The 2-kilobase intron of the herpes simplex virus type 1 latency-associated transcript has a half-life of approximately 24 hours in SY5Y and COS-1 cells

Abstract

The herpes simplex virus type 1 (HSV-1) 2-kb latency-associated transcript (LAT) is a stable intron, which accumulates in cells both lytically and latently infected with HSV-1. We have used a tetracycline-repressible expression system to determine the half-life of the 2-kb LAT RNA intron in the human neuroblastoma cell line SY5Y. Using Northern hybridization analyses of RNA isolated from transiently transfected SY5Y cells over time after repression of LAT expression, we measured the half-life of the 2-kb LAT to be approximately 24 h. Thus, unlike typical introns that are rapidly degraded in a matter of seconds following excision, the 2-kb LAT intron has a half-life similar to those of some of the more stable cellular mRNAs. Furthermore, a similar half-life was measured for the 2-kb LAT in transiently transfected nonneuronal monkey COS-1 cells, suggesting that the stability of the 2-kb LAT is neither cell type nor species specific. Previously, we found that the determinant responsible for the unusual stability of the 2-kb LAT maps to the 3' terminus of the intron. At this site is a nonconsensus intron branch point located adjacent to a predicted stem-loop structure that is hypothesized to prevent debranching by cellular enzymes. Here we show that mutations which alter the predicted stem-loop structure, such that branching is redirected, either reduce or abolish the stability of the 2-kb LAT intron.

FIG. 1.

Half-lives of α-globin and IL-2 transcripts as measured with a tetracycline-repressible transient-transfection system. SY5Y cells were transfected with pTet-tTAk and pTet-globin or pTet-IL-2 and incubated for 24 h. Transcription was repressed with tetracycline, and RNA was isolated at the times indicated postrepression. (A and C) RNA was analyzed by Northern hybridization using either an α-globin-specific probe (A) or an IL-2-specific probe (C). The half-lives of α-globin and IL-2 were quantitated as described in Materials and Methods. (B and D) The mean levels of α-globin (n = 2 for t = 3 and 9 h; n = 4 for t = 0, 6, and 12 h) (B) and IL-2 (n = 2) (D) over time relative to 0 h or 0 min post-tetracycline repression are presented. Error bars represent standard deviations.

FIG. 2.

LAT locus of the HSV-1 genome. (A) The linear, 152-kb HSV-1 genome with the unique long (U_L) and unique short (U_S) regions flanked by inverted repeat elements, called terminal repeat long (TR_L), terminal repeat short (TR_S), internal repeat long (IR_L), and internal repeat short (IR_S). (B) Enlargement of the LAT region of HSV-1 to show the different transcripts that map to this locus. (C) The pcDNA3/PstI-MluI minigene expression cassette. The PstI-MluI fragment harboring part of the LAT gene was cloned into the pcDNA3 expression vector as described previously (50). Arrows represent splice donor and acceptor sites used to produce the 2-kb LAT. Relevant restriction enzyme sites and the DNA fragments used as probes in Northern hybridization analyses are indicated.

FIG. 3.

Half-life of the 2-kb LAT intron in transiently transfected SY5Y cells. SY5Y cells were transfected with plasmids pTet-tTAk and pTet-LAT. LAT expression was repressed by the addition of tetracycline at 24 h posttransfection, and total RNA was isolated from cells at 0, 6, 12, 18, 24, 30, and 36 h post-tetracycline repression. (A) RNA was analyzed by Northern hybridization, and the amount of 2-kb LAT detected at each time point was determined and analyzed as described in Materials and Methods. (B) Mean level of 2-kb LAT (n = 3) over time relative to that at 0 h postrepression. Regression analysis of these data (r² value) is indicated. Error bars represent standard deviations.

FIG. 4.

Half-life of the 2-kb LAT intron in transiently transfected COS-1 cells. COS-1 cells were transfected as described for Fig. 3. (A) After 16 h, transcription was repressed and RNA was isolated and analyzed by Northern blotting and quantitated as described for Fig. 3. (B) Mean level of 2-kb LAT (n = 4, except for t = 36 h, where n = 2) over time relative to that at 0 h postrepression. Error bars represent standard deviations.

FIG. 5.

Structures of the pCons and pBam mutants of the 2-kb LAT intron. Nucleotide differences are shaded. The branch points of the wild-type (wt) 2-kb LAT and of the two mutants, which are indicated with arrows (major branch point, thick arrows; minor branch points, thin arrows), were determined previously (21). The predicted stem-loop structure of the wild-type 2-kb LAT is illustrated. This stem-loop is conserved in both mutants but is not shown. The figure is adapted from reference .

FIG. 6.

Half-life analyses of the Cons and Bam mutants of the 2-kb LAT intron. SY5Y cells were transfected with pTet-tTAk and pTet-Cons or pTet-Bam, and transcription was repressed with tetracycline as described for Fig. 3. (A and B) For the Cons mutant, Northern blot analyses for the 2-kb LAT intron were performed on RNAs isolated at 0, 0.5, 1, 2, and 3 h postrepression (A). The blot was then stripped and rehybridized with a LAT exon-specific probe (see Fig. 2) to verify transcription (B). (C and D) For the Bam mutant, Northern blot analyses for the 2-kb LAT intron were performed on RNAs isolated at 0, 3, 6, 12, 24, and 32 h postrepression (C). LAT-specific bands were analyzed and quantitated as described in Materials and Methods, and the mean level of the 2-kb LAT (n = 4) over time relative to that at 0 h postrepression is presented (D). Error bars represent standard deviations.