Neither LAT nor open reading frame P mutations increase expression of spliced or intron-containing ICP0 transcripts in mouse ganglia latently infected with herpes simplex virus - PubMed (original) (raw)
Comparative Study
Neither LAT nor open reading frame P mutations increase expression of spliced or intron-containing ICP0 transcripts in mouse ganglia latently infected with herpes simplex virus
Shun-Hua Chen et al. J Virol. 2002 May.
Abstract
Latent infections by herpes simplex virus are characterized by repression of productive-cycle gene expression. Several hypotheses to explain this repression involve inhibition of expression of the immediate-early gene activator ICP0 during latency. To address these hypotheses, we developed quantitative reverse transcriptase-PCR assays that detected spliced and intron-containing ICP0 transcripts in mouse ganglia latently infected with wild-type virus. In these ganglia, the numbers of spliced ICP0 transcripts correlated better with the numbers of transcripts from the immediate-early gene encoding ICP4 than with those from the early gene encoding thymidine kinase. There were fewer spliced than intron-containing ICP0 transcripts on average, with considerable ganglion-to-ganglion variation. We then investigated whether ICP0 expression in latently infected ganglia is reduced by the latency-associated transcripts (LATs) and whether splicing of ICP0 transcripts is inhibited by the product of open reading frame (ORF) P. A LAT deletion mutation which essentially eliminates expression of the major LATs did not appreciably increase levels of ICP0 transcripts. LAT deletion mutants did, however, appear to express reduced levels of intron-containing ICP0 transcripts. ORF P mutations did not alter levels of ICP0 transcripts in a manner consistent with inhibition of ICP0 splicing by ORF P. Although these results argue against antisense inhibition of ICP0 expression by LATs or inhibition of ICP0 splicing by ORF P, they are consistent with the possibilities of a block between immediate-early and early gene expression and regulation of spliced versus intron-containing ICP0 transcripts in latently infected ganglia.
Figures
FIG. 1.
Locations of relevant HSV transcription units. (A) Diagram of the prototype arrangement of the HSV genome, with the unique long (UL) and unique short (US) sequences (lines) bracketed by the terminal repeat (TR) and internal repeat (IR) regions (boxes). (B) Expanded diagram of the internal repeat region showing relative sizes, locations, and orientations of transcripts encoded in this region, with bent lines indicating sequences removed by splicing and arrowheads indicating the 3′ ends of the transcripts. The locations and orientations of the 0-1, 0-2, and 0-C2 oligonucleotides used as primers (indicated as 1, 2, and C2, respectively) and the 0-3 oligonucleotide used as a probe (indicated as 3) are shown. Major and a presumed minor LAT species are indicated with thick and thin lines, respectively, and the locations of the two most abundant L/ST species are shown (49). The stippled box encompassing the 5′ ends of the LATs and extending upstream represents the location of the ≈1.8-kb deletion in LAT mutants _dl_LAT1.8 and K_dl_LAT1.8, which is restored in KFSLAT+. This deletion removes the promoter, transcriptional start site, and 1,015 bp of transcribed sequences of LATs. The location of ORF P is shown as a hatched box. The positions of the 4BS mutation upstream of the L/STs and the n38 mutation in ORF P are shown by open arrows.
FIG. 2.
Quantitative RT-PCR assays of ICP0 transcripts. Phosphorimages of assays of spliced ICP0 transcripts (A) and intron-containing ICP0 transcripts (B). RT-PCR products generated using the 0-2 and 0-1 primer pair (A) or 0-2 and C2 primer pair (B) were separated on polyacrylamide gels, blotted, and probed with radiolabeled 0-3 oligonucleotide. Equivalent aliquots of either synthetic transcript mixes derived by in vitro transcription of pKS+ICP0-cDNA (A) or pBKS+ICP0-DNA (B), prepared with 5 μg of mouse brain RNA (A and B, left, under Standard) or individual ganglion RNAs (A and B, right, under Sample) were reacted with (+) or without (−) RT, and the products are displayed in adjacent lanes. In each of the left panels, the number of synthetic transcript molecules added to mouse brain RNA is indicated above the lanes. The B lane indicates where water was added instead of transcripts. The sizes of the RT-PCR products are indicated between the left and right panels. In each of the right panels, samples were derived from individual ganglia harvested at 30 days postinoculation from mock-infected mice (mock) or from mice infected with each of the indicated viruses. Lane M, molecular size markers (φX174 DNA digested with _Hin_fI and end-labeled with 32P). (C) Linearity of RT-PCR assays. The phosphorimages shown on the left of panels A and B were quantified, and the phosphorimager units (P.I.U.) were plotted versus the synthetic RNA molecules added to mouse brain RNA. The best-fit lines were generated by linear regression analysis, and the correlation coefficients (_r_2) are displayed adjacent to the lines. Solid diamonds, spliced ICP0 transcripts; solid squares, intron-containing ICP0 transcripts.
FIG. 2.
Quantitative RT-PCR assays of ICP0 transcripts. Phosphorimages of assays of spliced ICP0 transcripts (A) and intron-containing ICP0 transcripts (B). RT-PCR products generated using the 0-2 and 0-1 primer pair (A) or 0-2 and C2 primer pair (B) were separated on polyacrylamide gels, blotted, and probed with radiolabeled 0-3 oligonucleotide. Equivalent aliquots of either synthetic transcript mixes derived by in vitro transcription of pKS+ICP0-cDNA (A) or pBKS+ICP0-DNA (B), prepared with 5 μg of mouse brain RNA (A and B, left, under Standard) or individual ganglion RNAs (A and B, right, under Sample) were reacted with (+) or without (−) RT, and the products are displayed in adjacent lanes. In each of the left panels, the number of synthetic transcript molecules added to mouse brain RNA is indicated above the lanes. The B lane indicates where water was added instead of transcripts. The sizes of the RT-PCR products are indicated between the left and right panels. In each of the right panels, samples were derived from individual ganglia harvested at 30 days postinoculation from mock-infected mice (mock) or from mice infected with each of the indicated viruses. Lane M, molecular size markers (φX174 DNA digested with _Hin_fI and end-labeled with 32P). (C) Linearity of RT-PCR assays. The phosphorimages shown on the left of panels A and B were quantified, and the phosphorimager units (P.I.U.) were plotted versus the synthetic RNA molecules added to mouse brain RNA. The best-fit lines were generated by linear regression analysis, and the correlation coefficients (_r_2) are displayed adjacent to the lines. Solid diamonds, spliced ICP0 transcripts; solid squares, intron-containing ICP0 transcripts.
FIG. 3.
Correlation of expression of spliced ICP0 transcripts and ICP4 transcripts (A) and spliced ICP0 and tk transcripts (B) in ganglia latently infected with KOS. The numbers of each transcript in each individual ganglion (TG) were plotted against the amount of the other transcript in the same ganglion. The best-fit lines were generated by linear regression, and the correlation coefficients (_r_2) are displayed in each panel.
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