Regulation of herpes simplex virus immediate-early gene promoters in mouse neuroblastoma cells

Neuroscience Letters, 118 (1990) 185-188 Elsevier Scientific Publishers Ireland Ltd.

185

07213

Regulation of herpes simplex virus immediate-early gene promoters in mouse neuroblastoma cells L . M . K e m p l, I . H . G e l m a n 2, S.J. Silverstein 2 a n d D.S. L a t c h m a n 1 IMedical Molecular Biology Unit, Department of Biochemistry, University Collegeand Middlesex School of Medicine, The Windeyer Building, London, (U.K.) and 2Departmentof Microbiology, Collegeof Physicians and Surgeons, Columbia University, NY 10032 (U.S.A.)

(Received 30 April 1990; Revised version received 22 June 1990; Accepted 25 June 1990) Key words: Herpessimplexvirus; Immediate-earlygene; Neuronal cell; Latency; Gene regulation

The non-permissivityof C1300 mouse neuroblastoma cells for herpes simplexvirus (HSV) infection is due to a failure of such cells to transcribe the immediate-early(IE) genes following viral infection. We have transfected both C1300 cells and permissivecells with constructs in which each of the 5 IE promoters drives expression of the readily assayable chloramphenicol acetyl transferase (CAT) gene. These experiments show that the lack of IE gene transcription in C1300 cells is due to the weak activity of the five IE promoters in these cells compared to that observed in a range of permissive cell types. This effect is mediated both by up-stream elements and by sequences present in the minimal promoter. The different effects of DNA concentration on the activities of the minimal and complete promoters suggests that the up-stream sequences act by binding a repressor factor present in C1300 cells whilst the weak activity of the minimal promoter results from the absence of a positive factor in such cells.

Although herpes simplex virus (HSV) can productively infect a wide variety of different cell types both in vitro and in vivo, infection of neuronal cells in vivo resuits in asymptomatic latent infections whose periodic reactivation causes considerable difficulties in the treatment of herpetic disease (reviewed in refs. 13, 17). Studies on latently infected ganglia from a range of species, have established that they do not contain detectable levels of the m R N A s encoding the viral immediate-early (IE) proteins indicating that the lytic cycle is aborted in these cells at a very early stage [4, 18]. The small numbers of latently infected cells in vivo and their inaccessibility have thus far prevented a molecular analysis o f the processes responsible for the failure of the lytic cycle in these cells. To bypass this problem we have investigated the interaction o f HSV with a cell line of neuronal origin, the mouse C 1300 neuroblastoma line originally described by Augusti-Tocco and Sato [2]. These cells can be grown in large amounts in culture and are non permissive for HSV [19, 20] no viral IE proteins being synthesized following infection [1]. The cells thus exhibit a block to lytic infection which is similar to that observed in latently infected Correspondence: D.S. Latchman, Medical Molecular Biology Unit, Department of Biochemistry, University College and Middlesex School of Medicine, The Windeyer Building, Cleveland Street, London W1P 6DB, U.K.

0304-3940/90/$ 03.50 © 1990 Elsevier ScientificPublishers Ireland Ltd.

cells and may hence offer insights into the processes regulating HSV infection of neuronal cells. We have previously shown that the absence of the viral IE proteins following infection of these cells with HSV type l, strain F results from failure to transcribe their corresponding genes, the first time such a block to viral IE gene expression has been demonstrated in cells of neuronal origin [12]. In the case of the IE gene encoding ICP4, we have also shown that this lack of transcription is due to the weak activity of the IE promoter in C1300 cells. Thus a construct in which the IE-4 promoter regulates the expression of the chloramphenicol acetyl transferase (CAT) gene (Cat-: - 8) was expressed approximately 40-fold less well in C1300 cells compared to permissive B H K cells [11]. In order to extend these studies to the other IE promoters and to compare other permissive cell types with C 1300 cells, we used a series of constructs [5, 6] in which the C A T gene is regulated by each of the IE promoters. A construct in the same vector in which the C A T gene is regulated by the IE-4 promoter (from - 790 to + 33) was also included in these studies both for comparison and to confirm and extend our previous study which used a smaller fragment of the IE promoter (from - 330 to + 33) in a different plasmid vector. The levels o f expression directed by these constructs [5, 6] in C1300 cells were compared to that observed following their introduction into permissive cell types from a range of dif-

186 TABLE I ACTIVITY OF THE IE PROMOTERS IN VARIOUS CELL LINES Values indicate the percentages of chloramphenicol acetylated in lysates prepared from transfected cells, all samples having been equalized for protein content by the method of Bradford [3]. Cells were transfected by the method of Gorman [7] with 2 ,ug of DNA per 90 mm plate, n.d. = not determined. Constructs used are described by Gorman et al. [8, 9], and Gelman and Silverstein [5]. RSV, Rous Sarcoma virus; SV, SV40; IE, immediate-early; CAT, chloramphenicol acetyl transferase. Promoter

Plasmid plGA

Cell line BHK Vero

LTA

C1300

65 102 95 101

16 9 25 55 14 80

9 nd 45 30 12 25

13 8 3 2 1 2.5

RSV-CAT SV2-CAT IEO-CAT IE4-CAT IE27-CAT IE22/47-CAT

ll nd nd 38 I1 64

ferent species. The results of this experiment (Table I) indicated that each of the IE promoters had only a very weak activity in the C1300 line, although they were highly active in permissive cells including the mouse LTA cell line. The levels of activity directed by the Rous sarcoma virus promoter or that of SV40 [8, 9] were similar in the C 1300 cells to that observed in other cell types demonstrating that low transfection efficiency was not

a

responsible for the weak activity of the IE promoters in these cells. To study this effect further we carried out titration experiments by transfecting different amounts of IEDNA into the cells, the total amount of DNA transfected being equalized with pAT 153 plasmid vector. In this experiment activity of the IE-4 promoter in plGA 102, increased as more DNA was introduced into the C 1300 cells, exhibiting a threshold level after which the amount of chloramphenicol acetylated dramatically increased, to a point beyond which further increases in DNA transfected produced no increase in activity (Fig. 1). Despite this effect, the level of activity of the promoter was considerably lower in C1300 cells than in permissive cell types regardless of the amount of DNA introduced. The weak activity of the IE promoters in C1300 cells observed here, parallels the lack of IE gene transcription which we previously observed [12] following HSV infection of C1300 cells and suggests that the weak activity of the IE promoters in such cells is responsible for the failure of IE gene transcription and hence of the lytic cycle following infection of C 1300 cells. To investigate the basis of this weak activity, we introduced constructs containing progressively truncated IE promoters into C1300 cells and permissive BHK-21 cells (clone 13: - 14) and compared their relative levels of expression in the two cell types (Table II). In analyzing

13

IE-4 Full Promoter 100

IE-4 Minimal Promoter 100 •

BHK c13oo

[] rO

80

80 O m

60

60

r~

O

40

40 O O

O.

O.

20

,0 j_ILJ 0

0.5

1

2

4

ug DNA

8

10

2

5

7.5

10

20

ug DNA

Fig. 1. a: activity of the full IE-4 promoter in BHK and C1300 cells. Cells were transfected with the indicated amount of the IE-4 construct plGA 102 by the method of Gorman [7]. Amounts of DNA transfected were equalized to 20 gg per 90 mm plate with pAT 153 plasmid vector. Samples were equalized for protein content by the method of Bradford [3]. The figure indicates the percentage of chloramphenicol acetylated by each sample in a 30 min incubation. A negative control sample which had been transfected with plasmid vector alone was included in each experiment to test for any background CAT activity. No such activity was detected however. The amount of CAT activity in BHK cells transfected with 0.5 gg of DNA was not determined, b: activity of the truncated IE-4 promoter contained in plGA 91 in BHK and C 1300 cells. As before the negative control of plasmid vector alone gave no background activity, indicating that the low level of activity observed in C1300 cells represents genuine promoterdriven activity rather than background in the assay. The amount of CAT activity in BHK cells transfected with 20 #g of DNA was not determined.

187 TABLE II ACTIVITY OF TRUNCATED IE-PROMOTERS Figures indicate the percentagesof chloramphenicolacetylatedin each case and were obtained as in the legend to Table I. The value B/C is the relativeactivityof each construct in BHK comparedto C 1300cells. IE, immediate-early;CAT, chloramphenicolacetyltransferase. Promoter plGA

IE4 IE4 IE4 IE4 IE27 IE27

102 104 72 91 95 98

Promoter boundary

CAT activity

Relative activity

5'

3'

BHK

C1300

B/C

-790 - 330 -290 - 108 -240 - 84

+33 + 33 +33 + 33 +1 +1

55 45 75 7.5 14 30

2.0 1.5 2.5 1.0 1.0 5.2

27.5 30 30 7.5 14 6

these results it is instructive to consider first the IE-4 promoter. Truncation of this promoter from - 7 9 0 to - 330 results in a fall in activity in both cell lines but the relative activity of the promoter in the two cell lines remains similar indicating that although this region is important for activity in general, it is not responsible for the relative weakness of the promoter in C1300 cells. Similarly further truncation of the promoter to - 290, increases activity by a similar proportion in each cell type indicating that the region from - 330 to - 290 has a net negative effect on gene expression which is similar in both cell types. These results are in agreement with previous observations made in other permissive cell types [5]. Further truncation of the promoter to - 108 however, results in a dramatic decrease in activity in BHK cells in agreement with the presence in the - 290 to - 1 0 8 region of both T A A T G A R A T and Spl elements necessary for high-level expression of the promoter [10, 15, 16]. In C1300 cells, however, the effects of this truncation were much smaller, resulting in a higher relative activity of this truncated construct in C 1300 cells compared to B H K cells. The activity of the truncated construct was clearly above background in 3 replicate experiments. A similar effect of the up-stream region containing T A A T G A R A T and Spl elements was also observed on the IE 27 promoter. Here removal of this region (from - 240 to - 84) increased the activity of the promoter in both cell types indicating that this region contains a silencer element. The increase was considerably larger in C 1300 cells however, resulting as with the IE-4 promoter in increased relative activity of the minimal promoter in C 1300 compared to BHK cells. Clearly therefore one element responsible for the weak

activity of the IE promoters in C1300 cells lies in their up-stream regions. This effect could result from either the absence or low level in C1300 cells of a factor required for expression of the IE gene or the presence in C1300 cells of a negative factor which binds to this region. This latter possibility is more consistent with the dose dependent expression of the IE promoters in C 1300 cells (see Fig. 1) since the removal of this factor by titration would account for the dramatic increase in activity occurring at a specific concentration of IE promoter elements. Similarly the existence of such a factor binding to viral T A A T G A R A T elements would explain our observation that the activity of the IE-4 promoter can be increased by co-transfection of plasmids containing isolated T A A T G A R A T elements [11]. Whatever their mechanism, effects mediated through up-stream sequence elements cannot entirely explain the weak activity of the IE promoters in C1300 cells. Thus the removal of these elements does not entirely abolish the differential activity of these promoters in C 1300 and BHK cells, even the minimal promoters being expressed more weakly in C1300 cells (Table II) regardless of the amount of D N A transfected. Interestingly in experiments with the minimal IE-4 promoter although the amount of chloramphenicol acetylated in B H K cells increased as more D N A was transfected, no effect on the low, but detectable, activity seen in C1300 cells was observed as the amount of D N A was increased. This concentration-independent expression differs significantly from that of the intact promoter suggesting that the system is readily saturated and that a factor required for expression from the minimal IE promoter is present at only very low levels in C1300 cells. Therefore increasing the amount of D N A transfected does not increase activity. Interestingly low activity in B H K compared to C1300 cells was also observed with the minimal promoter of IE-27 which apparently contains only a T A T A box [6] indicating that this effect must be mediated through a previously unrecognized transcriptional control element or involves a particular T A T A box binding protein required for activity of the IE promoters. In summary multiple effects acting both on up-stream elements and the minimal promoter mediate the weak activity of the IE promoters in C 1300 cells and hence the non-permissivity of these cells for HSV. We are currently investigating the proteins interacting with this region in these cells in order to determine the mechanisms responsible for these effects.

We thank John Estridge for excellent technical assistance. This work was supported by grants from Action

188 R e s e a r c h f o r the C r i p p l e d C h i l d a n d t h e C a n c e r R e search Campaign

to D . S . L . a n d b y a g r a n t f r o m the

U S P H S C A 1 7 4 7 7 to S.J.S.

1 Ash, R.J., Butyrate-induced reversal of Herpes simplex virus restriction in neuroblastoma cells, Virology, 155 (1986) 584-592. 2 Augusti-Tocco, G. and Sato, G., Establishment of functional clonal lines of neurons from mouse neuroblastoma, Proc. Natl. Acad. Sci. U.S.A., 64 (1969) 311-315. 3 Bradford, M., A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. 4 Croen, K.D., Ostrove, J.M., Draguvic, L.J., Smialek, J.E. and Straus, S.E., Latent herpes simplex virus in human trigeminal ganglia. Detection of an immediate-early gene anti-sense transcript by in situ hybridization, New Eng., J. Med., 317 (1987) 1427-14322. 5 Gelman, I.H. and Silverstein, S., Herpes simplex virus immediateearly promoters are responsive to virus and cell trans acting factors, J. Virol., 61 (1987) 2286-2296. 6 Gelman, I.H. and Silverstein, S., Dissection of immediate-early gene promoters from Herpes simplex virus: sequences that respond to virus transcriptional activators, J. Virol., 61 (1987) 3167-3172. 7 Gorman, C.M. High efficiency gene transfer into mammalian cells. In: D.M. Glover (Ed.), DNA Cloning, a Practical Approach, Vol. 2, IRL Press, 1985, pp. 143-190. 8 Gorman, C.M., Moffat, L.F. and Howard, B.H., Recombinant genomes which express chloramphenicol actyltransferase in mammalian cells, Mol. Cell. Biol., 2 (1982) 1044-1051. 9 Gorman, C.M., Merlino, G.T., Willingham, M.C., Pastan, I. and Howard, B., The rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by transfection, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 67776781.

10 Jones, K.A. and Tjian, R., Spl binds to promoter sequences and activates herpes simplex virus immediate-early gene transcription in vitro, Nature, 317 (1985) 179-182. 11 Kemp, L.M., Dent, C.L. and Latchman, D.S., The octamer motif mediates transcriptional repression of HSV immediate-early genes and octamer-containing cellular promoters in neuronal cells, Neuron, 4 (1990) 215-222. 12 Kemp, L.M. and Latchman, D.S., Regulated expression of herpes simplex virus immediate-early genes in neuroblastoma cells, Virology, 171 (1989)607~I0. 13 Latchman, D.S., Molecular biology of herpes simplex virus latency, J. Exp. Pathol., 71 (1990) 133-141. 14 Macpherson, I. and Stoker, M., Polyoma transformation of hamster cell clones - an investigation of genetic factors affecting cell competence, Virology, 16 (1962) 147-151. 15 O'Hare, P. and Goding, C.R., Herpes simplex virus regulatory elements and the immunoglobulin octamer domain bind a common factor and are both targets for virion transactivation, Cell, 52 (1988) 435-445. 16 Preston, C.M., Frame, M.C. and Campbell, M.E.M., A complex formed between cell components and a herpes simplex virus structural polypeptide binds to a viral immediate-early gene regulatory DNA sequence, Cell, 52 (1988) 425-434. 17 Roizman, B. and Sears, A.E., An inquiry into the mechanisms of herpes simplex virus latency, Annu. Rev. Microbiol., 41 (1987) 543-57 I. 18 Stevens, J.G., Wagner, E.K., Devi-Rao, G.B., Cook, M.L. and Feldman, L.T., RNA complementary to a herpes virus alpha gene mRNA is prominent in latently infected neurons, Science, 235 (1987) 1056-1059. 19 Vahlne, A. and Lycke, E., Herpes simplex virus infection of mouse neuroblastoma cells. Proc. Soc. Exp. Biol. Med., 156 (1977) 82-87. 20 Vahlne, A. and Lycke, E., Herpes simplex virus infection of in vitro cultured neuronal cells, J. Gen. Virol., 39 (1978) 321-332.