Comparison of [14C]FMAU, [3H]FEAU, [14C]FIAU, and [3H]PCV for Monitoring Reporter Gene Expression of Wild Type and Mutant Herpes Simplex Virus Type 1 Thymidine Kinase in Cell Culture

Bal31 nuclease was used to resect the herpes simplex virus type 1 thymidine kinase (tk) gene from its 3' end, and a plasmid, pTK206, was isolated that lacked the processing and polyadenylation signals normally found at the 3' end of the gene. The wild-type gene, pTK2, and pTK206 were each transferred to pSV010, a plasmid containing the simian virus 40 (SV40) origin of DNA replication, allowing replication and analysis of the patterns of transcription in Cos-1 cells. Fragments of DNA containing processing and polyadenylation signals from SV40 and polyoma virus were inserted into the 3' end of the resected tk gene, pTK206. We found that tk gene expression requires a processing and polyadenylation signal, that signals from SV40 and polyoma virus could substitute for the herpes simplex virus tk signal, and that considerable differences in the levels of tk mRNA were present in Cos-1 cells transfected by these gene constructs. In addition, tk gene expression was restored to a low level after the insertion of an 88-base-pair fragment from the middle of the SV40 early region. Processing and polyadenylation do not occur in the vicinity of this fragment in SV40, even though it contains the hexanucleotide 5'-AAUAAA-3'.

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A mutant herpes simplex virus type 1 thymidine kinase reporter gene shows improved sensitivity for imaging reporter gene expression with positron emission tomography

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.97.6.2785 ◽

2000 ◽

Vol 97 (6) ◽

pp. 2785-2790 ◽

Cited By ~ 311

Author(s):

S. S. Gambhir ◽

E. Bauer ◽

M. E. Black ◽

Q. Liang ◽

M. S. Kokoris ◽

...

Keyword(s):

Gene Expression ◽

Positron Emission Tomography ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Reporter Gene ◽

Herpes Simplex Virus Type ◽

Emission Tomography ◽

Positron Emission ◽

Simplex Virus

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Utilization of the herpes simplex virus type 1 latency-associated regulatory region to drive stable reporter gene expression in the nervous system.

Journal of Virology ◽

10.1128/jvi.71.4.3197-3207.1997 ◽

1997 ◽

Vol 71 (4) ◽

pp. 3197-3207 ◽

Cited By ~ 67

Author(s):

R H Lachmann ◽

S Efstathiou

Keyword(s):

Gene Expression ◽

Nervous System ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Reporter Gene ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Regulatory Region ◽

Simplex Virus

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Analysis in Cos-1 cells of processing and polyadenylation signals by using derivatives of the herpes simplex virus type 1 thymidine kinase gene

Molecular and Cellular Biology ◽

10.1128/mcb.3.2.267-279.1983 ◽

1983 ◽

Vol 3 (2) ◽

pp. 267-279

Author(s):

C N Cole ◽

G M Santangelo

Keyword(s):

Gene Expression ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Thymidine Kinase ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Polyoma Virus ◽

Simplex Virus ◽

Polyadenylation Signals

Bal31 nuclease was used to resect the herpes simplex virus type 1 thymidine kinase (tk) gene from its 3' end, and a plasmid, pTK206, was isolated that lacked the processing and polyadenylation signals normally found at the 3' end of the gene. The wild-type gene, pTK2, and pTK206 were each transferred to pSV010, a plasmid containing the simian virus 40 (SV40) origin of DNA replication, allowing replication and analysis of the patterns of transcription in Cos-1 cells. Fragments of DNA containing processing and polyadenylation signals from SV40 and polyoma virus were inserted into the 3' end of the resected tk gene, pTK206. We found that tk gene expression requires a processing and polyadenylation signal, that signals from SV40 and polyoma virus could substitute for the herpes simplex virus tk signal, and that considerable differences in the levels of tk mRNA were present in Cos-1 cells transfected by these gene constructs. In addition, tk gene expression was restored to a low level after the insertion of an 88-base-pair fragment from the middle of the SV40 early region. Processing and polyadenylation do not occur in the vicinity of this fragment in SV40, even though it contains the hexanucleotide 5'-AAUAAA-3'.

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Mutation Spectra of Herpes Simplex Virus Type 1 Thymidine Kinase Mutants

Journal of Virology ◽

10.1128/jvi.76.11.5822-5828.2002 ◽

2002 ◽

Vol 76 (11) ◽

pp. 5822-5828 ◽

Cited By ~ 13

Author(s):

Qiaosheng Lu ◽

Ying T. Hwang ◽

Charles B. C. Hwang

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Thymidine Kinase ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Wild Type Virus ◽

Exonuclease Activity ◽

Wild Type ◽

Simplex Virus

ABSTRACT To examine whether the exonuclease activity intrinsic to the polymerase (Pol) of herpes simplex virus type 1 can influence the mutational spectra, we applied the denaturing gradient gel electrophoresis (DGGE) system combined with sequencing to characterize thymidine kinase mutants isolated from both the wild-type virus and a mutant deficient in exonuclease activity, Y7. Wild-type viruses produced predominately frameshift mutations (67%), whereas Y7 replicated a significantly lower proportion of frameshifts (21%; P < 0.005). Furthermore, the majority of substitutions were transitional changes in both groups, although they distributed differently. The implications of these findings are discussed.

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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

Journal of Virology ◽

10.1128/jvi.72.12.9889-9896.1998 ◽

1998 ◽

Vol 72 (12) ◽

pp. 9889-9896 ◽

Cited By ~ 15

Author(s):

Glen C. Otero ◽

Thomas J. Hope

Keyword(s):

Gene Expression ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Thymidine Kinase ◽

Herpes Simplex Virus Type ◽

Cytoplasmic Localization ◽

Independent Gene ◽

Cytoplasmic Accumulation ◽

Simplex Virus

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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