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89,2076-2080.
21. DiIanm, C. L., Drier, D. A., Deckman, I C., McCann III, P J., Liu, F , Roizman,
B., Colonno, R J , and Cordingly, M. G. (1993) Identification of the herpes sim-
plex vuus-1 protease cleavage sues by direct sequence analysis of autoproeolytic
cleavage products J Blol Chem 268,2048-205 1
22. DiIanm, C. L , Mapelli, C., Drier, D. A , Tsao, J , NatarJan, S , Riexmger, D ,
Festm, S. M., Bolgar, M., Yamanaka, G., Weinheimer, S P., Meyers, C A ,
Colonno, R. J., and Cordmgley, M G. (1993) In uztro activity of the herpes simplex
virus type 1 protease with peptide substrates, J. Blol. Chem 268,25,449-25,454.
23 McGeoch, D. J and Davison, A J. (1986) Alpha herpesviruses possess a gene
homologous to the protein kmase family of eukaryotes and retrovnuses Nucleic
Aczds Res 14, 1765-1777
24. Frame, M. C., Purves, F. C., McGeoch, D. J , Marsden, H. S , and Leader, D P.
(1987) Identification of the herpes simplex vu-us protein kmase as the product of
the viral gene Us3 J Gen Vwol. 68,2699-2704.
25. Purves, F. C , Longnecker, R. M., Leader, D. P., and Roizman, B (1987) The
herpes simplex virus 1 protein kinase is encoded by open readmg frame 3 which is
not essential for virus growth m cell culture J. Vwol 61,2896-2901
26. Purves, F. C., Donella Deana, A., Marchrori, F., Leader, D P., and Pinna, L.
A. (1986) The substrate specificity of the protein kinase induced m cells
Infected with herpesviruses. studies with synthetic substrates indicate struc-
tural requirements distmct from other protein kinases. Blochem Bzophys
Acta 889, 208-2 15.
27. Katan, M., Stevely, W. S., and Leader, D. P. (1985) Partial purification and char-
acterization of a new phosphoprotem kmase from cells infected with pseudora-
bies virus. Eur J. Biochem. 152,57-65.
Cunningham, C., Davison, A. J., Dolan, A., Frame, M. C., McGeoch, D. J.,
28.
Meredith, D. M., Moss, H. W. M., and Orr, A. C. (1992) The UL 13 vu-ion protein
of herpes simplex virus type 1 is phosphorylated by a novel vu-us-induced protein
kmase. J Gen. Vwol 73, 303-3 11.
29. Purves, F. C. and Roizman, B. (1992) The UL 13 gene of herpes simplex vu-us 1
encodes the functions for posttranslational processing associated with phosphory-
latton of the regulatory protein ˜22. Proc Nat1 Acad. Scz. USA 89, 73 10-73 14.
30. Purves, F. C., Ogle, W. O., and Roizman, B. (1993) Processing of the herpes
simplex vu-us regulatory protein a22 mediated by the UL 13 protein kmase deter-
mmes the accumulatton of a subset of a and y mRNAs and protems m Infected cells.
3 1. Coulter, L. J., Moss, H. W., Lang, J., and McGeoch, D. J. (1993) A mutant of
256 Blaho and Roizman

herpes simplex virus 1 in which the U,13 protein kinase gene IS dtsrupted. J Gen
Vwol 74,387-395
32. Overton, H. A , McMillan, D J., Klavmskis, L S , Hope, L., Ritchte, A. J , and
Wong-Kai-In, P. (1992) Herpes simplex virus type 1 gene UL13 encodes a phos-
phoprotein that IS a component of the vtrton Virology 190, 184-l 92
33 LeMaster, S. and Roizman, B. (1980) Herpes stmplex vu-us phosphoprotems II
Charactertzatron of the vtrton protein kmase and of the polypepttdes phosphory-
lated in the vtrton J Vu-01 35, 798-811
34 Hohhman, P J. and Cheng, Y -C (1979) DNase induced after mfectton of KB
cells by herpes stmplex virus type 1 or type 2 II. Characterization of an assoct-
ated endonuclease acttvtty J. Vwol 32,449-457
35 Crute, J. J , Tsuruml, T , Zhu, L , Weller, S. K , Ohvo, P. D , Challberg, M D ,
Mocarskt, E. S , and Lehman, I R (1989) Herpes simplex virus 1 heltcase-
prtmase* a complex of three herpes-encoded gene products Proc Nat1 Acad Scl
USA 86,2186-2189
36. Boehmer, P. E , Dodson, M S , and Lehman, I R. (1993) The herpes simplex type
1 ortgm bmdmg protein DNA hehcase acttvtty. J Bzol Chem 268, 1220-1225
37 Dodson, M S. and Lehman, I. R (1993) The herpes simplex virus type 1 ortgm
bmdmg protein DNA-dependent nucleostde trtphosphatase activity J Bzol
Chem. 268,1213-1219
38. Crute, J. J. and Lehman, I R. (1991) Herpes simplex virus-1 heltcase-prtmase
physical and catalytic properties J Blol Chem 266,4484-4488
39 Bruce, C B and Pearson, C K (1983) A helix-destabilizing protem form herpes
simplex virus type 1 infected cells which spectfically stimulates the vtrus induced
DNA polymerase activity in vitro. Biochem Bzophys Res. Comm. 116,327-334
40 Caradonna, S. J and Cheng, Y (198 1) Induction of uractl-DNA glycosylase and
dUTP nucleotidohydrolase acttvtty in herpes simplex virus-infected human cells.
J Blol. Chem 265,9834-9837
41. Mullaney, J., McL Moss, H W., and McGeoch, D. J. (1989) Gene UL2 of herpes
simplex virus type 1 encodes a uractl-DNA glycosylase J Gen Vu-01 70,449-454.
42. Wohlrab, F. and Francke, B. (1980) Deoxyrtbopyrimidme triphosphatase activity
specific for cells infected with herpes simplex virus type 1 Proc Natl Acad Scz
USA 77,1872-l 876
43 Frame, M C., Marsden, and Dutta, B M (1985) The ribonucleotide reductase
induced by herpes simplex vtrus type 1 mvolves mmtmally a complex of two
polypeptides (136K and 38K) J Gen Vzrol 66, 1581-1587
44. Bacchettt, S., Evelegh, M. J., and Mmrhead, B. (1986) Identtfication and separa-
tion of the two subunits of the herpes simplex vn-us rtbonuclease reductase J.
VzroZ 57, 1177-l 18 1
45. Langelier, Y. and Buttm, G (198 1) Charactertzatton of rtbonucleottde reductase
mductton m BHK-2 l/C 13 Syrian hamster cell line upon infection by herpes stm-
plex virus (HSV). J Gen Vwol 57,21-3 1.
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human adenovtrus 5 DNA Virology 52,456-467.
HSV-Cellular Protein Interactions
David S. Latchman


1. Introduction
The herpes simplex virus (HSV) lytic cycle is dependent on a precise
temporal pattern of viral gene expression with the initial expression of
the Immediate-early (IE) genes, followed by the early genes, and finally
late gene expression (I). Although such a temporal cascade of viral gene
expression mvolves the action of virally encoded regulatory protems,
such factors act, at least in part, by interacting with cellular transcrip-
tion factors that are present m the uninfected cell. Thus, although the HSV
vlrion protein Vmw65 1sessential for transactivation of the viral IE genes
in lytic infection by binding to the TAATGARAT sequences in the pro-
moters (2), it can only achieve this by forming a complex with the cellular
transcription factor Ott-1 (3,#) and other cellular factors (5). Similarly,
the IE promoters contam binding sites for other cellular transcription fac-
tors such as Spl (6) and this 1s also observed m the promoters for viral
genes of other kinetic classes, such as the early gene encoding thymidine
kinase (7).
In addition to their role in the viral lytic cycle, cellular factors binding
to viral promoters also are likely to play a critical role In producing
asymptomatic latent infections of neuronal cells with HSV. Thus, viral IE
gene expression is undetectable during latent infections (8,9), and these
infections can be established by viral mutants unable to express one or
more of the viral IE genes (20,Zl). Hence, latent infection 1s likely to
involve a failure of viral IE gene expression leading to an abortion of the
lytic cycle at an early stage. Although it was originally thought that the
absence of IE gene expression could arise from the failure of Vmw65 to
reach gangliomc neurons (22) this 1snow known not to be the case, since
From Methods m Molecular Medune, Vol 10 Herpes Simplex Vws Protocols
E&ted by S M Brown and A R MacLean Humana Press Inc , Totowa, NJ

257
Latchman
258
latency can be established readily m transgemc mice expressing Vmw65
in every cell (13). Hence the failure of IE gene expression in neuronal
cells must arise from an absence of a positively actmg cellular transcrip-
tion factor required for IE gene expression or from the spectfic expression
of a negatively acting cellular factor that inhibits IE gene expression.
Hence an understanding of the processes whereby HSV promoters are regu-
lated by cellular transcriptron factors 1sessential for our understanding of lytic
and latent mfections. In this chapter, I describe techniques that allow the iden-
ttfication of the regulatory elements in viral promoters that produce a particu-
lar pattern of expression. Subsequently, I describe the manner in which the
cellular transcription factors binding to such sites can be identified. By usmg
these methods, we have been able to identify a neuronally expressed cellular
transcription factor, Ott-2, which is responsible for the mhtbition of HSV IE
gene expression m these cells (Z4).
2. Assays
2.1. Promoter Assays
1. HEPES-buffered saline (HBS) 10X stock, 8.18% NaCl (w/v), 5 94%
HEPES (w/v), 0.2% Na*HPO, (w/v). Prior to transfection make a 2X HBS
solution and adJust to pH 7 12 with 1M NaOH Falter sterilize. The pH IS
absolutely critical
2. Phosphate-buffered salme (PBS)* 8 g NaCl, 2 g KCl, 1.5 g Na2HP04, 2 g
KH,P04/L
3 Dye reagent 100 mg Coomassie brtlhant blue G, 30 mg SDS, 50 mL 95% (v/v)
ethanol, 100 mL 85% (v/v) phosphortc acid/L.

2.2. DNA-Binding Assay
1. Buffer A* 10 mMHEPES, pH 7.9, 1.5 mMMgC1, 10 nnl4 KCl, 0.5 mMdtthto-
threitol (DTT)
2. Buffer C. 20 &HEPES, pH 7.9,25% glycerol, 1.5 mMMgCl,, 0.25 n1A4ethyl-
enedtamme tetra-acetic acid (EDTA)
3. STE. 10 mMTrrs-HCl, pH 7 6,1 mMEDTA, 100 mMNaC1.
4 TBE 10 mMTrts-HCI, 10 n&I boric acid, 2 UEDTA, pH 8 3.
5 Buffer F* 50 mM NaCl, 20 mM HEPES pH 7.9, 5 mA4 MgCI,, 0 1 mM EDTA,
20% glycerol, 1 mA4 CaCl,, 1 n-nI4 DTT.
6. Sample loading buffer: 950 pL formamtde, 25 uL 1% bromophenol blue, 25 uL
1% xylene cyanol.
7. Renaturationbuffer 1OmMHEPES 7 9,1 tnMDTT, lOOmMKCl,O.l%NP40
pH
8 Blockmg buffer: 10 mM HEPES pH 7 9, 1 m&I DTT, 5% nonfat dried milk
9 Hybridization buffer: 10 rmI4 HEPES pH 7 9, 50 mM NaCl, 0.1 mA4 EDTA, 1
mM DTT, 0 25% nonfat drted milk
10 Washing buffer 10 mMTris-HCl, pH 7.5, 50 mMNaC1.
259
US V-Cellular Pro tern Interactions
3. Methods
3.1. Promoter Assays (see Section 4.1.)
3.1,1. Transfection
In order to test the features of an HSV promoter that result m rt having cell-
type specific activity or that allow it to be actrvated by a particular Inducer or in
response to viral infection, it must be linked to a marker gene encoding a readily
assayable product, such as chloramphenicol acetyl transferase (15) or P-galac-
tosrdase (Z6). A number of plasmrd vectors containing the coding regions of
these genes are now avatlable and contain multrple cloning sites upstream of
the coding region to facilitate insertion of a heterologous promoter (2 7). Once
this has been done, the hybrid construct is introduced by transfection mto dlf-
ferent cell types or into the same cell type treated m different ways, for example,
with or without superinfectron with HSV and any effect of the regulatory
sequences on production of the assayable product 1s assessed.
In order to test promoter activity, tt 1s necessary to mtroduce the construct
containing it into cultured cells. A number of techniques exist for doing this,
including treatment with calcium phosphate (15). DEAE dextran (28), and
electroporation (19). We have found the calcium phosphate procedure to be
effective for many cell types and it IS therefore presented here.
1. On the day before transfection (d l), replate the cells to be used at a density of
104/cm2
2 On d 2, replace the culture medium with 5 mL of fresh medium containing 10%
fetal calf serum. DNA is added to the cells 2 h later.
3 To prepare the calcium phosphate-DNA precipitate for a go-mm dish containing
5 mL of medium, set up the following solutions. In tube A, place a solution con-
taming 5-20 ng of DNA together with 3 1 mL 2M CaCl, and bring the final vol-
ume to 0.25 mL with water. To tube B, add 0.25 mL of 2X HBS.
4. To make the precipitate, the contents of tube A must be added to the HBS in tube
B. The order of addition 1scrucial. Add the DNA solution dropwise to the HBS.
The precipitate will form immediately.
5. Pipet the precipitate onto the cells by slightly tiltmg the dish and adding the pre-
cipitate to the medium. Put the cells back into the incubator immediately to ensure
that the pH does not change.
6. Incubate the cells for 4-12 h. The longer incubation IS sometimes required for
promoters that are expressed weakly
7. Wash the cells m serum-free medium and then feed them with complete medium.
8. Harvest the cells on d 4. A test of transient expression can be carried out at this stage
3.1.2. Assay of Promoter Activity
Once the transfectlon protocol has been carried out, the cells can be har-
vested and promoter activity determined by assaying the activity of the enzyme
Latchman
260

encoded by the test gene. The activity of the enzyme followmg transfectlon of
different cell types or in differently treated cells provides a measure of the
relative promoter activity under these conditions. In experiments of this sort,
however, it 1s necessary to control for differences m the efficiency of DNA
uptake between different cell types or under different conditions. This can be
achieved by transfecting with constructs containing another promoter whose
activity 1sunchanged m the different cell types. The constructs containing this
promoter are transfected in parallel with those contaming the regulated pro-
moter and the activity in the different samples compared. However, it 1sprefer-
able to transfect each cell sample with both the regulated and control promoter
constructs. Hence, the actlvlty of each promoter can be assessedm the same
sample, controllmg for variations in transfectlon efficiency between different
plates of cells. To do thrs, the control and regulated promoters must drive the
expression of different assayable proteins. We therefore give protocols for
assaying the activity of chloramphemcol acetyl transferase and P-galactosl-
dase in the same extract. All assaysare carried out on samples that have been
equalized for their content of total protein as described. The choice of which
enzyme should be expressed from the control promoter and which from the
regulated one is entirely arbitrary and ˜111depend on the availability of control
promoter constructs, vectors, and so on.
3 1.2.1. CHLORAMPHENICOL ACETYL TRANSFERASE ASSAY
This assay rehes on allowing the enzyme to acetylate [˜4C]-chloramphenl-
co1and assaying the level of acetylated chloramphenicol by thin-layer chroma-
tography (TLC).
1 Following transfection, wash the cells with PBS,harvest and transfer them to a
1 5-mL microcentrifuge tube
2. Add 100 pL of 0 25MTns-HCl, pH 7.5 to the cell pellet
3. Disrupt the cells by freezing and thawing. To freeze-thaw, immersethe tubes m
liquid nitrogen for 2 mm, andthen transfer them to a 37°C water bath Repeatthe
cycle three times.
4. Spin down the cell debris and save the supernatantto test for enzymeactivity
Samplesmay be savedat this point by storageat -20°C.
5. Depending on the cell type and promoter to be assayed, amount of extract
the
assayed vary.
may
The reaction mixture contains:
a. 70 yL 0.25MTris-HCI, pH 7.5.
b 35 yL Water
c, 20 PL Cell extract
d 1 yL [˜4C]-chloramphenlco1 (40-50 Cl/mmol) (Amersham, Arlington
Heights, IL)
e. 20 pL 4 rnM acetyl CoA.
HS V-Cellular Protein Interactions 261

6. Incubate the reactton mtxture for 30 min at 37°C The mcubatton time can be
increased up to 60 mm, provided enough active acetyl-CoA is added to keep the
assay linear.
7. Extract the chloramphemcol with 1 mL ethyl acetate, by vortexmg for 30 s.
8 Spm for 2 mm in a mtcrocentrifuge tube and save the top organic layer which
will contain all forms of chloramphenicol.
9. Dry down the ethyl acetate under vacuum. This will take approx 2 h
10. Resuspend the chloramphenicol samples in 15 pL ethyl acetate and spot them on-
to silica gel TLC plates.
11. These plates are subjected to ascending chromatography with a 95.5 mixture of
chlorofonmmethanol.
12 After air-drying, expose the chromatography plate to X-ray film After exposure,
the regions correspondmg to acetylated and nonacetylated chloramphemcol can
be cut out and counted.
13. The percentage of total chloramphenicol converted to the monoacetate form gives
an estimate of the transcriptional activity.

3.1.2.2. BETA-GALACTOSIOASE ASSAY (16)

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