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10. Agarose ultra-pure electrophorests grade (54˜55 1OUB BRL Ltfe Technologies)
11 DNA electrophorests mol-wt markers
12 Ethidtum bromtde made up in water at 10 mg/mL stock.
13 M-MLV Reverse Transcrtptase (cat. no. 28025-013 Gibco-BRL)
14 Random Hexamers (Pharmacta Btotech cat no. 27-2166) 100 pmol/uL
15. Dtethyl pyrocarbonate-treated (DEPC) H,O. Add DEPC 0 1% to UHP water, and
stand at room temperature for 4 h Then autoclave.
16. RNasm ribonuclease inhibttor (Promega, cat. no N2511)

3. Methods
Many cell types can potenttally be used for the production of complemen-
tary cell lines. The parental lme must be permissive for virus growth and pref-
erably give high titers The cell lme must also be suscepttble to one or the other
of the transfection techniques available. Vero (African Green monkey ktdney
ECACC no. 84113001), BHK21 (Syrian hamster ktdney ECACC no. 85011433),
or MRCS (Human Fetal Lung Frbroblast ECACC. no. 84101801) cells are
appropriate parental lmes for many herpes viruses and will transfect with
high effictency. The amount of selective antibiotic or drug required to kill
untransfected cells should be determined prior to transfection, for example,
G418 should be titrated onto growing cells at concentrations rangmg from
200 pg/mL to 2 mg/mL. A suttable selection concentratton will give com-
plete destruction of the monolayer after IO-14 d. Cells are media changed
every 3 or 4 d.
3.7. Cat& Transfection of Vero Cells
1 Asptrate the growth media from a semiconfluent 80-cm2 flask of vero cells, wash
twice with 5 mL of PBS, and add 2 mL of trypsin. Incubate the flask at 37°C for
2-5 mm until the cells begm to lift off
2. Neutralize the trypsm by adding 5 mL of growth medta, and gently resuspend the
cells. Count the cells and adJust the concentration to give 1 x 1O5cells/ml.
3. Seed 60-mm Petri dishes (tissue-culture grade) at 5 x IO5 cells/plate
4 Incubate at 37°C 5% CO, for 24 h.
5 Aliquot 30 pg of plasmtd DNA mto an Eppendorf. This is suffictent DNA to
transfect three 60-mm plates Precipitate the DNA by adding 2 5 vol of ethanol
and ˜/to vol 3M sodmm acetate. Store at -80°C for 5 mm (or until frozen solid)
Spin Eppendorfs at 12K for 10 mm
6. Transfer the Eppendorf to a tissue-culture cabmet, and aspirate the ethanol. Air-
dry the DNA pellet Resuspend the DNA carefully m 60 pL of TE buffer. Trans-
fer to a universal contaming a further 675 uL of TE and 75 pL of CaCl,
7 Add dropwtse 750 uL of 2X HBS A fine precipitate should form Allow to stand
for 10 mm at room temperature.
8 Aspirate the medmm from the cells, and replace with 500 uL of the DNA precipt-
tate Incubate at 37°C 5% CO2 m a humtdtfied incubator for 4 h.
Cell Lines Expressing HSV Genes 93
9 Add 3 mL of growth medium (GM) to eachplate, andaspirateoff. Add 500 pL of
glycerol shocksolution, andallow to standat room temperaturefor 2 min Add 3
mL of GM, and aspirate Washonce,and then adda further 3 mL GM and return
to the incubator.
10. Forty-eight hours later, the cells are changedinto selectionmedium
11. At the concentrationsuggested, selectionin G418 will take about 2 wk. Split or
refeed the cells every 3 or 4 d, always maintaining the selection.
Single-cell clones can be isolated by dilution cloning either from a fully
selected mass culture or from transfected, but unselected cells 24-48 h
posttransfection. Both proceduues ˜111give similar results, There may be
advantages with selecting early if the gene is particularly poorly expressed,
smce a greater diversity of clones with fewer sister colonies will be Isolated.
3.1.7. Dilution Cloning of Cell Lmes
Plate cells in 96well plates at concentrations rangmg from 0.1-5 cells/well/
100 pL. Independent clones will appear after l-2 wk. Scan the plates under the
microscope to select wells that contain single colonies. Clones can be picked
and transferred to 24-well plates and expanded. It 1sadvisable to maintam the
selection pressure throughout the cloning process to minimize reversion of the
clone. As a precaution against losing the transfected cell lines from fungal or
bacterial contammation, allquots should be frozen at frequent intervals and
transferred to liquid nitrogen storage containers. A freeze medium of 10%
DMSO and 90% serum 1ssuitable for most cell types. When recovering cells
from frozen storage, replating in medium containing the selective drug will
often decrease cell viability. Selection should be applied 24 h postrecovery.
3.2. Screening of Cell Lines by PCR
Unfortunately not all clones isolated will express the transfected protein.
This is sometlmes owing to rearrangements and deletions within the plasmld
construct following or during Integration. A more frequent reason may be inef-
ficient application of the selection. Excess cells can be screened by PCR or
Southern blot to check plasmid integrity and to estimate copy number. Protein
expression is confirmed by RT-PCR or more frequently Western blot. If a null
mutant virus 1s available, the most precise screening method for “essential
gene” expression in a complementing cell line is by plaque assay or comple-
mentation assay.
3.2.1. Screening by DNA Analysis
The DNA prepared by this method is suitable for analysis by PCR and South-
ern or slot-blot probing.
1. Pellet l-5 x lo6 cells m a stenle Eppendorf, asplrate the me&a, and resuspend
Entwisle
94
the cell pellet m 730 uL of dtgestion buffer Add 20 pL of a stock solution of
protemase K Incubate at 56™C for 2-4 h
2 When completely digested, add 500 pL of equthbrated phenol/chloroform Vor-
tex briefly (15 s) Spm for 10 mm at 12K m an Eppendorf centrtfuge. Transfer
500 uL of the aqueous phase (top layer) to a clean Eppendorf Avoid transferrmg
any protein material present at the interface
3. Add 750 pL of ethanol to each sample, spin samples for 10 mm at 12K, and
carefully aspnate the ethanol without disturbing the DNA pellet Wash the pellet
m 70% ethanol, centrifuge, and aspirate Au-dry under vacuum
4. Resuspend the pellet m 50 pL of TE buffer Heat at 65°C for 2 h to macttvate
contammatmg DNases and atd m resuspension
5 Measure the absorbance at 260 nm (1 OD = 50 ug/mL) Dilute to give a working
stock of 100 pg/mL
Prepare a stock PCR mix as follows The volumes are scaled up depending
on the number of samples to be screened. For 20 PCR reactions wtth a final vol
of 20 pL/reactton, the mtx is made up as follows. Use sterile plasttcware
throughout,
1 Reaction mix
40 pL of 10X PCR buffer
64 pL of dNTP mtx
Primer 1 8 uL
Primer 2 8 pL
76 uL of LJHP
4 uL Taq polymerase
Mix well and aliquot 10 pL into sterile 0 5-mL PCR tubes Store on ice
2 Add 1 uL (60-400 ng) of genomtc template DNA, and make up to 20 IJL with
UHP H20. Overlay with 20 pL of light mineral oil.
3. Following PCR, the mineral 011is aspirated from the reaction and 2 uL of loading
buffer 1sadded to the sample
4. Prepare a 1% agarose gel in 1X TBE containing ethidium bromtde at 1 pg/mL.
5 Load samples alongside appropriate mol-wt markers and standard samples
Suggested PCR protocol
Step 1 Denature 5 mm 1x
94°C
Step 2 Denature 1 mm 94°C 25X
Annealmg 1 mm 40-55°C
Elongatton 2 mm 72™C
Step 3 5 mm 1X
72°C
Hold 4OC
Negative control reactions should be prepared m parallel contammg genomtc
DNA from untransfected cells Appropriate plasmid samples are also prepared as
postttve controls and spiked into control genomic DNA A dilution series rang-
95
Cell Lines Expressing HSV Genes
ing from 100 pg to 100 ng of plasmtd DNA 1s a useful range to check the effi-
ciency of the PCR reaction The PCR reaction may be Improved by the addttton
of DMSO to the reaction mtx at a final concentration of 7.5%.
3.2.2. Screening by RNA Analysis
Screening for protein expression by Western blot may be approprtate if a sutt-
able antibody is available and if the protem is expressed at detectable levels
Screening by RT-PCR can be a quick alternative. Total RNA can be prepared by
several methods (9), and many commercial kits are available (Stratagene RNeasy
cat. no. 74104). The RNA sample must be free of contaminatmg DNA.
Prepare a stock PCR mtx as follows. The volumes are scaled up dependmg
on the number of samples to be screened. For 10X RT-PCR reacttons with a
final volume of 100 pL/reaction, the mix IS as follows. Quantities can be scaled
down approprtately.
1 Reverse transcriptase reaction mrx (A).
20 pL PCR buffer
32 PL dNTP mrx (1 25 mM)
0.25 PL RNasin
10 pL random hexamers
5 pL enzyme
32 75 yL DEPC-treated H,O
Prepare the above mix m duplicate (B) omitting the RT enzyme
2. Dilute RNA samples into DEPC-treated H,O to a final vol of 10 yL Use approx
1 pg/reactton. Prepare samples in duplicate Set up negative controls omtttmg
template Add 10 pL of either RT mix A or B to each duplicate
3 Incubate at room temperature for 10 min, and then at 42°C for 1 h.
4. Heat-treat at 90°C for 5 min to inactivate the enzyme
5 Prepare PCR mix: 20X PCR reactions.
20 pL of prtmer 1 at a cone of 50 pmol/pL
20 pL of primer 2 at a cont. of 50 pmol/pL
200 pL 10X PCR buffer
8 PL Taq
1352 pL of DEPC-treated HZ0
6. Prepare approprtate water only controls. Overlay with 100 pL of mineral 011.
7 Suggested PCR protocol*
Step 1 Denature 5 mm 90°C 1X
1 min 90°C 1X
Step 2 Denature
Annealmg 1 mm 55°C 25X
Extension 1 mm 72°C 1X
Step 3 5 mm 72™C 1X
Hold 4°C
Entwisle
96
8 Prepare a 1% agarose gel m TBE 1X buffer, containing ethidmm bromtde at
1 ug/mL.
9 Load the samples alongstde appropriate mol-wt markers.

References
1 Gao, M., Colonno, R. J., et al (1994) The protease of Herpes Simplex Virus Type
1 IS essential for functional capstd formation and viral growth. J V˜rol 68, 6
3702-3712
2. Johnson, P. A., Best, M. G , Friedmann, T., and Parru, D. S. (199 1) Isolation of a
Herpes Simplex Virus Type 1 mutant deleted for the essenttal UL42 gene and
characterrzation of Its null phenotype J Vwol 65, 2 700-7 10.
3. Nguyen, L. H., Kmpe, D M., and Fmberg, R. W (1992) Rephcatton-defective
mutants of Herpes Simplex Vrrus (HSV) induce cellular lmmumty and protect
against lethal HSV infection J Vu-01 66, 12 7067-7072
4 Farrell, H. E., Mclean, C. S , Harley, C , Estathtou, S , Inghs, S., and Mmson, A
C (1994) Vaccine potential of a Herpes Simplex Vu-us Type 1 mutant wtth an
essential glycoprotem deleted J. Vzrol 68, 2 927-932,
5 Lokensgard, J R., Bloom, D. C., Dobson, A T., and Feldman, L T. (1994) Long
term promoter activity during Herpes Simplex Virus latency. J Vzrol 68, 11
7148-7158
6. Geller, A. L , Keyomarst, K., Bryan, J , and Pardee, A.B (1990) An efficient
deletion mutant packaging system for defective Herpes Stmplex Vuus vectors.
potential apphcattons to human gene therapy and neuronal physiology. Proc Nat1
Acad Scz USA 87,22 8950-8954
7 Smith, C A and DeLuca, N A (1992) Transdommant mhtbttton of Herpes Stm-
plex Virus growth m transgemc mice. Vzrology 191, 581-588.
8 Murray, E J., ed (1991) Gene transfer and expresston protocols, Methods in MOE-
ecular Bzology (Walker, J. M., ed ), Humana, Totowa, NJ
9. Extraction, purtficatlon and analysts of mRNA from eucaryotm cells, m Molecu-
lar Clonzng. A Laboratory Manual, vol. 1, Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY, p. 7.3-7 30
7
Analysis of HSV Polypeptides
Lars Haarr and Nina Langeland

1. Introduction
Lytic infection by herpes simplex virus (HSV) efficiently inhibits the syn-
thesis of most cellular proteins while a large number of viral protems 1sproduced,
including a host shut off protein (J-3). The inhibitory effect of the protem obvi-
ously needs a certain period of time to be fully efficient, but some cellular proteins
involved m vn-us replication still are produced. Examples are those mteractmg
wtth the regulatory viral protems VM,+,65 (65KTIF) and IE 110 (VMW 1IO, ICPO)
(4-8). Certain quiescent cellular genes are activated after mfectton to express
their proteins (9).
These basic facts have implicattons for analysis of HSV proteins. First, one
should distingutsh between virus-induced and virus-encoded proteins. Second,
extracts from infected cells always will contain a mixture of cellular proteins
and newly synthesized virus-encoded proteins, although the proportion of the
former ISmarkedly reduced at late times of infection. Compartson of uninfected
and infected material therefore is crucial for detection of virus-induced proteins.
Analysis of proteins is a large subject that can be divided into a number of
different topics. Here we will focus on separationof the proteins in polyacrylamide
gels, their identification and localization, and someposttranslational modtficattons
2. Methods
2.1. Separafion of Proteins by Electrophoresis
in Polyacrylamide Gels
2. I. I. Separation Capacities of One- Dimensional
and Two- Dimensional Gel Electrophoresis
Since the introduction by Laemmli (10) 25 yr ago, electrophorests m a dis-
contmuous polyacrylamide slab gel system has been one of the most widely
From Methods In Molecular Me&me, Vol IO Herpes Simplex Vws Protocols
&Wed by S M Brown and A R MacLean Humana Press Inc , Totowa, NJ

97
Haarr and Lange/and
98
used methods m experimental biology. The solubtlized proteins, covered with
the negatively charged detergent sodium dodecyl sulfate (SDS), are separated
according to then M,.s(SDS-PAGE). Under optimal conditions one can detect
a maximum of 50-70 distinct protein bands. The HSV genome has a coding
capacity for at least 76 different proteins (11-14). A product from a single
gene may exist in several forms because of posttranslatlonal processing. The
total number of viral and cellular proteins and then different forms IS far beyond
the separating capacity of SDS-PAGE, although the separation within a certain
range of A4,scan be markedly Improved by mampulatton with the polyacryla-
mrde concentratton
By two-dimensional (2D) gel electrophoresrs, as described by O™Farrell
(15,16) the proteins are separated tn a pH gradient according to then charges
before runnmg m a SDS-PAGE system. By this technique more than 1000 spots
can be separated easily in material from HSV-infected cells (17). Maximal
separation is obtained by adjustmg the condrtrons for electrophoresis either
in the first or in the second drmensron, depending on the molecular charges and
weights of the proteins of interest. A variety of ampholyte mixtures are avail-
able such that almost any area in the range pH 3.0-l 1.Ocan be expanded m the
first dimension. To avoid artifactual charges, a neutral detergent like Nomdet
P40 (NP-40) has to be used in the first dimension. The pH gradient gradually IS
broken down during electrophorests such that proteins may be lost both from
the acidic and the basic end of the gel This IS avoided by stopping the electro-
phorests before all protems have reached their isoelectrrc points, a method re-
ferred to as NEPHGE (nonequrhbrmm pH gel electrophoresrs) (16). Recent
advances m 2D gel electrophoresrs include replacement of the ampholyte sys-
tem with immobihzed pH gradients that do not drift during focusing (18-21)
2.1.2. Solubillzation of the Proteins and Denaturation
Proteins to be analyzedby SDS-PAGE areefficrently solubrlized by borltng m the
presenceof SDS and 2-P-mercaptoethanol.The ongmal solubihzation procedure for
2D gel electrophoresrsused NP-40 in combmattonwith high concentrattonsof urea
(9SM) and 2-mercaptoethanol (5%) (IS). Some hydrophobic membrane proteins,
however, are not drssolved under theseconditions (22). To overcome this problem a
combination of NP-40 and SDS or 3-([3-cholamtdopropyl] dtmethylammonlo)- l-

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