Departamento de Medicina Preventiva, Salud Pública y Microbiología, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo 4, E-28029 Madrid, Spain
Correspondence
E. Tabarés
enrique.tabares{at}uam.es
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ABSTRACT |
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MAIN TEXT |
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Pseudorabies virus (PRV) has only one IE gene (IE180), which encodes a protein of 180 kDa (IE180; Ihara et al., 1983). IE180 has a high level of similarity to the IE proteins of other alphaherpesviruses, such as ICP4 [herpes simplex virus type 1 (HSV-1)], IE40 [varicella-zoster virus (VZV)], IE1 (equid herpesvirus 1) and p180 (bovine herpesvirus 1) (Vlcek et al., 1989
). These proteins have been divided into five collinear regions based on their predicted amino acid sequences, with a high level of similarity in regions 2 and 4 and little similarity in regions 1, 3 and 5 (Cheung, 1989
; Wu & Wilcox, 1991
). The IE180 protein has a DNA-binding domain that binds specifically to the consensus sequence of the alphaherpesviruses' transcription initiation site (Wu & Wilcox, 1991
), implying that it has a role in the negative regulation of its own gene.
The purpose of this study was firstly to construct and characterize recombinant PRV by using the HSV-1 4TK gene as a selection marker, and then to use the recombinants to study the role of the PRV IE180 protein in HSV-1
4 promoter regulation and in the possible complementation of ICP4-deficient HSV-1.
Two PRV recombinants, gIS8 and N1aHTK, were constructed by insertion of the HSV-1 chimeric gene 4TK (Post et al., 1981
; Poffenberger et al., 1983
), which is included in the 3·6 kbp PvuII fragment. This fragment has the
4 gene promoterregulatory region juxtaposed in appropriate transcriptional orientation to the HSV-1 TK gene; it is used widely for genetic manipulation of HSV-1 (Roizman & Jenkins, 1985
) to supply TK activity to recombinant viruses (Post et al., 1981
). The gIS8 virus was obtained by insertion of the
4TK gene into NcoINcoI sites in the PRV BamHI-7 fragment (Fig. 1a
), which also includes gI, gE and US9 (Fernández et al., 1999
). This insertion produced an increase in mobility in the PRV BamHI-7 fragment compared to that of the BamHI-6 fragment (Fig. 1b and c
, lanes 3 and 4). The N1aHTK recombinant was made by insertion of the
4TK gene into the BalI site at codon 264 of the PRV TK gene sequence, which is located in the BamHI-11 fragment (Fig. 1a
).
4TK insertion produced a mobility increase in the PRV BamHI-11 fragment, compared to that of the BamHI-7 fragment (Fig. 1b and c
, lanes 5 and 6).
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Detection of TK activity in the recombinant viruses correlated with HSV-1 TK gene mRNA expression. By using Northern blot analysis, we studied expression of IE mRNA (-RNA) and early mRNA (
-RNA) in recombinant viruses by using cycloheximide and phosphonoacetic acid (PAA) treatment, respectively, and late RNA in the absence of inhibitors (Fig. 1d
). Cytoplasmic RNA (Camacho & Tabarés, 1996
) was fractionated on a 1 % formaldehyde agarose gel, blotted onto nitrocellulose and hybridized [according to Cistué & Tabarés (1992)
] with 32P-labelled 0·3 kbp TK DNA corresponding to the SacIPstI fragment, nt 447748 of the coding sequence (McKnight, 1980
). HSV-1 TK mRNA was detected as a transcript of approximately 1·3 kbp (McKnight, 1980
). TK behaved as an early gene in HSV-1, its natural infection context, as it was expressed in the presence of PAA (Fig. 1d
, lane C) and in the absence of cycloheximide or PAA (Fig. 1d
, lane D). In contrast, HSV-1 TK mRNA derived from the
4TK chimeric gene was expressed as an IE transcript in recombinant PRVs N1aHTK and gIS8, showing overaccumulation in the presence of cycloheximide (Fig. 1d
, lanes E and H) and was inhibited strongly in early or late infection phases (Fig. 1d
, lanes F, G, I and J). The HSV-1
4 promoter thus behaves as an IE promoter in the PRV recombinant genomic context, with strong inhibition of expression in the early and late phases, implying homologous regulation of the HSV-1 promoter by PRV proteins.
Autorepression of gene expression by ICP4 involves physical interaction between the protein and DNA (Michael & Roizman, 1993). As the ICP4 and IE180 proteins share extensive identity in the domains termed regions 2 and 4, autoregulation of HSV-1 ICP4 and PRV IE180 gene transcription may occur through a similar mechanism. Region 2 domains of both ICP4 and IE180 expressed in Escherichia coli bind to DNA sequences that overlap the transcription start site in their respective gene promoters (Wu & Wilcox, 1991
). Our results suggest that the high binding affinity observed in vitro of PRV IE180 domain 2 to the DNA sequences that flank the HSV-1 ICP4 promoter transcription start site (Wu & Wilcox, 1991
) may be functional in vivo and could be implicated in negative regulation of the HSV-1 ICP4 promoter of PRV recombinants.
As autorepression of ICP4 on its own promoter is affected by binding-site orientation and its distance from the TATA box (Leopardi et al., 1995), it was unclear whether the similarity of IE180 and ICP4 domain 2 regions and their target DNA sequences would be reflected by functional equivalence. To determine whether IE180, the unique PRV IE protein, is responsible for negative regulation of the HSV-1
4 promoter, we performed transfection experiments in 293T cells with pRTKC, which encodes the chimeric
4TK gene, either alone or together with a plasmid that encodes the PRV IE180 protein (pE180). As a control for positive regulation of the HSV-1
4 promoter, we cotransfected pRTKC with pVP16, which encodes HSV-1 VP16 protein. The pE180 plasmid was constructed by insertion of the BamHI-8 fragment, which contains the PRV IE180 coding region, into pCDNA3 (Invitrogen). Plasmid pVP16 consists of an EcoRVFokI fragment that contains the HSV-1 VP16 coding region, inserted into the vector pCDNA 3.1HisA (Invitrogen). Plasmid pRTKC was obtained by cloning the SalIKpnI fragment from pHSV-1-TK into the SalIKpnI site in the vector pRPE4 (Invitrogen) without the RSV promoter. pHSV-1-TK contains
4TK in the pUC18 vector.
To ensure that all DNA, RNA and protein expression measurements could be compared directly, experiments were performed with the same transfection cultures of 293TpRTKC cells, either alone or cotransfected with plasmid pEGFP, pE180 or pVP16. 293T cells (9x106) were put in a 150 cm2 flask 18 h before transfection and treated with 25 µM chloroquine (Sigma) in 6 ml medium, 5 min before transfection (Abad et al., 2002). Plasmid construct (60 µg) in 3 ml 204 mM CaCl2 was mixed with 3 ml 2x HBS (274 mM NaCl, 50 mM HEPES, 1·5 mM Na2HPO4, pH 7). Medium was changed after 8 h. DNA, RNA and IE180 and VP16 proteins were analysed at 48 h post-transfection. Cellular DNA was isolated by SDS/proteinase treatment (Tabarés, 1987
); RNA was isolated with the Total Isolation system (Promega). RNA synthesis from transfected cells was analysed by RT-PCR. PCR was carried out with primers HTK5A (5'-ACTGCGGGTTTATATAGACGG-3') and HTK6 (5'-ATGAGGGCCACGAACGCCAG-3') for the HSV-1 TK gene, IE180-S (5'-CTTCAGCCAGCTCCTGGCGG-3') and IE180-AS (5'-GGCCGAAGAGGAGATCCTCG-3') for the IE180 gene, VP16-S (5'-CGCGCTATGTACCATGCTCG-3') and VP16-AS (5'-CCATTCCACCACATCGCTGG-3') for the VP16 gene and GAPDH-S (5'-CCACCCATGGCAAATTCC-3') and GAPDH-AS (5'-TCTAGACGGCAGGTCAGG-3') for the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as an internal control. For RT-PCR, RNA was reverse-transcribed to cDNA by using retrotranscriptase (GeneAmp Gold RNA; Applied Biosystems). PCR and RT-PCR products were analysed by 1 % agarose gel electrophoresis. RNA analysis showed that HSV-1 TK RNA increased 2·16-fold in the presence of VP16 protein [Fig. 2a
(iii), lane 5], whereas it was inhibited in the presence of IE180 protein [Fig. 2a
(iii), lane 4] compared to the amount of HSV-1 TK mRNA expressed by pRTKC alone [Fig. 2a
(iii), lane 3]. The amount of cellular RNA was similar in all experiments, as determined by the GADPH control [Fig. 2b
(iiiiv), lanes 1, 3, 4 and 5]. Presence of the
4TK gene and the control GAPDH gene was determined by PCR [Fig. 2a
(i), b (i)]; these products were characterized by hybridization [Fig. 2a
(ii), b (ii)]. DNA amplification products were characterized by sequencing and used as probes to analyse TK [Fig. 2a
(ii, iv)] and GAPDH [Fig. 2b
(ii), iv)] in transfected cell samples. IE180 and VP16 protein synthesis was detected by Western blot analysis [Fig. 2a
(v)]. We conclude that the presence of the PRV IE180 gene product inhibits transcription of the HSV-1 TK gene by downregulating the HSV-1
4 promoter to levels that were undetectable by the RT-PCR assay.
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ACKNOWLEDGEMENTS |
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Received 18 March 2004;
accepted 19 May 2004.
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