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HSV-Cellular Protein Interactions
David S. Latchman
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
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
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).
2.1. 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
2. Phosphate-buffered salme (PBS)* 8 g NaCl, 2 g KCl, 1.5 g Na2HP04, 2 g
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-
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
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.
US V-Cellular Pro tern Interactions
3.1. Promoter Assays (see Section 4.1.)
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
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
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 [â€˜4C]-chloramphenl-
co1and assaying the level of acetylated chloramphenicol by thin-layer chroma-
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 37Â°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 -20Â°C.
5. Depending on the cell type and promoter to be assayed, amount of extract
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 [â€˜4C]-chloramphenlco1 (40-50 Cl/mmol) (Amersham, Arlington
e. 20 pL 4 rnM acetyl CoA.
HS V-Cellular Protein Interactions 261
6. Incubate the reactton mtxture for 30 min at 37Â°C The mcubatton time can be
increased up to 60 mm, provided enough active acetyl-CoA is added to keep the
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
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.
18.104.22.168. BETA-GALACTOSIOASE ASSAY (16)