The gene that encodes the herpes simplex virus type 1 latency-associated transcript influences the accumulation of transcripts (Bcl-x(L) and Bcl-x(S)) that encode apoptotic regulatory proteins - PubMed (original) (raw)

The gene that encodes the herpes simplex virus type 1 latency-associated transcript influences the accumulation of transcripts (Bcl-x(L) and Bcl-x(S)) that encode apoptotic regulatory proteins

Weiping Peng et al. J Virol. 2003 Oct.

Abstract

The herpes simplex virus type 1 latency-associated transcript (LAT) inhibits apoptosis. We demonstrate here that LAT influences the accumulation of the Bcl-x(L) transcript versus the Bcl-x(S) transcript in Neuro-2A cells. Bcl-x(L) encodes an antiapoptotic protein, whereas Bcl-x(S) encodes a proapoptotic protein. Promoting the accumulation of Bcl-x(L) in neurons may inhibit apoptosis, thus enhancing the latency-reactivation cycle.

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Figures

FIG. 1.

Analysis of Bcl-x RNA expression in infected Neuro-2A cells. Using a multiplicity of infection of 4, Neuro-2A cells were infected with strain _d_LAT2903 (dLAT) or wt HSV-1 (McKrae strain) (WT). As controls, cultures were mock infected (Mock). At 24 h after infection (A) or at 8 and 24 h after infection (B), RNA was prepared using Trizol reagent and the RNA was subsequently treated with RNase-free DNase I for 30 min at room temperature. First-strand cDNA was synthesized using 2 μg of total RNA as the template, 0.5 μg of oligo(dT)12-18 as a primer, and a SuperScript preamplification kit (Stratagene, La Jolla, Calif.). For each sample, 1/10 of the cDNA reaction mixture was used with the indicated primers (Table 1). Using 35 (A) or 31 (B) cycles, amplification was conducted as described in Table 1. The positions of the Bcl-xL (xL) and Bcl-xS (xS) bands are indicated. Omitting reverse transcriptase from the reaction eliminated amplification of the specific bands (data not shown). A 100-bp ladder was used as a marker (M) to estimate the size of the amplified cDNA products. These results are representative of at least five independent experiments.

FIG. 2.

Analysis of apoptosis regulatory genes in infected Neuro-2A cells. Using a multiplicity of infection of 4, Neuro-2A cells were infected with strain _d_LAT2903 (dLAT) or wt HSV-1 (McKrae strain) (WT). As controls, cultures were mock infected (Mock). At 6, 16, or 24 h after infection, RNA was prepared using Trizol reagent and the RNA was subsequently treated with RNase-free DNase I for 30 min at room temperature. Samples containing total RNA (1 μg) were electrophoresed on a 1.2% formaldehyde agarose gel, and the position of the rRNA was determined. First-strand cDNA was synthesized using 2 μg of total RNA as the template, 0.5 μg of oligo(dT)12-18 as a primer, and a SuperScript preamplification kit (Stratagene). For each sample, 1/10 of the cDNA reaction mixture was used with the indicated primers (Table 1). Amplification was conducted as described in Table 1. The closed circles denote the positions of amplified products. Omitting reverse transcriptase from the reaction eliminated amplification of the specific bands (data not shown). A 100-bp ladder was used as a marker (M) to estimate the size of the amplified cDNA products. These results are representative of at least four independent experiments.

FIG. 3.

LAT inhibits splicing of Bcl-xS in transfected Neuro-2A cells. Neuro-2A cells were transfected with the plasmids described below. Lane 1, 4 μg of LAT3.3-1 μg of pcDNA3.1; lane 2, 4 μg of LAT3.3-1 μg of pCMVBax; lane 3, 4 μg of pcDNA3.1-1 μg of pCMVBax; lane 4, 5 μg of pcDNA3.1. LAT3.3 contains the LAT promoter plus the first 1.5 kb of LAT coding sequences (19). pCMVBax contains the full-length Bax cDNA downstream of the human cytomegalovirus promoter; the plasmid was obtained from Upstate Biotechnology (Lake Placid, N.Y.). At 48 h after transfection, using primers that amplify Bcl-x (A), Bcl-2 (C), or Bak (D), RNA was prepared and RT-PCR was performed as described for Fig. 1. (B) cDNA reactions that did not contain reverse transcriptase (No RT) were amplified using the Bcl-X primers. In the No RT reactions, the Bcl-x primers did amplify the expected products. The No RT reaction also yielded no bands when the Bcl-2 or Bak primers were used (data not shown).

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The gene that encodes the herpes simplex virus type 1 latency-associated transcript influences the accumulation of transcripts (Bcl-x(L) and Bcl-x(S)) that encode apoptotic regulatory proteins - PubMed (original) (raw)