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Splicing-independent expression of the herpes simplex virus type 1 thymidine kinase gene is mediated by three cis-acting RNA subelements - PubMed

Splicing-independent expression of the herpes simplex virus type 1 thymidine kinase gene is mediated by three cis-acting RNA subelements

G C Otero et al. J Virol. 1998 Dec.

Abstract

Herpes simplex virus genes are predominantly intronless. We identified cis-acting elements in the intronless herpes simplex virus type 1 thymidine kinase (TK) gene that facilitate intron-independent gene expression. TK sequences functionally replaced the hepatitis B virus (HBV) posttranscriptional regulatory element (PRE) by inducing the expression of the intronless HBV surface message. TK also activated the pDM138 assay by inducing the cytoplasmic accumulation of intron-containing RNA. Multiple cis-acting RNA sequences, or subelements, that induce cytoplasmic localization of unspliced RNA were mapped within the TK gene. The presence of multiple RNA subelements within the TK gene is reminiscent of the multiple subelements in the HBV PRE required for the cytoplasmic accumulation of intronless HBV RNAs. Similar to HBV PRE subelements, duplication of a single TK subelement resulted in greater-than-additive increases in activity. A reporter chimera containing a single TK subelement juxtaposed to an HBV PRE subelement demonstrated a commensurate increase in activity. These results suggest that viral intronless genes utilize a similar strategy for intron-independent gene expression that requires multiple cis-acting RNA signals. Furthermore, like HBV PRE-containing RNA, TK cytoplasmic localization is not sensitive to leptomycin B, a drug that inhibits the export of proteins containing nuclear export signals. From this, we conclude that proteins that bind TK and facilitate its cytoplasmic accumulation do not travel through a CRM1-dependent RNA transport pathway.

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Figures

FIG. 1
FIG. 1

(A) Schematic illustration of HBV surface vector ΔR5 and derivative reporter constructs containing the WPRE and the TK gene in the sense and antisense (AS) orientations. (B) Surface protein assay demonstrating the effect of TK gene sequences on surface protein expression. The TK gene induces surface gene expression to levels comparable to those produced by WPRE and the HSAg control which contains the HBV PRE. The TK gene in the antisense orientation has no effect over that of empty vector ΔR5.

FIG. 2
FIG. 2

(A) Schematic illustration of the pDM138 constructs containing the TK gene, the HBV PRE, and the RRE. Base pairs are numbered from the transcription start site (27). SD, splice donor; SA, splice acceptor; LTR, long terminal repeat. (B) pDM138 CAT assay demonstrating induction of the cytoplasmic localization of unspliced, intron-containing RNA by TK gene sequences. The TK gene induces CAT expression to levels comparable to those induced by the HBV PRE. (C) Northern blot analysis of nuclear and cytoplasmic RNAs from 293 cells transiently transfected with 138TK and 138RRE. The blot was hybridized with a probe that detects CAT-containing RNAs. Both the TK- and RRE-containing CAT RNAs are present in the nucleus and cytoplasm at the predicted sizes for full-length, unspliced RNAs. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) pDM138 CAT assay demonstrating the ability of TK gene sequences to induce cytoplasmic localization of intron-containing RNA when positioned outside the 138 intron. The TK gene induces CAT expression to similar levels when positioned outside or within the intron. SEM, standard error of the mean.

FIG. 3
FIG. 3

pDM138 CAT assay demonstrating the effect a PPE mutation has on the level of TK-induced CAT activity. The reporter containing the PPE-mutated TK gene, 138TK119LSO, has reduced activity (84%) compared to 138TK. SEM, standard error of the mean.

FIG. 4
FIG. 4

(A) Schematic illustration of the pDM138 constructs containing the TK gene and several 3′ deletions. (B) pDM138 CAT assay demonstrating the stepwise decrease in activity of the 3′ TK deletions. (C) Schematic illustration of the pDM138 constructs containing the TK gene and several 5′ deletions. (D) pDM138 CAT assay demonstrating the stepwise decrease in activity of the 5′ TK deletions. For definitions of abbreviations, see the legend to Fig. 2.

FIG. 5
FIG. 5

(A) Schematic illustration of the pDM138 constructs containing several TK internal deletions. (B) pDM138 CAT assay demonstrating the activity of several TK internal deletions used to map the remaining cis-acting elements in TK. 138TK AS contains TK in the antisense orientation. (C) pDM138 CAT assay demonstrating the activity of the three TK minimal cis-acting RNA subelements mapped by deletion analysis. For definitions of abbreviations, see the legend to Fig. 2.

FIG. 5
FIG. 5

(A) Schematic illustration of the pDM138 constructs containing several TK internal deletions. (B) pDM138 CAT assay demonstrating the activity of several TK internal deletions used to map the remaining cis-acting elements in TK. 138TK AS contains TK in the antisense orientation. (C) pDM138 CAT assay demonstrating the activity of the three TK minimal cis-acting RNA subelements mapped by deletion analysis. For definitions of abbreviations, see the legend to Fig. 2.

FIG. 5
FIG. 5

(A) Schematic illustration of the pDM138 constructs containing several TK internal deletions. (B) pDM138 CAT assay demonstrating the activity of several TK internal deletions used to map the remaining cis-acting elements in TK. 138TK AS contains TK in the antisense orientation. (C) pDM138 CAT assay demonstrating the activity of the three TK minimal cis-acting RNA subelements mapped by deletion analysis. For definitions of abbreviations, see the legend to Fig. 2.

FIG. 6
FIG. 6

HBV surface gene expression assay demonstrating that one TK gene subelement, nt 641 to 841, is sufficient to induce intronless surface gene expression.

FIG. 7
FIG. 7

(A) Schematic illustration of the TK(641-841) subelement duplication construct in pDM138. (B) pDM138 CAT assay demonstrating that the TK(641-841) subelement duplication, TK(641-841x2), induces more than twice the CAT activity of a single subelement. For definitions of abbreviations, see the legend to Fig. 2.

FIG. 8
FIG. 8

(A) Schematic illustration of the TK(641-841) subelement and HBV PREβ subelement chimeric construct in pDM138. (B) pDM138 CAT assay demonstrating that the TK(641-841)PREβ chimeric construct induces CAT activity greater than the sum of the activities induced by individual subelements. For definitions of abbreviations, see the legend to Fig. 2.

FIG. 9
FIG. 9

(A) Effect of LMB on TK-induced expression of intron-containing RNA in the pDM138 CAT assay. 138RRE was transfected with and without pRSVRev in the presence and absence of LMB. 138TK was transfected in the presence and absence of LMB. LMB had no significant effect on TK-induced CAT expression, while LMB inhibited Rev-induced CAT expression 72%. (B) Dose-response curve of LMB effects on Rev-dependent and TK-mediated gene expression in the CAT assay. 138TK (gray) and 138RRE and pRSVRev (dark gray) were transfected in the presence of increasing concentrations of LMB. As reported in reference , Rev-dependent CAT expression decreased until maximally inhibited at 2 nM LMB. TK-mediated CAT expression was not adversely affected between 0 and 10 nM LMB. SEM, standard error of the mean.

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