing the latency-associated transcript of herpes simplex virus type 1 interferes with and delays interferon expression in productively infected neuroblastoma cells and trigeminal Ganglia of acutely infected mice

Weiping Peng  1 , Gail Henderson, Melissa Inman, Lbachir BenMohamed, Guey-Chuen Perng, Steven L Wechsler, Clinton Jones
Affiliations
Comparative Study

The locus encompassing the latency-associated transcript of herpes simplex virus type 1 interferes with and delays interferon expression in productively infected neuroblastoma cells and trigeminal Ganglia of acutely infected mice

Weiping Peng et al. J Virol. 2005 May.

Abstract

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is the only abundant viral transcript expressed in latently infected neurons. LAT inhibits apoptosis, suggesting that it regulates latency by promoting the survival of infected neurons. The LAT locus also contains a newly described gene (AL), which is antisense to LAT and partially overlaps LAT encoding sequences. When human (SK-N-SH) or mouse (neuro-2A) neuroblastoma cells were infected with a virus that does not express LAT or AL gene products (dLAT2903), beta interferon (IFN-beta) and IFN-alpha RNA expression was detected earlier relative to the same cells infected with HSV-1 strains that express LAT and AL. Infection of neuro-2A cells with dLAT2903 also led to higher levels of IFN-beta promoter activity than in cells infected with wild-type (wt) HSV-1. In contrast, IFN RNA expression was the same when human lung fibroblasts were infected with dLAT2903 or wt HSV-1. When BALB/c mice were infected with dLAT2903, IFN-alpha and IFN-beta RNA expression was readily detected in trigeminal ganglia (TG) 4 days after infection. These transcripts were not detected in TG of mice infected with wt HSV-1 or dLAT2903R (marker-rescued dLAT2903) until 6 days postinfection. When TG single-cell suspensions from infected BALB/c mice were prepared and incubated in vitro with wt HSV-1 as a source of antigen, TG cultures prepared from mice infected with dLAT2903 produced and secreted higher levels of IFN protein than wt HSV-1 or dLAT2903R. Collectively, these studies suggest that the LAT locus interferes with and delays IFN expression.

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Figures

FIG. 1.
FIG. 1.
Morphology of SK-N-SH cells infected with HSV-1. (A) TRL and IRL indicate the terminal and inverted long repeats in the HSV-1 genome. IRS and TRS indicate the inverted and terminal short repeats. UL and US indicate the unique long and unique short regions. The IRL is expanded. The TRL contains the same genes except they are flipped left to right (indicated by the closed triangle). (B) Schematic of genes within the long repeats. The large arrow indicates the primary LAT transcript. The solid rectangle represents the very stable 2-kb LAT intron. The LAT TATA box is indicated by TATA. The start of LAT transcription is indicated by the arrow at +1 (genomic nucleotide 118801). The relative position of the AL transcript is also presented, and the positions of the AL transcript are given using the numbering system of LAT. Several restriction enzyme sites and the relative locations of the ICP0 and ICP34.5 transcripts are shown for reference. The position of the deletion in dLAT2903 (−161 to +1499) is denoted by the Xs. This deletion is present in both copies of the repeats (indicated by the black triangle in the TRL); thus, dLAT2903 is a LAT null mutant because the core promoter and LAT coding sequences are deleted (46, 47). In addition, dLAT2903 does not express AL gene products (42). (C) SK-N-SH cultures were infected with dLAT2903 or wt HSV-1 (multiplicity of infection [MOI] = 8). One hour after inoculation with the virus at 37°C, the inoculum was removed and the cells were washed three times (10 ml each wash) with phosphate-buffered saline to remove residual virus. Fresh medium was then added to the cultures. At 24 h after infection, Hoechst 33412 staining was performed. Apoptotic cells were counted, and the percentage of apoptotic cells is given. At least 200 cells from each culture were examined from three different experiments. Means were compared by the Tukey-Kramer test at a 5% significance level. The differences between dLAT2903 and LAT+ (wt or dLAT2903R) or mock infected versus virus infected were significant. At 48 h after infection, the amount of virus was determined by freeze-thawing the infected cells and pelleting the debris. The amount of virus on rabbit skin cells was calculated. The results are the mean of three independent studies.
FIG. 2.
FIG. 2.
Gene expression following infection of SK-N-SH cells with HSV-1. SK-N-SH cultures were infected with an LAT mutant (dLAT2903) or a LAT-expressing strain of HSV-1 (wt McKrae or dLAT2903R) at an MOI of 8. Shown in this study are cells infected with wt HSV-1. The results for dLAT2903R and wt McKrae were the same. Total RNA was prepared at the indicated time (hours) after infection using procedures described in Materials and Methods. Two micrograms of total RNA was used for cDNA synthesis using random primers. PCR was then performed using 1/10 of the cDNA reaction mixture and the primers described in Table 2. (A) Analysis of IFN expression in SK-N-SH cells after infection. All IFN-α subtypes were amplified with primers corresponding to the conserved regions of the respective human IFN-α subtypes. The IFN-amplified products were excised and sequenced to confirm they were in fact IFN-amplified bands. The panel marked Total RNA contains 1 μg of total RNA electrophoresed on a 1% formaldehyde gel. The number of PCR cycles used to amplify cDNA for IFN-α was 36 and the number of cycles used to amplify IFN-β or IFN-γ was 34. (B) Analysis of viral gene expression after infection. A total of 31 PCR cycles were used to amplify the respective viral genes. The primers used for PCR amplification are described in Table 1. These results are representative of three independent experiments. vhs, virion host shutoff.
FIG. 3.
FIG. 3.
Induction of different IFN-α subtypes in SK-N-SH cells infected with McKrae or dLAT2903. (A) The IFN-α cDNAs generated by RT-PCR from SK-N-SH cells infected with McKrae or dLAT2903 (Fig. 2A) were cloned as described in Materials and Methods. Randomly selected clones were sequenced and the sequence analyzed to identify which IFN-α subtype was present. (B) Primers that specifically amplify IFN-α5 or IFN-α6 were synthesized (Table 2) and used for RT-PCR as described in Materials and Methods.
FIG. 4.
FIG. 4.
Pretreatment of SK-N-SH cells with IFN inhibits HSV-1 plaque formation. SK-N-SH cells were seeded in six-well plates, and the cells reached 80 to 90% confluence. On the next day, SK-N-SH cultures were treated with IFN-β (0.1 to 1,000 U) (Calbiochem 407297) for 6 h and then infected with HSV-1 (MOI = 0.01). One hour later, the medium was replaced with Earl's modified Eagle's medium containing 10% fetal calf serum and the same concentration of IFN used as in the 6-h pretreatment. The number of plaques was counted 48 to 72 h after infection. The number of plaques without IFN-β was set at 100%. Although IFN-β treatment had a slight effect on the size of plaques, we did not observe dramatic differences between dLAT2903 and LAT+ strains (data not shown). These results are the average of five different experiments.
FIG. 5.
FIG. 5.
Induction of IFN-α in HEL or neuro-2A cells infected with McKrae or dLAT2903. Neuro-2A cells (A) or HEL cells (B) were infected with dLAT2903 or a LAT-expressing strain of HSV-1 (wt McKrae or dLAT2903R) at an MOI of 8. Total RNA was prepared at the indicated time (hours) after infection. Two micrograms of total RNA was used for cDNA synthesis using random primers. PCR was then performed using 1/10 of the cDNA reaction mixture and the primers described in Table 2.
FIG. 6.
FIG. 6.
Induction of IFN-α in TG of mice infected with McKrae or dLAT2903. (A) BALB/c mice were infected with HSV-1 as described in Materials and Methods. At the indicated times after infection, TG were harvested and total RNA prepared (TG from 2 mice/time point). RT-PCR using the designated primers was performed (Table 2). All mouse IFN-α subtypes were amplified using consensus primers that are located within the conserved region of the IFN-α RNA. The top panel shows the corresponding levels of β-actin mRNA as a loading control. (B) The IFN-α cDNA band that contains a mixture of the IFN-α subtypes was excised and cloned, and the insert was sequenced. The band at 4 days after infection (McKrae) did not yield clones, which was expected because we were unable to amplify IFN-α cDNAs at this time.
FIG. 7.
FIG. 7.
Expression of IFN in TG of mice infected with dLAT2903 or LAT+ strains of HSV-1. BALB/c mice were infected with HSV-1 as described in Materials and Methods. At the indicated times after infection, TG were harvested, and total RNA was prepared. RT-PCR using the designated primers (Table 1) was performed. As loading controls, the expression levels of β-actin and p53 are shown in the bottom panels. The results are representative of two different experiments.
FIG. 8.
FIG. 8.
Decreased IFN secretion in TG from mice infected with wt HSV-1 or dLAT2903R viruses. TG single-cell suspensions were obtained from age- and sex-matched mice infected with dLAT2903, wt HSV-1, or dLAT2903R at 5 days postinfection. Noninfected mice were used as a negative control. For each reaction, an equivalent number of cells from two TG were either left untreated (white bars) or incubated in vitro at 37°C with heat-inactivated wt HSV-1 as a source of antigen (black bars). The secreted IFN was measured 96 h later by a standard sandwich ELISA assay. The results are representative of two different experiments.
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