Herpes Simplex Virus Type 1 TK Research Articles

Inhibition of Transient Gene Expression with Plasmids Encoding Herpes Simplex Virus Type 1 UL55 and Alpha Genes

Herpes simplex virus type 1 (HSV-1) subgenomic sequences from 0.743 to 0.782 map units have been molecularly cloned as plasmid AT1 and shown to inhibit stable DNA-mediated gene transformation of Ltk- cells with the HSV-1 thymidine kinase (tk) gene. Here it is shown that AT1 also inhibits transient gene expression. Expression from the chloramphenicol acetyltransferase (CAT) gene under the control of either the HSV-1 tk gene or the Rous sarcoma virus (RSV) promoter was inhibited when cotransfected into Ltk- and CV-1 cells with equimolar amounts of AT1. AT1 was subcloned as three overlapping plasmids called AT1a, alpha 27 and AT1b. The alpha 27 plasmid encodes the HSV-1 immediate early gene, alpha 27; AT1a possesses sequences that specify an open reading frame in HSV-1 strain KOS used in these studies, although the significance of this open reading frame is unknown; AT1b possesses the sequences for UL55 and UL56, also genes for which no function has been reported. No single subclone or pair of subclones demonstrated significant inhibition of transient gene expression. Cotransfection of all three subclones did result in inhibition of RSV-CAT gene expression, suggesting that information from each subclone is necessary. One of the three subclones, alpha 27, contains the HSV-1 immediate early gene, alpha 27, so the possibility that other immediate early genes could substitute for alpha 27 was tested. Inhibition of RSV-CAT gene expression was also achieved by cotransfection of AT1a and AT1b with either an alpha 0- or alpha 4-containing plasmid, suggesting that the role of the alpha 27-containing plasmid can be replaced by other alpha genes with trans-regulating capability. Finally, AT1a and AT1b linker insertion mutants have been constructed and used to study the role these plasmids play in mediating inhibition. These results suggest that AT1 contains HSV-1 functions in addition to that of alpha 27 that interfere with gene expression.

Differential regulation by varicella-zoster virus (VZV) and herpes simplex virus type-1 trans-activating genes

Transient expression assays were performed in Vero cells in order to compare varicella-zoster virus (VZV)-encoded trans-activating proteins [defined by the products of open reading frames (ORF) 4 and 62) with herpes simplex virus type-1 (HSV-1) trans-activating proteins, ICP4 and ICP0, with respect to activation of gene expression. We demonstrate that the product of VZV ORF4 and ORF62 (which are the HSV-1 analogs of ICP27 and ICP4, respectively) stimulate a variety of viral and cellular gene promoters, including the HSV-1 thymidine kinase (tk) promoter. On the other hand, expression of a recombinant vector containing the VZV tk promoter could not be stimulated, by HSV-1 infection or by the HSV-1 ICP4 or ICP0 proteins expressed during cotransfection experiments. These data suggest different mechanisms of activation of the VZV and the HSV-1 tk gene promoters by “ trans-activating” factors.

Sequences of herpes simplex virus type 1 that inhibit formation of stable TK+ transformants.

We have identified two regions of the herpes simplex virus type 1 (HSV-1) genome that inhibit DNA-mediated transformation of thymidine kinase-less L (Ltk-) cells by the cloned HSV-1 tk gene. When plasmids containing the EcoRI fragments EK or JK were mixed at 30 fmol/ml with the tk gene and transfected into Ltk- cells, the frequency of transformation was inhibited 80 to more than 90% relative to the control. Of the remaining 10 EcoRI fragments of the HSV-1 genome, 8 were inactive and 2 were weakly active. A 6.1-kilobase PstI subclone between 0.743 and 0.782 map units was isolated from pEK. This clone, pEK-P3P4, exhibited antitransformation activity toward HSV-1 tk and also the bacterial genes gpt and neo. pEK-P3P4 contains the alpha 27 gene, and restriction endonuclease inactivation and subcloning studies established that alpha 27 alone did not inhibit transformation. However, alpha 27 plus sequences both upstream and downstream of alpha 27 did inhibit transformation. In addition, alpha 0 or alpha 4 could substitute for alpha 27 in effecting antitransformation with these sequences. Therefore, an alpha gene and two additional loci in pEK-P3P4 are required for antitransformation. A second antitransforming locus in the reiterated sequences common to EK and JK and distinct from those in pEK-P3P4 was also identified but not characterized in detail. How antitransformation may be an expression of regulation of viral and host cell gene expression is discussed.

Mapping of the transcriptional initiation site of the herpes simplex virus type 1 ICP8 gene in infected and transfected cells.

The initiation site for transcription of the herpes simplex virus type 1 (HSV-1) gene encoding the major DNA-binding protein. ICP8, was mapped by nuclease S1 analysis of RNA-DNA hybrids. When RNA isolated from cells infected with HSV-1 was used, one major start site of ICP8 gene transcription was mapped at 89 base pairs to the right of the BstEII site at 0.409 map units. In cells transfected with a cloned ICP8 gene, the same major start site was detected either in the presence or absence of the immediate-early (alpha) genes encoding ICP4 or ICP0, which have been shown to stimulate ICP8 gene expression in transfected cells. Both ICP4 and ICP0 stimulated the accumulation of the ICP8 gene transcripts in the transient expression system, and their effects were synergistic. By comparison of the sequence of the putative promoter region of the ICP8 gene with the promoter of the HSV-1 TK gene, a significant similarity was detected between the three transcriptional regulatory signals of the TK gene and the upstream sequences of the ICP8 gene. Analysis of promoters of other delayed-early (beta) genes showed that they all contained regions of significant homology with the distal signals of the upstream sequences of the TK or ICP8 gene.

Production of herpes simplex virus type 1 thymidine kinase in the presence of thymidine analogues

Treatment of herpes simplex virus type 1 (HSV-1) infected Vero, BHK, BHKtk − and LMtk − cells with 5-iodo-5′-amino-2′,5′-dideoxyuridine (AIdUrd) caused increased synthesis of ICP36 and an increase in HSV-1 thymidine kinase (tk) activity at late times of infection. The overproduced ICP36 was identified as the HSV-1 encoded tk protein by immunoprecipitation. Whereas the thymidine analogue 5′-amino-5′-deoxythymidine (AdThd) caused an increase in HSV-1 tk synthesis and activity in wild type Vero and BHK cells, 5-iodo-2′-deoxyuridine (IdUrd) caused a similar increase only in tk − cells (LMtk −, BHKtk −). In vivo and in vitro stabilization studies using a [ 35S]methionine pulse-chase experiment or heat inactivation studies with purified HSV-1 tk revealed that stabilization of tk by the analogues could not account for the extent of the observed increase. Since overproduction of tk is observed only at late times of infection, it is suggested that the presence of these thymidine analogues in either the viral DNA or the cellular nucleotide pools is responsible for the observed differential effects.

Inhibition by interferon of herpes simplex virus thymidine kinase and DNA polymerase in infected and biochemically transformed cells.

The induction of thymidine kinase (TK) and DNA polymerase was inhibited by interferon (IFN) in mouse L-cells infected with herpes simplex virus type 1 (HSV-1). The inhibitory activity of IFN at this early stage of HSV-1 replication was followed by a reduced virus yield and was dependent on the multiplicity of infection. The expression of a cloned thymidine kinase (tk) gene of HSV-1, in biochemically transformed L-cells (LTK+), was not affected by IFN. These same LTK+ cells, however, developed an antiviral state since, upon HSV-1 infection, the induction of TK and DNA polymerase of the replicating virus was inhibited by IFN. Furthermore, IFN inhibited the transactivation of the HSV-1 tk gene in the biochemically transformed LTK+ cells, which followed infection by a virus mutant defective in the tk gene (HSV-1 TK-). This transactivation is dependent on expression of immediate-early HSV-1 alpha-genes. These results indicate that IFN inhibits HSV-1 replication at an early step prior to DNA synthesis. In addition, IFN displays a differential effect on the HSV-1 thymidine kinase gene, either when part of the replicating virus or when expressed as a cellular gene in biochemically transformed cells.

Mapping functional domains in the promoter region of the herpes thymidine kinase gene.

The cloned herpes simplex virus type 1 (HSV-1) thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene can be used to transform TK- cells to a TK+ phenotype. Transformants generated in this way express TK at a basal constitutive level that is inducible to a higher level by infection with TK- herpes virus. We have studied the effect of mutations generated in vitro on both the constitutive and virus-induced expression of TK in transformants. Four Xho I linker insertions and two deletions in the 5' untranscribed region of the cloned HSV-1 TK gene were generated in vitro. A deletion that removed all but nine base pairs of the 5' untranscribed region virtually eliminated constitutive expression and completely prevented induction by herpes virus infection. Two of the insertions have particularly interesting properties. One, nine base pairs upstream from the cap site, inactivates constitutive expression without stopping induction. The other, 50 base pairs upstream from the cap site has the opposite effect (i.e., normal constitutive expression but no induction). Analysis of these results leads us to propose that the 5' untranscribed region of the HSV-1 TK gene is quite complex with several functional domains having differential roles in the constitutive and herpes-induced expression of the TK gene.

Herpes simplex virus thymidine kinase expression in trigeminal ganglion infection: Correlation of enzyme activity with ganglion virus titer and evidence of in vivo complementation

Experimental trigeminal ganglion and corneal infection in mice was studied with three thymidine kinase-positive (TK +) strains of herpes simplex virus type 1 (HSV-1) and eight TK − HSV-1 mutants. Viruses were extensively tested in cell culture to determine whether any were temperature sensitive ( ts) for virus replication or for TK activity. TK − mutants were no more ts than were TK + viruses. By arabinosylthymine testing and measurement of thymidine phosphorylation, it was apparent that some TK − mutants were, in fact, intermediate for TK activity (TK ±). After corneal inoculation of individual viruses it was observed with one exception that TK −, TK ±, and TK + viruses replicated in ocular tissues (3 days postinoculation, 2.2 × 10 3–1.1 × 10 5 PFU/eye). However, TK − mutants were rarely isolated from trigeminal ganglia (<2.5–<5.6 × 10 −1 PFU/mg), whereas TK + and to a lesser degree TK ± viruses were frequently (1.2 × 10 3–1.2 × 10 4 PFU/mg and 3.2 × 10 0–2.1 × 10 3 PFU/mg). In vivo complementation studies were performed by corneal inoculation of TK −−TK − and TK −−TK − virus mixtures. TK + HSV complemented TK − virus since significant amounts of TK − HSV were isolated from trigeminal ganglia (complementation indices were >60–>1600). In addition, after inoculation of certain TK −–TK − pairs, complementation in ganglia was observed (complementation indices were >2.4–>120). These studies support the hypothesis that HSV-1 TK expression is necessary for sensory ganglion (neuron) infection in three ways: HSV-1 TK mutants that replicated in ocular tissues and were not ts mutants did not replicate in vivo in trigeminal ganglia; (ii) there was a correlation between level of viral TK activity and trigeminal ganglion virus titer; and (iii) when complemented by TK + or TK − HSV, TK − virus replicated in trigeminal ganglia.

Herpes simplex virus thymidine kinase activity of thymidine kinase-deficient Escherichia coli K-12 mutant transformed by hybrid plasmids.

A hybrid plasmid (pAGO) that contains the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in the form of a 2-kilobase-pair (kbp) Pvu II fragment inserted at the Pvu II site of plasmid pBR322 was used to transform TK- Escherichia coli K-12 strain KY895. pAGO-transformed KY895 cells exhibited partially restored ability to incorporate [3H]dThd into DNA and an HSv-1-specific TK activity. Bacteria cured of plasmid pAGO (or transformed by plasmid pBR322) did not show enhanced incorporation of [3H]dThd into DNA or HSV-1 TK activity. Plasmid pMH1A was derived from pAGO by deletion of 2067 bp of DNA sequence from pBR322 and 105 bp from the HSV-1 TK gene. E. coli K-12 strain KY895 cells transformed by pMH1A did not show enhanced incorporation of [3H]dThd into bacterial DNA, although pMH1A DNA isolated from transformed KY895 cells, like pAGO DNA, did transform TK- mouse fibroblast [LM(TK-)] cells to the TK+ phenotype. The expression of HSV-1 TK activity by E. coli K-12 suggests that intervening sequences may be absent from the coding region of HSV-1 tk or that the coding region of the gene possesses short intervening sequences which do not disrupt the translational reading frame.

Hybrid plasmids containing an active thymidine kinase gene of Herpes simplex virus 1

The gene for the thymidine kinase (TK) of Herpes simplex virus type 1 (HSV-1) is located in the KpnI m and BamHI p fragments of the genome (Wigler et al., Cell 11, 223-232 (1977)). These fragments have been inserted into the EcoRI and BamHI sites, respectively, of plasmid pBR322, and propagated in E.coli. The TK gene contained in the recombinant plasmids was shown to be biologically active when introduced into TK- mouse L cells. Detailed restriction site maps of the BamHI p fragment have been constructed and the approximate location of the TK gene has been determined. Mouse cells transformed with cloned HSV-1 tk+ DNA produced HSV-1-specific thymidine kinase; superinfection with HSV-1 tk- virus increased the level of TK activity tenfold, suggesting that the BamHI p sequences present in transformed cells respond to virus-encoded regulatory gene product(s).

Herpes simplex virus gene expression in transformed cells. I. Regulation of the viral thymidine kinase gene in transformed L cells by products of superinfecting virus

In this paper we show that the expression of the herpes simplex virus type 1 (HSV-1) gene for thymidine kinase (tk) in HSV-transformed cells is subject to regulation by two viral products synthesized during productive infection of these cells with a tk- mutant of HSV-1. The cell line used in this study is a derivative of tk-deficient mouse L cells that, after exposure to UV-inactivated HSV-1, had acquired the HSV-1 gene for tk (which we term a resident viral gene) and consequently expressed the tk+ phenotype (LVtk+ cells). Productive infection of these cells with HSV-1(tk-) at appropriate multiplicities caused significant enhancement of the viral tk activity. The results of several experiments allow us to conclude that this enhancement was due to increased synthesis of tk specified by the HSV-1 gene resident in the LVtk+ cells and that a specific protein made early after infection with HSV-1(tk-) mediated the enhancement, probably by increasing the production of mRNA from the viral tk gene resident in the LVtk+ cells. Our data also indicate that another HSV-1(tk-) product acted to turn off tk synthesis. The finding that tk activity continued to increase for a longer time after infection of the LVtk+ cells at 2 PFU/cell than after infection at higher multiplicities suggested the synthesis of a product which inhibited tk synthesis and whose concentration reached critical levels earlier at higher multiplicities of infection. Inhibition of DNA synthesis after infection, a treatment that depresses the synthesis of late viral proteins, prolonged the synthesis of tk in LVtk+ cells infected at either 2 or 5 PFU/cell. Infection of the LVtk+ cells with HSV-2(tk-) resulted in only small increases in tk activity, indicating some type specificity in recognition of viral products that can modify the expression of the HSV-1 tk gene resident in these cells.