Neuron,
Vol. 4, 215-222,
February,
1990, Copyright
0 1990 by Cell Press
Octamer Motif Mediates Transcriptional Repression of HSV Immediate-Early Genes and OctamerContaining Cellular Promoters in Neuronal Cells 1. M. Kemp, C. 1. Dent, and D. S. latchman Medical Molecular Biology Unit Department of Biochemistry University College and Middlesex School The Windeyer Building London WIP 6DB England
of Medicine
Summary Cl 300 mouse neuroblastoma cells are nonpermissive for infection with herpes simplex virus owing to a failure of viral immediate-early gene transcription following infection. The weak activity of the immediate-early gene promoters in these cells is mediated by the binding of a repressor factor to the octamer-related TAATGARAT motifs in these promoters. This repressor activity is specific to cells of neuronal origin (being absent in a range of permissive nonneuronal cells) and is also able to repress the activity of cellular octamer-containing promoters introduced into Cl300 cells. The role of this repressor in the regulation of octamer-containing cellular genes in neuronal cells and in the control of latent infections with herpes simplex virus is discussed. Introduction Herpes simplex virus (HSV) establishes lifelong latent infections of sensory neurons in human dorsal root ganglia (reviewed by Roizman and Sears 1987; Latchman, 1990). Although these infections are entirely asymptomatic, their periodic reactivation followed by migration of the virus to the periphery and subsequent lytic infection of epithelial cells results in the facial and genital sores suffered by many individuals. Such differences in susceptibility of different cell types to viral infection are also observed in vitro; a wide variety of different cell types readily support viral replication, whereas primary cultures of sensory neurons are relatively nonpermissive for lytic infection (Kennedy et al., 1983; Wilcox and Johnson, 1988). Following infection of susceptible cells in vivo or in vitro, the first stage in the lytic cycle is the transcription of the viral immediate-early (IE) genes, whose protein products play an essential role in the subsequent stages of viral gene expression (Preston, 1979; Watson and Clements, 1980). In contrast, IE mRNAs cannot be detected by in situ hybridization in latently infected ganglia (Croen et al., 1987; Stevens et al., 1987), and hence the lytic cycle is aborted at an early stage. Most interestingly, IE gene expression in lytic infection is dependent upon the interaction of constitutively expressed cellular transcription factors, notably, Spl and the octamer binding protein OTF-1, with binding sites in the IE promoters (Jones and Tjian 1985;
O’Hare and Goding, 1988). The failure of IE gene expression in neuronal cells is therefore likely to be caused by differences in the nature of these cellular transcription factors in such cells, which may in turn reflect differences in the regulation of cellular gene expression mediated by these factors in neuronal cells. Hence, a study of the cellular factors interacting with IE promoters in neurons maythrow light not only on the processes mediating latent infection, but also on cellular gene regulation in neuronal cells. Unfortunately, the amounts of material available from primary cultures of sensory neurons are insufficient for such a study; we have therefore used the Cl300 mouse neuroblastoma cell line (Augusti-Tocco and Sato, 1969) as a model system for studying the interaction of HSV with cells of neuronal origin. Thus, these cells are nonpermissive for lytic infection with HSV (Vahlne and Lycke, 1977, 1978). By using nuclear run-on assays, we have recently shown (Kemp and Latchman, 1989) that this effect is mediated by a failure to transcribe the viral IE genes following infection and that this transcriptional block can be relieved by pretreating the cells with sodium butyrate, which is known to increase their permissivity for HSV infection (Ash, 1986). Here we report that this transcriptional repression is mediated by the octamer motif in the IE promoters. Such repression is also observed when octamer-containing cellular promoters are introduced into Cl300 cells and can be relieved by mutation or deletion of the octamer element, suggesting that this effect is important in cellular as well as viral gene regulation in neuronal cells. Results To investigate the mechanisms responsible for the failure of viral IE gene expression in Cl300 cells, we used aconstruct (IE-CAT) in which the promoter of the major HSV-1 IE gene (IE3, which encodes ICP4) regulates the expression of the gene encoding the chloramphenicol acetyltransferase protein (CAT; Gorman et al., 1982a). In this experiment IE-CATwas expressed approximately 40-fold less well when transfected into Cl300 cells than in permissive BHK cells, as assayed by CAT activity, although the levels of expression driven by the Rous sarcoma virus promoter (Gorman et al., 1982b) were similar in both cell types (Figure 1). In five replicate experiments, the IE-CATconstruct was consistently expressed between 30 and 50 times less well in Cl300 compared with BHK cells. No activity was observed in either cell type with CAT vector that lacked any promoter element. To confirm that the difference in the activity of IECAT in BHK and Cl300 cells was mediated at the level of transcription of the CAT gene, we carried out a nuclear run-on assay (Pate1 et al., 1986) to directly measure transcription in the transfected cells. In these ex-
Neuron 216
2
16 Figure
1. Assay
3
13
4
75
of CAT Activity
Assay was carried out according to the method of Corman (1985). Cells were transfected using the calcium phosphate procedure (Corman, 1985) with either an RSV-CAT construct (lanes 1 and 2) or an HSV IE-CAT construct (lanes 3 and 4). Lanes 1 and 3 show the results obtained in permissive BHK cells; lanes 2 and 4 show results obtained in Cl300 cells. Numbers on the bottom indicate the percentage of available chloramphenicol acetylated in each case.
periments (Table 1) a high level of IE-driven CAT gene transcription was detected in BHK cells compared with Cl300 cells, although the levels of CAT gene transcription in cells transfected with the RSV-CAT construct were similar in both cell types. As expected, endogenous cellular genes such as that encoding histone H2B showed similar levels of transcription in all samples. A high level of activity of the IE promoter (as assayed by CAT activity) was also detectable in a range of nonneuronal human and rodent cell lines including HeLa, Vero, 3T3, and L cells (data not shown). Hence, the failure of HSV to express its IE genes following infection of Cl300 cells is due to the weak activity of the IE promoters in such cells. This weak pro-
Table 1. Nuclear Run-On Assay CAT Gene in Cells Transfected Transfected Cl300
to Measure Transcription of the with Various CAT Constructs
DNA
Cells
BHK
Cells
Gene
RSV-CAT
IE-CAT
RSV-CAT
I E-CAT
CAT H2B 123
40 (4) 48 (5) 180 (15)
5 t-1 41 (3) 175 (12)
41 (6) 46 (7) 150 (14)
160 (15) 45 (5) 170 (11)
Figures (average of two determinations whose range is given in parentheses) indicate the counts per minute binding to the indicated clone in nuclear run-on assays in cells transfected with the indicated DNA. Clone 123 is derived from a cellular gene whose RNA level does not change in HSV infection (Kemp et al., 1986).
moter activity compared with that observed in BHK cells was observed in three separate experiments regardless of the amount of IE-CAT DNA that was transfected (Figure 2). Interestingly, however, as the amount of IE-CAT DNA transfected into Cl300 cells was increased, a significant increase in CAT activity was observed at one point in the concentration series (between 2 gg and 5 PLg of DNA in Figure 2), after which CAT activity remained relatively constant with increasing DNA concentration. Such a pattern of activity suggested that the weak activity of the IE promoters in Cl300 cells might be due to the presence of a trans-acting repressor in neuronal cells that binds to the IE promoter. Thus, if this repressor is present in relatively low amounts, then as the amount of [E-CAT DNA is increased, the repressor will be progressively titrated out, and a sudden increase in CAT activity will occur at the point in the concentration series when all repressor has been bound. If such a repressor does indeed exist, it should be possible to increase IE-CAT activity by cotransfecting with IE-CAT the cloned binding site for the repressor unlinked to any promoter element. This would serve to bind out the repressor and allow increased IE-CAT activity. In three replicate experiments in which various fragments of the IE promoter were cotransfected with IE-CAT, we were able to produce increased CAT activity (Figure 3) using a plasmid (pF) containing a fragment of the IE promoter with a single TAATGARAT (R = purine) DNA sequence element and no other characterized DNA binding motifs. This viral regulatory motif is related at the DNA sequence level to the octamer motif found in a number of cellular gene promoters (for review see Falkner et al., 1986), and in lytic infection, binding of the constitutively expressed cellular transcription factor OTF-1 to this sequence is essential for high level activity of the IE promoters (Baumruker et al., 1988; O’Hare and Coding, 1988). In Cl300 cells, by contrast, it is clear that this motif binds a trans-acting repressor of IE gene activity. Thus, cotransfection of pF with the IE-CAT plasmid resulted in an increase in CAT activity in Cl300 cells (Figure 3), which can only be explained by postulating that pF binds out such a trans-acting repressor, allowing IECAT activity to increase. In contrast, cotransfection of pF into permissive cells resulted only in a small decrease in IE-CAT activity (Figure 3). This is likely to be due to competition between pF and IE-CAT for binding of the OTF-1 factor required for IE gene expression; such competition was previously observed in permissive cell types (Latchman et al., 1989). A similar competition between pF and IE-CAT for the OTF-1 present in Cl300 cells, which would occur following binding out of the repressor, is likely to be responsible for the observation (Figure 3) that the increase in IE-CAT activity obtained by cotransfection of pF into C1300cells is reduced when high levels of pF DNA are used. Hence, the weak activity of IE promoters in c-1300
Octamer-Mediated 217
Repression
a
b
Figure Cl300
1
2
cells is mediated by a trans-acting repressor that binds to the TAATGARAT motif. In turn, this weak activity of the IE promoters causes the observed failure of IE gene transcription following infection of these cells. Interestingly, previous studies (Vahlne et al., 1981; Kemp and Latchman, 1989) have shown that as the amount of virus added to Cl300 cells is progressively increased (from 1 to 50 pfu per cell), a greater than linear increase in the number of cells expressing viral IE proteins and supporting the lytic cycle is observed. A similar effect is also seen in infections of primary sensory neuron cultures (Wilcox and Johnson, 1988). As with the strong concentration dependence of IE-CAT expression, this effect is likely to be mediated by the binding out of the repressor by virally born TAATCARAT elements, allowing high level activity of the excess IE promoters. If this is the case, it should be possible to increase the permissivity of Cl300 cells by first transfecting TAATGARAT-containing plasmids to bind out the repressor and subsequently infecting with low levels of
%
600,
400 -
200 +
‘;
5
102:
Figure 3. Percentage Change in IE-CAT Activity in Cl300 Cells in Which the [E-CAT Plasmid Had Been Cotransfected TAATGARATContaining pF DNA Cl300 cells (circles); BHK cells (squares). Amount containing pF DNA is indicated in micrograms. are the average of three determinations whose by the bars.
or BHK with
of TAATGARAT Values plotted range is shown
of CAT Activity
in BHK or
BHK (a) or Cl300 (b) cells were transfected with the indicated amounts (in micrograms) of the IE-CAT plasmid.
virus. When Cl300 cells were transfected with the TAATGARAT-containing IE-CAT or pF plasmids and infected 24 hr later with HSV-I, a clear increase in permissivity (as assayed by the number of cells staining with an antibody to ICP4) was observed in cells transfected with IE-CAT (Table 2) or pF (Figure 4) compared with those transfected with carrier DNA (PAT 153 vector or salmon sperm DNA) lacking TAATCARAT elements. Three replicate experiments gave similar results. Hence, the binding out of repressor by TAATGARAT elements can relieve the block on IE gene expression in virally infected Cl300 ceils, confirming that this repressor is responsible for the failure of lytic infection in these cells. Most importantly, the repressor activity is specific to neuronal cells; no increase in permissivity was observed when IE-CATor pF was introduced into nonneuronal cells such as 3T3 fibroblasts (Table 2 and Figure 4). There was no increase in permissivity even when low levels of virus were used so that only a relatively small proportion of untransfected cells stained with the antibody, and thus any increase in permissivity would have been readily detected. Indeed, in these cells introduction of IE-CAT or pF results in a decrease in permissivity probably because, as in the cotransfection experiments, they compete for OTF-1, which is required for IE gene transcription (O’Hare and Coding, 1988). A similar phenomenon is presumably responsible for the decreased enhancement in permissivity observed when high levels of IECAT are transfected into Cl300 cells (Table 2). Some enhancement of permissivity is observed in Cl300 cells regardless of the amount of IE-CAT transfected, however, indicating that in these cells the binding of repressor always predominates. Hence, increased permissivity can be produced by binding out of the neuronal repressor by excess TAATCARAT motifs. The relationship of this motif to the octamer element found in a variety of cellular gene promoters and the ability of the TAATGARAT sequence to bind the constitutively expressed octamer binding protein OTF-1 (Baumruker et al., 1988; O’Hare and Coding, 1988) suggested that the repressor we have
-l-=-+-pG
200 J
2. Assay Cells
Table
2. Effect
DNA
Transfected
Carrier
DNA
0 IO 8 6 4
of Transfecting
IE-CAT
fug) IE-CAT 0 0 2 4 6
DNA
on the
Permissivity
HSV-1
Infection
5 pfu
per Cell
Cl300
Cells
ND 13% 30% 36% 27%
of Cells
(271210) (64/210) (75/203) (55/205)
to Viral
Infection
1 pfu
per
3T3 Cells
Cl300
Cells
ND 87% 86% 90 % 76%
4.5% (9/201) 5.3% (12/226) 4.6% (10/216) 11% (22/200) 9.6% (191205)
(921106) (122/141) (49/54) (72/95)
Cell 3T3 Cells 14% (311219) 16.7% (351209) 10% (18/178) 7.4% (14/187) 13.1% (24/183)
Values indicate the total numbers and percentages of cells staining with the 58s monoclonai antibody to the HSV-1 IE protein ICP4 (Showalter et al., 1981). Cells were transfected by the method of Gorman (1985) with the amounts (in ug) of IE-CAT DNA indicated, and the amounts of DNA applied to each plate were equalized with carrier DNA lacking TAATGARAT elements (total salmon sperm DNA or PAT 153); 24 hr after transfection cells were infected with HSV-1 and then stained 16 hr after infection with the 5% antibody. ND, not determined.
identified might be a neuronal cell-specific octamer binding protein. To investigate this possibility we tested whether increased permissivity of Cl300 cells could be produced by transfection of a plasmid (PO) containing a cloned 15 bp oligonucleotide that included the consensus octamer motif (ATGCAAAT). As shown in Table 3, this was indeed the case, confirming that the repressor is an octamer binding protein present in CL300 cells and exerts its effect by binding to the related TAATGARAT elements in the IE promoters. To provide physical evidence for the existence of the repressor protein, we carried out DNA mobility shift assays (Fried and Crothers, 1981) using the identical octamer oligonucleotide employed in the competition experiments. Using extracts from Cl300 cells,
three protein-DNA complexes formed on this probe (Figure 5, lane a). In contrast, only the highest mobility complex formed on an oligonucleotide containing the mutant sequence ATAATAAT, which abolishes recognition by previously characterized octamer binding proteins (Lenardo et al., 1987; Figure 5, lane b). This complex (which also forms on nonoctamer oligonucleotides) thus represents a nonspecific DNA binding protein, whereas the other two bands are due to the binding of sequence-specific octamer binding proteins. When extracts from permissive cells were used (Figure 5, lane c), only the nonspecific complex and the largest complex were observed; the complex of intermediate mobility was absent. It is clear, therefore, that in addition to the ubiquitous octamer binding protein OTF-1 (which is responsible for the largest complex), Cl300 cells also contain an additional octamer binding protein that is absent in permissive cells and is likely to be the repressor factor identified in the competition experiments. The existence of such a factor raises the question of whether it plays any role in the regulation of octamer-containing cellular genes in neuronal cells. To test this possibility we transfected Cl300 cells with a construct in which the octamer-containing immunoglobulin heavy chain enhancer regulates the ex-
Table 3. Effect of Transfecting on the Permissivity of Cl300
Figure 4. Effect of Transfecting the TAATGARATContaining Plasmid pF on the Permissivity of Cl300 Cells or 3T3 Cells to Subsequent Infection with HSV-1 The change in the number of cells staining is plotted against the amount of pF DNA transfected. Values plotted are the average of three determinations whose range is shown by the bars. N indicates Cl300 cells.
DNA
Transfected
Carrier
DNA
(up) PO
an Octamer-Containing Cells to Viral Infection Cl300 1 pfu
Cells Infected per Cell
Plasmid
at
10 0 II/206 (5.4%) 6 4 15/208 (7.2%) 4 6 23/201 (11.4%) Figures indicate the numbers and percentages of cells staining with the monoclonal antibody to the viral IE protein ICP4 and were obtained as described in Table 2.
Octamer-Mediated 219
Repression
a
I a
3
4
Figure 6. Assay of CAT Activity in Cl300 Cells Transfected Various Octamer-Containing Cellular Promoters
Figure
5. DNA
Mobility
Shift
Assay
Lane a, extract from Cl300 cells with labeled wild-type octamer oligonucleotide (ATGCAAAT); lane b, extract from Cl300 cells with labeled mutant octamer oligonucleotide (ATAATAAT); lane c, extract from permissive 373 cells with wild-type octamer oligonucleotide. The arrow indicates the position of the additional sequence-specific octamer binding protein present in the Cl300 cells.
pression of a CAT gene driven by the B-globin promoter (Kadesch et al., 1986). In this experiment (Figure 6, lanes 1 and 2) the low level of activity of this construct was increased by deleting a 48 bp region of the enhancer that contained the octamer element. Similarly, the expression of a construct in which the immunoglobulin enhancer regulates the c-fos promoter was increased (Figure 6, lanes 3 and 4) by mutation of the octamer element from ATGCAAAT to ATAATAAT; this mutation abolishes recognition by the two octamer binding proteins present in C1300cells (see Figure 5) and results in decreased enhancer activity in most cell types (Lenardo et al., 1987). Nuclear run-on assays carried out on the transfected cells (Table 4) indicated that this effect was mediated by increased transcription of the CATgene in cells transfected with the mutant octamer construct. Hence, the repressor is capable of acting on cellular as well as viral promoter elements. Discussion In this report
we have shown
that the octamer
motif
with
Lane I, IV510 construct lacking the octamer element; lane2, IVS6 construct containing the octamer element; lane 3, A56construct with a wild-type octamer element (ATGCAAAT); lane 4, A56 octaconstruct containing a mutated octamer element (ATAATAAT). These constructs are described in Kadesch et al. (1986) and Lenardo et al. (1989).
is capable of mediating the repression of octamercontaining cellular promoters and of HSV IE genes in neuronal cells. This effect is in contrast to that seen in nonneuronal cells where the octamer element acts as a positive regulator of cellular gene promoters containing it, which include those of the small nuclear RNAs, the histone H2B gene, and the immunoglobulin genes (reviewed by Falkner et al., 1986). In each of these promoters, the octamer element is crucially required for its specific pattern of gene activity, mediating, for example, the cell cycle activity of the H2B promoter, the B cell specificity of the immunoglobulin promoters, as well as the constitutive ex-
Table 4. Nuclear Run-On Assay to Measure of the CAT Gene in Cl300 Cells Transfected Octamer-Containing CAT Constructs Transfected Gene
A56 Octa+
.CAT H2B 123
21 (3) 44 (7) 175 (18)
Transcription with
DNA A56 Octa45 (5)
46 (6) 179 (17)
Figures (average of two determinations whose range is given in parentheses) indicate the counts per minute binding to the indicated clone in nuclear run-on assays in cells transfected with the indicated DNA.
NW*Oll 220
pression of the small nuclear RNAs. The differences in the pattern of gene activity mediated by the OCtamer in these promoters is believed to be due to differences in its relationship to other elements in the promoter that affect its recognition by different octamer binding proteins (Tanaka et al., 1988). Thus, in addition to the constitutively expressed octamer binding protein OTF-1, a B cell octamer binding protein, OTF-2, has been described and purified (Scheidereit et al., 1987); more recently several other octamer binding proteins have been identified in different cell types, notably, embryonal carcinoma cells (Lenardo et al., 1989; Scholer et al., 1989), and in the brain (He et al., 1989). The additional octamer binding protein we have detected may therefore be identical to one of these tissue-specific proteins identified by others, or may represent a novel protein expressed specifically in a limited range of neuronal cell types. Of particular interest is the possibility that this factor may be related to one of the additional octamer binding proteins detected by several groups (Lenardo et al., 1989; Scholer et al., 1989) in embryonal carcinoma cells. Thus, these cells, like Cl300 cells, are nonpermissive for HSV (Bell et al., 1987), and both cell types express the gene encoding the small nuclear RNA Ulb, which has an octamer different from the consensus sequence and is expressed in only a limited number of cell types (Lund et al., 1985). This raises the possibility that the repressor activity we have identified might also act as a tissue-specific activator of certain octamer-containing promoters in which the octamer element has a sequence differing from the consensus or is located in a different position relative to other promoter elements. Whatever the case, it is clear that the activity we have described here can repress transcription of certain octamer-containing cellular promoters and TAATGARAT-containing viral promoters. As such, this factor is responsible for the failure of viral IE gene transcription in Cl300 cells (Kemp and Latchman, 1989) and the consequent failure of the viral lytic cycle (Vahlne and Lycke, 1977,1978). The presence of a similar factor in immortalized neurons from rat dorsal root ganglia, the natural site of latent infection with HSV in vivo (S. C. Wheatley, L. M. Kemp, J. N. Wood, and D. S. Latchman, unpublished data), suggests that it may also play a role in the establishment of latent infection. Similarly, modulation of its expression in response to nerve growth factor or stress might be responsible for reactivating the virus in response to these stimuli (Carton and Kilbourne, 1952; Wilcox and Johnson, 1988). It should be noted, however, that the fact that even after titration out of the repressor the activity of IE-CAT in Cl300 cells does not match that in BHK cells (Figures 2 and 3) suggests neuronal cells may also lack positive-acting factors needed for high level IE gene expression. It is clear, however, that the neuronal cell octamer
repressor factor we have identified here plays a major role in the nonpermissivity of neuronal cells. Further study of this factor should throw considerable light on the mechanisms regulating the clinically important problem of latent infection as well as vindicating the use of HSV as a means of probing the processes regulating cellular gene expression in neuronal cells. Experimental
Procedures
Cells and Viruses BHK-21 cells (clone 13; MacPherson and Stoker, 1962) and Cl300 cells (clone 41A3; Augusti-Tocco and Sam, 1969; Ash 1986) were grown in RPM1 medium supplemented with 10% fetal calf serum. Viral infection of transfected cells was carried out 24 hr after transfection (see below! using the indicated multiplicity of HSV-‘I straili F (Ejercito et al., 1968). Sixteen hours after infection cells were stained with the 58s antibody to the viral IE protein ICP4 (Showalter et al., 1981) as previously described (Kemp and Latchman, 1989). Plasmid DNAs The IE-CATconstruct contains the lE3 gene promoter (from -330 to +33) linked to the CAT gene (Stow et al., 19861, while the pF plasmid contains a 63 bp element from the IE4/5 promoter containing a single TAATGARAT element. The p0 plasmid contains a chemically synthesized decanucleotide, containing the consensus octamer sequence with a 5’-GATC overhang at either end cloned into the BamHl site of pUC 1813 (Kay and McPherson, 1987). DNA Transfection Transfection of plasmid DNA was carried out according to the method of Gorman (1985). Except when indicated otherwise, all transfections were carried out using ?O ug of DNA per 2 x IO6 cells on a 90 mm plate. The amounts of DNA in all samples in competition experiments were equalized by use of carrier DNA (PAT 153 or total salmon sperm DNA). Twenty-four hours after transfection, cells were either harvested for nuclear run-on or CAT assays or infected with HSV-1 as described above. CAT Assays Assays of CAT activity were carried out as described by Gorman (1985), extracts having been equalized for protein content as determined by the method of Bradford (1976). Nuclear Run-On Assays Nuclear run-on assays of transcriptional activity in transfected cells were carried out by allowing nuclei prepared from the cells to incorporate radiolabeled [3zP]CTP into nascent transcripts as previously described (Pate1 et al., 1986). The labeled products of such an assay were then used to probe replicate dot blots that had been spotted with 5 pg of plasmid DNAs containing the CAT gene or the control cellular genes histone H2B (Marashi et al., 1984) and clone 123 (derived from a cellular gene whose mRNA level does not change in HSV infection; Kemp et al., 1986). After hybridization the filters were autoradiographed, and labeled spots were cut out and counted by scintillation counting. DNA Mobility Shift Assays Whole cell extracts for use in mobility shift assays were prepared as described by Manley et al. (1980). Extracts were incubated at 30°C for 15 min with labeled octamer oligonucleotide prior to electrophoresis on a nondenaturing acrylamide gel. Acknowledgments We thank John Estridge for excellent technicai assistance and the foliowing for the very kind gifts of plasmid constructs: David Baltimore and Mike Lenardo (A56 and A56 octa-), Tom Kadesch
Octamer-Mediated 221
Repression
(pSVABCCAT IVS6 and IO), Janet Partridge (PO), and Chris Preston (IE-CATand pF). This work was supported by Action Research for the Crippled Child and the Cancer Research Campaign.
Kemp, L. M., Brickell, P M., La Thangue, N. B., and Latchman, D. S. (1986). Transcriptional induction of cellular genes during lytic infection with herpes simplex virus. Biosci. Rep. 6,945-951.
Received
Kennedy, P. G. E., Clements, G. E., and Brown, S. M. (1983). Differential susceptibility of human neural cell types in culture to infection with herpes simplex virus. Brain 706, 101-119.
August
8, 1989;
revised
November
3, 1989.
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Preston, C. M. (1979). Control of herpes simplex virus type 1 mRNA synthesis in cells infected with wild-type virus or the temperature sensitive mutant tsK. J. Virol. 29, 275-284. Roizman, B., and Sears, nisms of herpes simplex 543-571.
A. E. (1987). An inquiry into the mechavirus latency. Annu. Rev. Microbial. 47,
Scheidereit, C., Heguy, A., and Roeder, R. G. (1987). Identification and purification of a human lymphoid-specific octamer binding protein (OTF-2) that activates transcription of an immunoglobulin promoter in vitro. Cell 57, 783-793. Schiiler, H. R., Hatzopoulos, A. K., Balling, R., Suzuki, N., and Gruss, P. (1989). A family of octamer-specific proteins present during mouse embryogenesis: evidence for germline-specific expression of an Ott factor. EMBO J. 8, 2543-2550. Stevens, J. C., Wagner, E. K., Devi-Rao, C. B., Cook, M. L., and Feldman, L. T. (1987). RNA complementary to a herpes virus gene mRNA is prominent in latently infected neurons. Science 235, 1056-1059. Stow, N. D., Murray, M. D., and Stow, E. C. (1986). Cis-acting signals involved in the replication and packaging of herpes simplex virus type-l DNA. Cancer Cells 4, 497-507. Showalter, S. D., Zweig, M., and Hampar, B. (1981). Monoclonal antibodies to herpes simplex virus type 1 proteins including the immediate-early protein ICP4. Infect. Immun. 34, 684-692. Tanaka, M., Crossniklaus, U., Herr, W., and Hernandez, Activation of the U2 sn RNA promoter by the octamer
N. (1988). motif de-
Neuron 222
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A herpes for early
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Wilcox, C. L., and Johnson, E. M. (1988). Characterization nerve growth factor-dependent herpes simplex virus latency neurons in vitro. J. Viral. 62, 393-399.
of in
Vol. 4, 215-222,
February,
1990, Copyright
0 1990 by Cell Press
Octamer Motif Mediates Transcriptional Repression of HSV Immediate-Early Genes and OctamerContaining Cellular Promoters in Neuronal Cells 1. M. Kemp, C. 1. Dent, and D. S. latchman Medical Molecular Biology Unit Department of Biochemistry University College and Middlesex School The Windeyer Building London WIP 6DB England
of Medicine
Summary Cl 300 mouse neuroblastoma cells are nonpermissive for infection with herpes simplex virus owing to a failure of viral immediate-early gene transcription following infection. The weak activity of the immediate-early gene promoters in these cells is mediated by the binding of a repressor factor to the octamer-related TAATGARAT motifs in these promoters. This repressor activity is specific to cells of neuronal origin (being absent in a range of permissive nonneuronal cells) and is also able to repress the activity of cellular octamer-containing promoters introduced into Cl300 cells. The role of this repressor in the regulation of octamer-containing cellular genes in neuronal cells and in the control of latent infections with herpes simplex virus is discussed. Introduction Herpes simplex virus (HSV) establishes lifelong latent infections of sensory neurons in human dorsal root ganglia (reviewed by Roizman and Sears 1987; Latchman, 1990). Although these infections are entirely asymptomatic, their periodic reactivation followed by migration of the virus to the periphery and subsequent lytic infection of epithelial cells results in the facial and genital sores suffered by many individuals. Such differences in susceptibility of different cell types to viral infection are also observed in vitro; a wide variety of different cell types readily support viral replication, whereas primary cultures of sensory neurons are relatively nonpermissive for lytic infection (Kennedy et al., 1983; Wilcox and Johnson, 1988). Following infection of susceptible cells in vivo or in vitro, the first stage in the lytic cycle is the transcription of the viral immediate-early (IE) genes, whose protein products play an essential role in the subsequent stages of viral gene expression (Preston, 1979; Watson and Clements, 1980). In contrast, IE mRNAs cannot be detected by in situ hybridization in latently infected ganglia (Croen et al., 1987; Stevens et al., 1987), and hence the lytic cycle is aborted at an early stage. Most interestingly, IE gene expression in lytic infection is dependent upon the interaction of constitutively expressed cellular transcription factors, notably, Spl and the octamer binding protein OTF-1, with binding sites in the IE promoters (Jones and Tjian 1985;
O’Hare and Goding, 1988). The failure of IE gene expression in neuronal cells is therefore likely to be caused by differences in the nature of these cellular transcription factors in such cells, which may in turn reflect differences in the regulation of cellular gene expression mediated by these factors in neuronal cells. Hence, a study of the cellular factors interacting with IE promoters in neurons maythrow light not only on the processes mediating latent infection, but also on cellular gene regulation in neuronal cells. Unfortunately, the amounts of material available from primary cultures of sensory neurons are insufficient for such a study; we have therefore used the Cl300 mouse neuroblastoma cell line (Augusti-Tocco and Sato, 1969) as a model system for studying the interaction of HSV with cells of neuronal origin. Thus, these cells are nonpermissive for lytic infection with HSV (Vahlne and Lycke, 1977, 1978). By using nuclear run-on assays, we have recently shown (Kemp and Latchman, 1989) that this effect is mediated by a failure to transcribe the viral IE genes following infection and that this transcriptional block can be relieved by pretreating the cells with sodium butyrate, which is known to increase their permissivity for HSV infection (Ash, 1986). Here we report that this transcriptional repression is mediated by the octamer motif in the IE promoters. Such repression is also observed when octamer-containing cellular promoters are introduced into Cl300 cells and can be relieved by mutation or deletion of the octamer element, suggesting that this effect is important in cellular as well as viral gene regulation in neuronal cells. Results To investigate the mechanisms responsible for the failure of viral IE gene expression in Cl300 cells, we used aconstruct (IE-CAT) in which the promoter of the major HSV-1 IE gene (IE3, which encodes ICP4) regulates the expression of the gene encoding the chloramphenicol acetyltransferase protein (CAT; Gorman et al., 1982a). In this experiment IE-CATwas expressed approximately 40-fold less well when transfected into Cl300 cells than in permissive BHK cells, as assayed by CAT activity, although the levels of expression driven by the Rous sarcoma virus promoter (Gorman et al., 1982b) were similar in both cell types (Figure 1). In five replicate experiments, the IE-CATconstruct was consistently expressed between 30 and 50 times less well in Cl300 compared with BHK cells. No activity was observed in either cell type with CAT vector that lacked any promoter element. To confirm that the difference in the activity of IECAT in BHK and Cl300 cells was mediated at the level of transcription of the CAT gene, we carried out a nuclear run-on assay (Pate1 et al., 1986) to directly measure transcription in the transfected cells. In these ex-
Neuron 216
2
16 Figure
1. Assay
3
13
4
75
of CAT Activity
Assay was carried out according to the method of Corman (1985). Cells were transfected using the calcium phosphate procedure (Corman, 1985) with either an RSV-CAT construct (lanes 1 and 2) or an HSV IE-CAT construct (lanes 3 and 4). Lanes 1 and 3 show the results obtained in permissive BHK cells; lanes 2 and 4 show results obtained in Cl300 cells. Numbers on the bottom indicate the percentage of available chloramphenicol acetylated in each case.
periments (Table 1) a high level of IE-driven CAT gene transcription was detected in BHK cells compared with Cl300 cells, although the levels of CAT gene transcription in cells transfected with the RSV-CAT construct were similar in both cell types. As expected, endogenous cellular genes such as that encoding histone H2B showed similar levels of transcription in all samples. A high level of activity of the IE promoter (as assayed by CAT activity) was also detectable in a range of nonneuronal human and rodent cell lines including HeLa, Vero, 3T3, and L cells (data not shown). Hence, the failure of HSV to express its IE genes following infection of Cl300 cells is due to the weak activity of the IE promoters in such cells. This weak pro-
Table 1. Nuclear Run-On Assay CAT Gene in Cells Transfected Transfected Cl300
to Measure Transcription of the with Various CAT Constructs
DNA
Cells
BHK
Cells
Gene
RSV-CAT
IE-CAT
RSV-CAT
I E-CAT
CAT H2B 123
40 (4) 48 (5) 180 (15)
5 t-1 41 (3) 175 (12)
41 (6) 46 (7) 150 (14)
160 (15) 45 (5) 170 (11)
Figures (average of two determinations whose range is given in parentheses) indicate the counts per minute binding to the indicated clone in nuclear run-on assays in cells transfected with the indicated DNA. Clone 123 is derived from a cellular gene whose RNA level does not change in HSV infection (Kemp et al., 1986).
moter activity compared with that observed in BHK cells was observed in three separate experiments regardless of the amount of IE-CAT DNA that was transfected (Figure 2). Interestingly, however, as the amount of IE-CAT DNA transfected into Cl300 cells was increased, a significant increase in CAT activity was observed at one point in the concentration series (between 2 gg and 5 PLg of DNA in Figure 2), after which CAT activity remained relatively constant with increasing DNA concentration. Such a pattern of activity suggested that the weak activity of the IE promoters in Cl300 cells might be due to the presence of a trans-acting repressor in neuronal cells that binds to the IE promoter. Thus, if this repressor is present in relatively low amounts, then as the amount of [E-CAT DNA is increased, the repressor will be progressively titrated out, and a sudden increase in CAT activity will occur at the point in the concentration series when all repressor has been bound. If such a repressor does indeed exist, it should be possible to increase IE-CAT activity by cotransfecting with IE-CAT the cloned binding site for the repressor unlinked to any promoter element. This would serve to bind out the repressor and allow increased IE-CAT activity. In three replicate experiments in which various fragments of the IE promoter were cotransfected with IE-CAT, we were able to produce increased CAT activity (Figure 3) using a plasmid (pF) containing a fragment of the IE promoter with a single TAATGARAT (R = purine) DNA sequence element and no other characterized DNA binding motifs. This viral regulatory motif is related at the DNA sequence level to the octamer motif found in a number of cellular gene promoters (for review see Falkner et al., 1986), and in lytic infection, binding of the constitutively expressed cellular transcription factor OTF-1 to this sequence is essential for high level activity of the IE promoters (Baumruker et al., 1988; O’Hare and Coding, 1988). In Cl300 cells, by contrast, it is clear that this motif binds a trans-acting repressor of IE gene activity. Thus, cotransfection of pF with the IE-CAT plasmid resulted in an increase in CAT activity in Cl300 cells (Figure 3), which can only be explained by postulating that pF binds out such a trans-acting repressor, allowing IECAT activity to increase. In contrast, cotransfection of pF into permissive cells resulted only in a small decrease in IE-CAT activity (Figure 3). This is likely to be due to competition between pF and IE-CAT for binding of the OTF-1 factor required for IE gene expression; such competition was previously observed in permissive cell types (Latchman et al., 1989). A similar competition between pF and IE-CAT for the OTF-1 present in Cl300 cells, which would occur following binding out of the repressor, is likely to be responsible for the observation (Figure 3) that the increase in IE-CAT activity obtained by cotransfection of pF into C1300cells is reduced when high levels of pF DNA are used. Hence, the weak activity of IE promoters in c-1300
Octamer-Mediated 217
Repression
a
b
Figure Cl300
1
2
cells is mediated by a trans-acting repressor that binds to the TAATGARAT motif. In turn, this weak activity of the IE promoters causes the observed failure of IE gene transcription following infection of these cells. Interestingly, previous studies (Vahlne et al., 1981; Kemp and Latchman, 1989) have shown that as the amount of virus added to Cl300 cells is progressively increased (from 1 to 50 pfu per cell), a greater than linear increase in the number of cells expressing viral IE proteins and supporting the lytic cycle is observed. A similar effect is also seen in infections of primary sensory neuron cultures (Wilcox and Johnson, 1988). As with the strong concentration dependence of IE-CAT expression, this effect is likely to be mediated by the binding out of the repressor by virally born TAATCARAT elements, allowing high level activity of the excess IE promoters. If this is the case, it should be possible to increase the permissivity of Cl300 cells by first transfecting TAATGARAT-containing plasmids to bind out the repressor and subsequently infecting with low levels of
%
600,
400 -
200 +
‘;
5
102:
Figure 3. Percentage Change in IE-CAT Activity in Cl300 Cells in Which the [E-CAT Plasmid Had Been Cotransfected TAATGARATContaining pF DNA Cl300 cells (circles); BHK cells (squares). Amount containing pF DNA is indicated in micrograms. are the average of three determinations whose by the bars.
or BHK with
of TAATGARAT Values plotted range is shown
of CAT Activity
in BHK or
BHK (a) or Cl300 (b) cells were transfected with the indicated amounts (in micrograms) of the IE-CAT plasmid.
virus. When Cl300 cells were transfected with the TAATGARAT-containing IE-CAT or pF plasmids and infected 24 hr later with HSV-I, a clear increase in permissivity (as assayed by the number of cells staining with an antibody to ICP4) was observed in cells transfected with IE-CAT (Table 2) or pF (Figure 4) compared with those transfected with carrier DNA (PAT 153 vector or salmon sperm DNA) lacking TAATCARAT elements. Three replicate experiments gave similar results. Hence, the binding out of repressor by TAATGARAT elements can relieve the block on IE gene expression in virally infected Cl300 ceils, confirming that this repressor is responsible for the failure of lytic infection in these cells. Most importantly, the repressor activity is specific to neuronal cells; no increase in permissivity was observed when IE-CATor pF was introduced into nonneuronal cells such as 3T3 fibroblasts (Table 2 and Figure 4). There was no increase in permissivity even when low levels of virus were used so that only a relatively small proportion of untransfected cells stained with the antibody, and thus any increase in permissivity would have been readily detected. Indeed, in these cells introduction of IE-CAT or pF results in a decrease in permissivity probably because, as in the cotransfection experiments, they compete for OTF-1, which is required for IE gene transcription (O’Hare and Coding, 1988). A similar phenomenon is presumably responsible for the decreased enhancement in permissivity observed when high levels of IECAT are transfected into Cl300 cells (Table 2). Some enhancement of permissivity is observed in Cl300 cells regardless of the amount of IE-CAT transfected, however, indicating that in these cells the binding of repressor always predominates. Hence, increased permissivity can be produced by binding out of the neuronal repressor by excess TAATCARAT motifs. The relationship of this motif to the octamer element found in a variety of cellular gene promoters and the ability of the TAATGARAT sequence to bind the constitutively expressed octamer binding protein OTF-1 (Baumruker et al., 1988; O’Hare and Coding, 1988) suggested that the repressor we have
-l-=-+-pG
200 J
2. Assay Cells
Table
2. Effect
DNA
Transfected
Carrier
DNA
0 IO 8 6 4
of Transfecting
IE-CAT
fug) IE-CAT 0 0 2 4 6
DNA
on the
Permissivity
HSV-1
Infection
5 pfu
per Cell
Cl300
Cells
ND 13% 30% 36% 27%
of Cells
(271210) (64/210) (75/203) (55/205)
to Viral
Infection
1 pfu
per
3T3 Cells
Cl300
Cells
ND 87% 86% 90 % 76%
4.5% (9/201) 5.3% (12/226) 4.6% (10/216) 11% (22/200) 9.6% (191205)
(921106) (122/141) (49/54) (72/95)
Cell 3T3 Cells 14% (311219) 16.7% (351209) 10% (18/178) 7.4% (14/187) 13.1% (24/183)
Values indicate the total numbers and percentages of cells staining with the 58s monoclonai antibody to the HSV-1 IE protein ICP4 (Showalter et al., 1981). Cells were transfected by the method of Gorman (1985) with the amounts (in ug) of IE-CAT DNA indicated, and the amounts of DNA applied to each plate were equalized with carrier DNA lacking TAATGARAT elements (total salmon sperm DNA or PAT 153); 24 hr after transfection cells were infected with HSV-1 and then stained 16 hr after infection with the 5% antibody. ND, not determined.
identified might be a neuronal cell-specific octamer binding protein. To investigate this possibility we tested whether increased permissivity of Cl300 cells could be produced by transfection of a plasmid (PO) containing a cloned 15 bp oligonucleotide that included the consensus octamer motif (ATGCAAAT). As shown in Table 3, this was indeed the case, confirming that the repressor is an octamer binding protein present in CL300 cells and exerts its effect by binding to the related TAATGARAT elements in the IE promoters. To provide physical evidence for the existence of the repressor protein, we carried out DNA mobility shift assays (Fried and Crothers, 1981) using the identical octamer oligonucleotide employed in the competition experiments. Using extracts from Cl300 cells,
three protein-DNA complexes formed on this probe (Figure 5, lane a). In contrast, only the highest mobility complex formed on an oligonucleotide containing the mutant sequence ATAATAAT, which abolishes recognition by previously characterized octamer binding proteins (Lenardo et al., 1987; Figure 5, lane b). This complex (which also forms on nonoctamer oligonucleotides) thus represents a nonspecific DNA binding protein, whereas the other two bands are due to the binding of sequence-specific octamer binding proteins. When extracts from permissive cells were used (Figure 5, lane c), only the nonspecific complex and the largest complex were observed; the complex of intermediate mobility was absent. It is clear, therefore, that in addition to the ubiquitous octamer binding protein OTF-1 (which is responsible for the largest complex), Cl300 cells also contain an additional octamer binding protein that is absent in permissive cells and is likely to be the repressor factor identified in the competition experiments. The existence of such a factor raises the question of whether it plays any role in the regulation of octamer-containing cellular genes in neuronal cells. To test this possibility we transfected Cl300 cells with a construct in which the octamer-containing immunoglobulin heavy chain enhancer regulates the ex-
Table 3. Effect of Transfecting on the Permissivity of Cl300
Figure 4. Effect of Transfecting the TAATGARATContaining Plasmid pF on the Permissivity of Cl300 Cells or 3T3 Cells to Subsequent Infection with HSV-1 The change in the number of cells staining is plotted against the amount of pF DNA transfected. Values plotted are the average of three determinations whose range is shown by the bars. N indicates Cl300 cells.
DNA
Transfected
Carrier
DNA
(up) PO
an Octamer-Containing Cells to Viral Infection Cl300 1 pfu
Cells Infected per Cell
Plasmid
at
10 0 II/206 (5.4%) 6 4 15/208 (7.2%) 4 6 23/201 (11.4%) Figures indicate the numbers and percentages of cells staining with the monoclonal antibody to the viral IE protein ICP4 and were obtained as described in Table 2.
Octamer-Mediated 219
Repression
a
I a
3
4
Figure 6. Assay of CAT Activity in Cl300 Cells Transfected Various Octamer-Containing Cellular Promoters
Figure
5. DNA
Mobility
Shift
Assay
Lane a, extract from Cl300 cells with labeled wild-type octamer oligonucleotide (ATGCAAAT); lane b, extract from Cl300 cells with labeled mutant octamer oligonucleotide (ATAATAAT); lane c, extract from permissive 373 cells with wild-type octamer oligonucleotide. The arrow indicates the position of the additional sequence-specific octamer binding protein present in the Cl300 cells.
pression of a CAT gene driven by the B-globin promoter (Kadesch et al., 1986). In this experiment (Figure 6, lanes 1 and 2) the low level of activity of this construct was increased by deleting a 48 bp region of the enhancer that contained the octamer element. Similarly, the expression of a construct in which the immunoglobulin enhancer regulates the c-fos promoter was increased (Figure 6, lanes 3 and 4) by mutation of the octamer element from ATGCAAAT to ATAATAAT; this mutation abolishes recognition by the two octamer binding proteins present in C1300cells (see Figure 5) and results in decreased enhancer activity in most cell types (Lenardo et al., 1987). Nuclear run-on assays carried out on the transfected cells (Table 4) indicated that this effect was mediated by increased transcription of the CATgene in cells transfected with the mutant octamer construct. Hence, the repressor is capable of acting on cellular as well as viral promoter elements. Discussion In this report
we have shown
that the octamer
motif
with
Lane I, IV510 construct lacking the octamer element; lane2, IVS6 construct containing the octamer element; lane 3, A56construct with a wild-type octamer element (ATGCAAAT); lane 4, A56 octaconstruct containing a mutated octamer element (ATAATAAT). These constructs are described in Kadesch et al. (1986) and Lenardo et al. (1989).
is capable of mediating the repression of octamercontaining cellular promoters and of HSV IE genes in neuronal cells. This effect is in contrast to that seen in nonneuronal cells where the octamer element acts as a positive regulator of cellular gene promoters containing it, which include those of the small nuclear RNAs, the histone H2B gene, and the immunoglobulin genes (reviewed by Falkner et al., 1986). In each of these promoters, the octamer element is crucially required for its specific pattern of gene activity, mediating, for example, the cell cycle activity of the H2B promoter, the B cell specificity of the immunoglobulin promoters, as well as the constitutive ex-
Table 4. Nuclear Run-On Assay to Measure of the CAT Gene in Cl300 Cells Transfected Octamer-Containing CAT Constructs Transfected Gene
A56 Octa+
.CAT H2B 123
21 (3) 44 (7) 175 (18)
Transcription with
DNA A56 Octa45 (5)
46 (6) 179 (17)
Figures (average of two determinations whose range is given in parentheses) indicate the counts per minute binding to the indicated clone in nuclear run-on assays in cells transfected with the indicated DNA.
NW*Oll 220
pression of the small nuclear RNAs. The differences in the pattern of gene activity mediated by the OCtamer in these promoters is believed to be due to differences in its relationship to other elements in the promoter that affect its recognition by different octamer binding proteins (Tanaka et al., 1988). Thus, in addition to the constitutively expressed octamer binding protein OTF-1, a B cell octamer binding protein, OTF-2, has been described and purified (Scheidereit et al., 1987); more recently several other octamer binding proteins have been identified in different cell types, notably, embryonal carcinoma cells (Lenardo et al., 1989; Scholer et al., 1989), and in the brain (He et al., 1989). The additional octamer binding protein we have detected may therefore be identical to one of these tissue-specific proteins identified by others, or may represent a novel protein expressed specifically in a limited range of neuronal cell types. Of particular interest is the possibility that this factor may be related to one of the additional octamer binding proteins detected by several groups (Lenardo et al., 1989; Scholer et al., 1989) in embryonal carcinoma cells. Thus, these cells, like Cl300 cells, are nonpermissive for HSV (Bell et al., 1987), and both cell types express the gene encoding the small nuclear RNA Ulb, which has an octamer different from the consensus sequence and is expressed in only a limited number of cell types (Lund et al., 1985). This raises the possibility that the repressor activity we have identified might also act as a tissue-specific activator of certain octamer-containing promoters in which the octamer element has a sequence differing from the consensus or is located in a different position relative to other promoter elements. Whatever the case, it is clear that the activity we have described here can repress transcription of certain octamer-containing cellular promoters and TAATGARAT-containing viral promoters. As such, this factor is responsible for the failure of viral IE gene transcription in Cl300 cells (Kemp and Latchman, 1989) and the consequent failure of the viral lytic cycle (Vahlne and Lycke, 1977,1978). The presence of a similar factor in immortalized neurons from rat dorsal root ganglia, the natural site of latent infection with HSV in vivo (S. C. Wheatley, L. M. Kemp, J. N. Wood, and D. S. Latchman, unpublished data), suggests that it may also play a role in the establishment of latent infection. Similarly, modulation of its expression in response to nerve growth factor or stress might be responsible for reactivating the virus in response to these stimuli (Carton and Kilbourne, 1952; Wilcox and Johnson, 1988). It should be noted, however, that the fact that even after titration out of the repressor the activity of IE-CAT in Cl300 cells does not match that in BHK cells (Figures 2 and 3) suggests neuronal cells may also lack positive-acting factors needed for high level IE gene expression. It is clear, however, that the neuronal cell octamer
repressor factor we have identified here plays a major role in the nonpermissivity of neuronal cells. Further study of this factor should throw considerable light on the mechanisms regulating the clinically important problem of latent infection as well as vindicating the use of HSV as a means of probing the processes regulating cellular gene expression in neuronal cells. Experimental
Procedures
Cells and Viruses BHK-21 cells (clone 13; MacPherson and Stoker, 1962) and Cl300 cells (clone 41A3; Augusti-Tocco and Sam, 1969; Ash 1986) were grown in RPM1 medium supplemented with 10% fetal calf serum. Viral infection of transfected cells was carried out 24 hr after transfection (see below! using the indicated multiplicity of HSV-‘I straili F (Ejercito et al., 1968). Sixteen hours after infection cells were stained with the 58s antibody to the viral IE protein ICP4 (Showalter et al., 1981) as previously described (Kemp and Latchman, 1989). Plasmid DNAs The IE-CATconstruct contains the lE3 gene promoter (from -330 to +33) linked to the CAT gene (Stow et al., 19861, while the pF plasmid contains a 63 bp element from the IE4/5 promoter containing a single TAATGARAT element. The p0 plasmid contains a chemically synthesized decanucleotide, containing the consensus octamer sequence with a 5’-GATC overhang at either end cloned into the BamHl site of pUC 1813 (Kay and McPherson, 1987). DNA Transfection Transfection of plasmid DNA was carried out according to the method of Gorman (1985). Except when indicated otherwise, all transfections were carried out using ?O ug of DNA per 2 x IO6 cells on a 90 mm plate. The amounts of DNA in all samples in competition experiments were equalized by use of carrier DNA (PAT 153 or total salmon sperm DNA). Twenty-four hours after transfection, cells were either harvested for nuclear run-on or CAT assays or infected with HSV-1 as described above. CAT Assays Assays of CAT activity were carried out as described by Gorman (1985), extracts having been equalized for protein content as determined by the method of Bradford (1976). Nuclear Run-On Assays Nuclear run-on assays of transcriptional activity in transfected cells were carried out by allowing nuclei prepared from the cells to incorporate radiolabeled [3zP]CTP into nascent transcripts as previously described (Pate1 et al., 1986). The labeled products of such an assay were then used to probe replicate dot blots that had been spotted with 5 pg of plasmid DNAs containing the CAT gene or the control cellular genes histone H2B (Marashi et al., 1984) and clone 123 (derived from a cellular gene whose mRNA level does not change in HSV infection; Kemp et al., 1986). After hybridization the filters were autoradiographed, and labeled spots were cut out and counted by scintillation counting. DNA Mobility Shift Assays Whole cell extracts for use in mobility shift assays were prepared as described by Manley et al. (1980). Extracts were incubated at 30°C for 15 min with labeled octamer oligonucleotide prior to electrophoresis on a nondenaturing acrylamide gel. Acknowledgments We thank John Estridge for excellent technicai assistance and the foliowing for the very kind gifts of plasmid constructs: David Baltimore and Mike Lenardo (A56 and A56 octa-), Tom Kadesch
Octamer-Mediated 221
Repression
(pSVABCCAT IVS6 and IO), Janet Partridge (PO), and Chris Preston (IE-CATand pF). This work was supported by Action Research for the Crippled Child and the Cancer Research Campaign.
Kemp, L. M., Brickell, P M., La Thangue, N. B., and Latchman, D. S. (1986). Transcriptional induction of cellular genes during lytic infection with herpes simplex virus. Biosci. Rep. 6,945-951.
Received
Kennedy, P. G. E., Clements, G. E., and Brown, S. M. (1983). Differential susceptibility of human neural cell types in culture to infection with herpes simplex virus. Brain 706, 101-119.
August
8, 1989;
revised
November
3, 1989.
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