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m
I

A------ A----- d-----
F G -77
--T-T
fil ril

MIX with protem extract
m
1

AL@ EL ch
G G
P G G P
Protem bindz?
Protetn bmds


Gel electrophoresls
1




zi:
and
cleave
with
ptpendme
at
methylated
residues




- -
Absence of band
mdicates the




Fig. 3. Methylatron interference assay jn which the guanine resrdwes that are crm-
cal for binding of a protein to the DNA can be identified by the fact that then methy-
lation prevents protein bmdmg and hence the formatron of a retarded complex in a
DNA mobility shift assay

polyacrylamtde gel prepared as for the DNA mobrhty shift assay
7 After running, cover the gel with chngfilm and expose tt to X-ray film for approx 4 h
Latchman
268
8 Excise the retarded and unretarded bands and extract the DNA with 1M LiCl
Add 10 ug glycogen, phenol extract, and then ethanol precipitate the DNA
9. Redissolve the DNA m 10 uL 1Mpiperidme (freshly diluted) and heat it to 90°C
for 30 s
10 Cool the sample on ice, pulse-spur it m a microfuge, and freeze-dry the sample
11 Redissolve the DNA in 50 uL of water and freeze-dry again
12 Resuspend the DNA m 5 uL sample loadmg buffer, heat at 90°C for 3 mm and
freeze it until ready to load onto a denaturing polyacrylamide gel.
3.2.5. Southwestern Blotting
of DNA mobiltty shift, DNase I footprmtmg, and methyla-
The techniques
tion interference discussed m previous sections can provide considerable
information on the nature of the interaction between a particular DNA sequence
and transcription factors. They do not however, provide information on the pro-
tein itself and its characteristics. Ultimately, such mformatton can be obtained by
clonmg the gene encoding the transcriptton factor using one of a number of
different methods that are beyond the scope of this chapter (for a drscussron,
see refs. 32 and 33) Prior to this, however, it 1sposstble to determine the size
of the protein by using the technique of Southwestern blotting (34). In this
method, protein extracts are electrophoresed on a standard SDS-polyacryla-
mide gel and the separated proteins transferred to nitrocellulose membrane and
probed with a radioactively labeled oligonucleotide containing the DNA-
binding site of interest. A protein capable of binding to this specrfic site will do
so, producing a radioactive band, and its size can be determined by comparison
to marker protems of known size.
1 Electrophorese approx 50 pg of protein extract on a standard SDS-polyacryla-
mide gel and transfer onto a mtrocellulose membrane at 20 mA overnight in 25
mM Tris-HCI, 200 mM glycme, 20% methanol.
2 Denature the protein bound to the filter m 6A4 guamdme-hydrochloride for 30 mm
3, Renature the protein overnight in renaturation buffer
4 Incubate the filter m blocking buffer for 60 mm at room temperature with
gentle agitation.
5 Incubate the blot for at least 2 h (or overnight) m hybridization buffer with about
3.5 x 10™ cpm/mL of [32P]-labeled concatamerized ohgonucleotide probe.
6. Wash the blot in washing buffer, wrap it m clmgfilm, and autoradiograph.
4. Notes
4.1. Promoter Assays
1 The amount of DNA added to each transfection should be the same This can be
achieved by adding appropriate amounts of salmon sperm DNA
2 Great care is needed in making up the HBS buffer since the pH is very critical for
these experiments.
269
HS V-Cellular Protein Interactions

3. If the precipitate looks dense and opaque, rather than translucent, the HBS has
not been prepared at the correct pH
4.1.1. Chloramphenicol Acetyl Transferase Assay
4. Acetyl-CoA is very unstable and should be made up fresh or kept at -20°C for
not more than 10 d
5. The TLC tank should be lined with filter paper around the inside to assist equilib-
rium. The solvent should be made up fresh every day, smce chloroform is very volatile
4.1.2. P-Galactosidase Assay
6 It is important to remember that mammahan cells contain a eukaryotic isozyme
for /3-galactostdase. Therefore a blank containing a nontransfected cellular lysate
should be included m the experiment
4.2. Identification of Cellular Transcription Factors
4.2.1. Preparation of Whole-Ceil Extracts
7. To prepare extracts from tissues, grmd the tissue m liquid nitrogen to a fine pow-
der before resuspendmg in buffer C
8. For tissue extracts it may be necessary to use a tissue macerator instead of a
Dounce homogenizer to get efficient disruption of cells.
4.2.2, DNA Mobility Shift Assay
9 For fragment probes, the phosphates at the end of the purified fragment must be
removed wtth calf Intestinal phosphatase prior to kinase labelmg
10. For preparing concatamenzed oligonucleotide probes for use m Southwestern blottmg
kmase, treat the annealed oligonucleotide m 50 mMTris-HCI, pH 7.6, 10 mMMgCl,,
5 mMDTT, and then add DNA ligase and ATP to 5 mMand allow to ligate overnight
at room temperature. The kmased, ligated concatamers are then separated from
free nucleotides on a Sephadex G50 column by collectmg the first peak.
11. Prior to use, the protein concentration of different extracts is equalized based on
assays of then protein content
12. For competitor assay, competitor oligonucleotides are added to the mixture at
onefold, lo-fold, and loo-fold molar excess before addition of the extract,
13. For antibody assay, 1 uL of each of a series of dilutions of the antiserum under
test is added to the binding reaction before addition of the extract Similar dilu-
tions of preinnnune serum are added to parallel reactions as a control.
14. The gel is pre-electrophoresed before addmon of the samples until the current
drops from 3@-10 mA (approx 2 h).
15. The gel 1s run until bromophenol blue m a separate marker track has run approx
two-thirds of the way down the gel.
4.2.3. DNase I Footprinting Assay
16. In initial experiments it will be necessary to titrate the amount of extract used, the
amount of poly dIdC and the magnesium concentration m order to obtain the
appropriate level of digestion with DNase 1.
Latchman
270
17 In contrast to the DNA mob&y shift assay where the probe can be labeled at both
ends, the probe must be labeled at one end only. The probe IS therefore prepared m
the same way as before and the labeled fragment is then digested with a second restnc-
tton enzyme and gel purified to isolate a fragment wtth only one labeled end Use
approx 5 fmol of probe per reaction labeled to 50-100 counts per second.
18. The gel is prerun for approx 30 min prior to loading and then run at 1600 V/30 mA

References
1 Honess, R. W and Roizman, B (1974) Regulation of herpesvirus macromolecu-
lar synthesis. I Cascade regulation of the synthesis of three groups of viral pro-
tems J. Vu-01 14, 8-19.
2. Campbell, M. E M , Palfreyman, J W , and Preston, C. M. (1984) Identtfication
of herpes simplex vnus DNA sequences which encode a trans-acting polypeptide
responsible for stimulatton of immedtate-early transcription J A402 Bzol 180, l-l 9
3 O™Hare, P and Godmg, C R. (1988) Herpes simplex vu-us regulatory elements
and the nnmunoglobulm octamer domain bmd a common factor and are both tar-
gets for vtrion transactivation Cell 52,435-445
4 Preston, C M , Frame, M C , and Campbell, M E M (1988) A complex formed
between cell components and a herpes simplex virus structural polypepttde bmds
to a viral immediate-early gene regulatory DNA sequence Cell 52,425434
5 Werstuck, G H. and Capone, J P (1993) An unusual cellular factor potentiates
protein-DNA complex assembly between Ott- 1 and Vmw65 J Bzol Chem 268,
1272-1278
Jones, K A and TJian, R (1985) Spl binds to promoter sequencesand activates herpes
simplex vnus immediate-early gene transcriptton zn vztro Nature 317, 179-l 82
Jones, K A., Yamamoto, K. R , and TJian, R. (1985) Two distmct transcription
factors bmd to the HSV thymidme kinase promoter zn vztro Cell 42,.559-572
Croen, K D , Ostrove, J M., Dragovic, L. J , Smtalek, J E., and Straus, S E.
(1987) Latent herpes simplex vu-us m human trtgeminal gangha Detectton of an
immediate-early gene anti-sense transcript by in situ hybridtzatton. New Engl J
Med 317, 1427-1432
9 Stevens, J G., Wagner, E K., Devi-Rao, G. B , Cook, M L , and Feldman, L T.
(1987) RNA complementary to a herpes virus alpha gene mRNA is promment m
latently Infected neurons Sczence 235, 1056-1059
10 Valyt-Nagt, T , Deshmane, S. L., Sptvack, J. G , Steiner, I , Ace, C I , Preston, C.
M , and Fraser, N. W (1991) Investigation of herpes simplex virus type 1 (HSV-
1) gene expression and DNA synthesis during the establishment of latent mfec-
tton by an HSV-1 mutant m 1814 that does not replicate m mouse trigemmal
ganglia. J Gen Vzrol 72,641-649
11 Katz, J , Bodin, T., and Coen, D M (1990) Quantitative polymerase cham reac-
tion analysis of herpes simplex vu-us DNA m gangha of mice infected with repli-
cations incompetent mutants. J Vzrol 64,4288-4295.
12 Roizman, B. and Sears, A E (1987) An mqmry mto the mechanisms of herpes
simplex vuus latency Ann Rev Mzcrobzol 41, 543-571
271
HSV-Cellular Protein Interactions
13. Sears, A E., Hukkanen, V., Labow, M. A., Levine, A. J., and Roizman, B. ( 199 1)
Expression of the herpes simplex virus ICC. transinducing factor (VP16) does not
induce reactivation of latent virus or prevent the establishment of latency m mice
J Vwol 65,2929-2935
14. Lillycrop, IS. A , Howard, M. K , Estridge, J K , and Latchman, D S. (1994)
Inhibition of herpes simplex virus infection by ectopic expression of neuronal
splice variants of the Ott-2 transcrtption factor. Nucleic Aczds Res 22, 8 15-820.
15. Gorman, C M. (1985) High efficiency gene transfer mto mammalian cells, m
DNA Cloning* A Practzcal Approach, vol 2 (Glover, D M , ed ), IRL, Oxford,
UK, pp 143-l 90.
16. Herbomel, P , Bourachit, B., and Yaniv, M (1984) Two distmct enhancers with
different cell specificities co-exist in the regulatory region of polyoma Cell 39,
653-662
17. Frebourg, T and Brison, 0. (1988) Plasmid vectors with multiple clonmg sites
and CAT-reporter gene for promoter clonmg and analysis in animals Gene 65,
315-318.
18. Queen, C and Baltimore, D. (1983) Immunoglobulm gene transcrrption is acti-
vated by downstream sequence elements Cell 33,741-748
19. Potter, H., Weir, L , and Leder, P. (1984) Enhancer dependent expression of
human K immunoglobulm genes introduced mto mouse pre-B lymphocytes by
electroporation Proc Nat1 Acad 5™cz USA 81, 7161-7165
20 Bradford, M. (1976) A rapid and sensitive method for the quantitation of micro-
gram quantities of proteins utilizing the prmciple of protem-dye bmdmg Anal
Blochem 72,248-254
21 Sheng, M., Dougan, S T , McFadden, G , and Greenberg, M E (1988) Calcium
and growth factor pathways of c-fos transcriptional activation require distinct
upstream sequences. Mol Cell. Blol 8,2787-2796
22 Luckow, B and Schutz, G (1987) CAT constructions with multiple unique
restriction sites for the functional analysts of eukaryotic promoters and regulatory
elements Nucleic Acids Res 15, 5490.
23. Latchman, D. S. (1990) Eukaryotic Transcription Factors. Biochem J 270,28 l-289
24 Dignam, J. D., Lebovitz, R. M., and Roeder, R. G (1983) Accurate transcription
initiation by RNA polymerase II m a soluble extract from rsolated mammalian
nuclei. Nuclezc Aczds Res 11, 1475-1489.
25. Manley, J L., Fire, A, Cano, A, Sharp, P A., and Gefter, M L (1980) DNA-
dependent transcriptron of adenovirus genes m a soluble whole-cell extract. Proc
Nat1 Acad Scl USA 77,3855-3859
26. Fried, M. and Crothers, D M. (198 1) Equihbria and kmetics of Lac repressor-
operator interactions by polyacrylamide gel electrophoresis Nuclezc Acids Res 9,
6505-6525.
27 Garner, M. M and Revzm, A (198 1) A gel electrophoresis method for quantify-
ing the binding of proteins to specific DNA regions application to components of
the Eschertchza coli lactose operon regulatory system. Nucleic Acids Res 9,
3047-3060
272 Latchman

28. Galas, D and Schmitz, A. (1978) DNase footprmtmg: A simple method for the
detection of protein-DNA bmdmg specificity Nucleic Acids Res 5,3 157-3 170
29 Dynan, W. S and TJian, R. (1983) Isolation of transcription factors that discrimi-
nate between different promoters recognized by RNA polymerase II Cell 32,
669680.
30 Siebenhst, U. and Gilbert, W. (1980) Contacts between the RNA polymerase and
an early promoter of phage T7 Proc. Nat1 Acad Scl USA 77, 122-126.
3 1 Maxam, A. M and Gilbert, W. (1980) Sequencing end labelled DNA with base-
specific chemical cleavages. Methods Enzymol 65,499-560.
32 Latchman, D S (1995) Eukaryotrc Transcrzptzon Factors, 2nd ed , Academic,
London, UK
33 Latchman, D. S (ed ) (1993) Transcription Factors A Practical Approach IRL,
Oxford University Press, Oxford, UK
34. Sambrook, J., Fritsch, E. F., and Mamatis, T. (1989) Molecular Clonzng. A Labo-
ratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
19
Models of Recurrent Infection
with HSV in the Skin and Eye of the Mouse
Terry J. Hill and Carolyn Shimeld


1. Introduction
Animal models remain essential for studies of many aspects of the biology
of herpes simplex vnus (HSV). Such studies include basic experiments on
pathogenesis (including characterization of viral mutants), tests of antiviral
drugs, and methods of mmrumzation. With reference to models of recurrent
infection, high levels of recurrence and climcal disease have been achieved
with guinea pigs (parttcularly with genital infection) and rabbits (particularly
with ocular infection; reviewed m ref. 1). However, tn contrast to these ani-
mals, with the laboratory mouse there are many Inbred and congemc lines; a
major advantage for tmmunological studies. To thts can now be added the
growmg technology of transgenic and “knockout” animals. For these reasons
we have expended consrderable effort in developing various mouse models of
mfection, particularly with HSV type 1 (HSV-1).
This chapter focuses primarily on murme models of experimentally induced
recurrent infection m the skm or eye. The basrc pattern of these models is srmi-
lar and consist of two stages: first, the production of latently infected animals
by means of a primary infection, usually at a peripheral site; and second, m

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( 61 .)



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