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(36) and a triphosphatase activity (37). Although detailed biochemical studies
of the helicase-primase were done using purified protein (381, a convenient
technique for measuring the helix-destabtlizing activtty is to assay its abiltty to
decrease the melting temperature of DNA (39).
1. Infected cell nuclear extracts are prepared as described m Section 3.7. and dia-
lyzed against the extraction buffer to remove small molecules
2 The extract is passed over a Sephadex G-200 gel filtration column that pre-
eqmhbrated in extraction buffer and fractionated
3 The helix-destabilizing activity is determined by measuring the affect of the fractions
on the melting temperature (T,,) of poly(dT-dA), measured at 254 nm, usmg a tem-
perature-controlled microcuvet and spectrophotometer (e g., Varian, Palo Alto, CA).
4. Twenty-five microliters of each fraction is mixed with 25 ug of poly(dT-dA) in
200 pL of 10 &potassium phosphate, pH 7 8, 10% glycerol, and 50 pL of 100
mA4 MgC&. UV absorption measurements at 254 nm are taken each minute for
30 min at temperatures between 0 and 75°C
5 The reference sample contains buffer without DNA and control measurements
are done using the same conditions but without any added protein.
3.20. Uracil-DNA Glycosylase
Uracil-DNA glycosylase is encoded by the UL2 gene of HSV- 1. Its function
is to remove uracil residues from DNA generated by the insertion of dUTP or
the deaminatlon of dCTP (40,41). The activity is assayed by measuring the
release of trittated uracil from DNA by infected cell protein.
1. Infected cells are harvested at 12 h post infection m 0 2A4 potassium phosphate,
pH 8.0, I WEDTA, 2 mMMgC12, 2 mMDTT, 1% Triton X-100,1 mMPMSF,
and 20% glycerol, briefly sonicated, and dialyzed m 50 mMTris-HCI, pH 7.5, 1
mA4 DTT, 1 mM MgCl,, and 20% glycerol.
2. Infected cell extract (10 yL) is added to mixtures (200 PL) containing 50 mA4
Tris-HCl, pH 7 5,2 mM DTT, 100 pg/mL BSA, and 4 pg/mL of [3H]DNA (see
Note 15), and reacted at 37°C for varying times.
3. Reactions are terminated by rapid chillmg to 4°C followed by the addition of 25
PL of sheared calf thymus DNA (1 mg/mL) and 25 PL of 4Mperchloric acid.
4 After 10 mm at 4°C the samples are microcentrifuged, the supernatant is
removed, and the radioactivity present is measured by ltquid scmtillatton (see
Section 3.12.).
Blah0 and Roizman
250

3.21. dlJTPase
The herpesvn-us dUTPase is encoded by the T&50 gene and IS not essential
for virus replicatron m tissue culture cells. dUTPase was first described by
Wohlrab and Francke as a deoxyribopyrimidine trrphosphatase activity that
was specific for cells infected wtth HSV-1 (42). In this study, trlphosphatase ac-
tivity 1sassayed by measuring the productton of radiolabeled deoxynucleostde
monophosphates from the correspondmg trtphosphates.
1. Infected cell nuclei are prepared as described 1n SectIon 3 6 and are resuspended
1n 20 mM HEPES, pH 7 8, 1 mA4 DTT, 1 mA4 MgCl*, 80 mA4 potassium acetate
2. Approximately 3 x lo6 nuclei are added to reactions (50 uL) contalnmg 1 mA4
DTT, 3 mM EDTA, 2 mM ATP, and 50 pA4 [3H]dUTP (2 Cl/mmol)
3. Reactions are initiated by the addition of MgCl* to 80 mA4 and incubated at 4“C,
prior to termination with 20 uL of 0 IMEDTA and 125 uL cold methanol (4°C).
4. Four-microliter aliquots of the reaction mixture are mixed with unlabeled nucle-
ot1de markers (e g , 0 1 mM each of dUTP, dUDP, and dUMP) and spotted on
polyethyllmine(PEI)-cellulose thin-layer chromatography plates that were
prewashed with 100% methanol The plates may be dried following the methanol
wash, as well as prior to development without consequence
5. The TLC plates are developed with IMHCOOH-0 5ML1Cl at room temperature,
dried, and examined by illumination with UV light
6. Spots containing nucleoslde mono-, d1-, and triphosphates are excised and the
radioactivity contained 1n them 1sdetermined using 11qu1dsclntlllatlon (see Sec-
tion 3.12 )
3.22. Ribonucleofide Reducfese
The HSV-I ribonucleotrde reductase consists of two subumts, encoded by
the UL39 and UL40 genes, which are tightly combined m an a& complex;
both subunits are required for activity (43). The enzymatic assay requires a
high multtplicrty of infection m order to get consistent results (44,45).
1 Cells should be infected at an MO1 of at least 20 and the infection should proceed
for at least 7 h, prior to makmg cellular extracts (see Section 3.4.1.).
2 The infected cell extract 1spassed through a small cation exchange column (e g.,
AGI-X8, Bio-Rad), previously equlltbrated 50 mMTns-HCl, pH 8.0, 1 mA4DTT,
to remove nucleotldes
3. Approximately 0.5 mg of infected cell protein 1s added to mixtures (100 uL)
containing 5 mM Tns-HCl, pH 7.0, 50 mM FeC13, 4 mM NaF, 6.5 mM magne-
slum acetate, 3 m&I ATP, 5 mA4 dlthloerythntol, 5 mA4 CDP plus 1 x 10™ cpm of
[3H]CDP and incubated at 37°C for 60 mm
4 The reaction 1sstopped by the addition of 50 uL of 4M perchlorlc acid and boil-
ing for 10 min. During this step the nucleotldes are converted to monophosphates.
5. The mixture is then neutralized by adding 15-20 uL of 1OM KOH prior to
pelleting insoluble material by centrifugatlon
251
Analyses of HSV Proteins
6 Thirty microhters of the supernatant are apphed to the center of a PEI-cellulose
plates prespotted with 2 5 mM cold CMP and cCMP markers.
7. The plate is washed by ascending irrigation once with distilled water, dried, and
the buffer front at the top IS removed and discarded.
8 The plate is then turned 180™ and developed in the opposite direction in a solu-
tion of 20.40:100:0.5 (v:v) 5M ammonium acetate, pH 9.8:saturated sodium
tetraborate:95% ethanol:0.25MEDTA.
9 After drymg, the CMP and dCMP spots are identified by UV light illummation,
excised, and the radioactivity presentin eachis determinedby liquid scmtdlation
(see Section3.12.).
3.23. HSV-2 Ribonucleotide Reductase Protein Kinase
A protein kinase activity IS associated with the large subunit of rrbonucle-
otide reductase of HSV-2, but not that of HSV-1 (20). Although the kinase
autophosphorylates itself, the exact substrate of the kmase is not known. There-
fore, to study this kinase It is necessary to separate tt from the other viral
kmases. This is done readily by immunoprecipitation (see Note 8) using an
antibody spectfic for the large subunit.
1 Unlabeled infected cell extracts (25 pL) (see Section 3 4.1 ) are mixed with mono-
clonal antibody MAB30 and 20 pL of protein A-sepharose CL4B and incubated
for 30 mm at 4°C.
2. The beads are washed three times with 500 uL of O.l5MNaCl,20 mA4Tris-HCI,
pH 7.4, resuspended in 50 pL of 20 mM Tris-HCl, pH 7 4,5 mA4 MgCl,, 0.1 yM
[y32P]ATP (10 PCi), and Incubated at 30°C for 10 mm
3. The reaction is terminated by boiling in 0.1% SDS prior to denaturing gel elec-
trophoresis (see Section 3.1 ).

4. Notes
1. It is a standard and necessary practice to degas the separating gel solution prior to
adding SDS and TEMED for optimum resolution of electrophoretically sepa-
rated proteins m denaturing DATD gels. Note that 9.3% gels are aesthetically the
mostpleasing(!), but at timesit is necessary increase percentage polyacryla-
to the of
mide to as much as 17% to detect and resolve small mol-wt protems.
2. The major causeof poor quality viral protein gels is overloadmg. Always deter-
mine protein concentration of the sample prior to loadmg Never load more than
75 yg viral protein on a 0. l-mm gel.
3 Protein gel solutions A, B, and C should be made as stocks; store A and B at
room temperature and C in the dark at 4°C. Use a fresh ammonium persulfate
solution and running buffer
4 When labeling viral proteins m vivo with phosphate, especially the a proteins,
the trick is to keep the extent of labeling of host proteins to a minimum. The
easiest way to do this is to be sure that you get an excellent infection of your
cells; thus, be sure all cells are infected and the cell monolayer is at 70-80%
Blaho and Roizman

confluency at the trme of labeling. Another way to get the same result is to starve
the cells for phosphate prior to labeling, this helps when labeling early m infec-
tion. Late m Infection it may have little or no effect and it may even be detrimental
5. In all cases, cells are grown m Dulbecco™s modified Eagle™s mmtmal essenttal me-
dium (DMEM) supplemented with 5% newborn calf serum. HEp-2 and BHK cells
are the most commonly used cells for studies of viral proteins m this laboratory
HeLa cells are useful for studies on proteins in nuclear extracts but these cells are
partially restrictive to HSV replication Vero cells frequently express large amounts
of a specific protein, but they also contam rather active proteases that have a predilec-
tion for numerous denatured viral proteins. Although we recommend against using
cells other than HEp-2s and Helas, if you must, be certain that you solubihze the cells
m SDS and boil them immediately. We recommend the use of HeLa S3 cells for the
isolation of infected cell nuclei. Although infected cell nuclei from all cell types are
extremely fragile, nuclei from S3 cells are the most stable that we have found.
6. It is extremely difficult to identify specific viral proteins if one labels infected
cells with phosphate for a long period of time The preferred interval is less than
1 h at any time and two h late m mfection. A major cause of unmterpretable
results is the use of way too much [32P,] This also leads to radtoacttve waste
problems Even amounts as low as 10 @r/4 x lo6 cells requires at least three monlayer
washes with phosphate-buffered salme Caution should taken during this proce-
dure to reduce the amount of radioactive “splatter” during these manipulations
7. Most standard mnnunoblot protocols (Western blots) can be used for the analysis
of viral polypeptides. However, we have found that the lowest background levels
are obtained followmg blocking for at least 1 h with 5% nondairy lowfat milk
(e g , Carnation by Borden) m phosphate buffered salme (w v)
8. Two fluorographic techmques are used. The first mvolves impregnatmg denatur-
mg gels with fluors (either 20% sodmm sahcylate or 20% 2,5-diphenyloxazole
[PPO] in DMSO) prior to drying The second requires that the polypeptides are
electrically transferred to nitrocellulose and dried prior the spraying them with
En3Hance (NEN). In both cases, fluorography is done by placing either the dried
gel or membrane directly against X-ray film (Kodak X-OMAT) Depending on
the specific activity of the labeled proteins, the film may require exposure times
of 4 d to as long as 4 mo in order to get convmcmg signals.
9 In order to differentiate specific viral polypepttdes from other viral or cellular pro-
teins, it may be necessary to immunoprecipitate the proteins of interest pnor to ana-
lyzing them. In our hands, efficient precipitations require preclearing the infected cell
extracts prior to adding the antibody. In the case where a rabbit polyclonal antiserum
is used, add premnnune serum, protein A-sepharose, and goat antirabbit immunoglo-
bulin imtnunobeads directly to the extract, mix the slurry, pellet the beads, and fi-
nally, use the supematant for the munune precipitation reactions.
10 Bacteria seldom tolerate long viral polypeptides. Depending on the piece of viral
DNA used to make the fusion protein, varying extents of proteolysls will occur
during the growth of the bacteria. The major factor affecting this is the length of
the final fusion protein* thus, the length of viral peptide added should be kept as
Analyses of IiSV Proteins 253
short as possible. Although incubation of the bacterial cultures at a lower tem-
perature (28-30°C) reduces nonspecific proteolysts sometimes at the expense of
longer incubation times, the best way to reduce proteolysis IS to delay the mduc-
tton by IPTG as long as possible and to keep the induction period short; as little
as 30 min of induction IS sufficient for a late log phase culture
11 Although the mtrocellulose filter binding assay is able to pick up proteins labeled
to low specific activities, one drawback 1s that the protem of Interest must be
pure Thus, it is ideal for use with viral fusion proteins that can be highly purified
and can be compared easily to the parental (GST) protein. However, Intact viral
proteins must be purified partially either by immunological or chromatographic
techniques prior to doing the filter bindmg assay.
12 Since the actual number of TK assays that are done at one time is significantly
large, the process of washing the filters can be quite laborious. A convement trick
is to attach the numerous filters to a 1-mL glass pipet using paper clips and then
dangle the filters mto a plastic tray that contains the various wash solutions. In
this manner, as many as 80 filters can be handled easily at one time.
Although most transfectron techniques appear to give satisfactory results, we prefer
13
the procedure described by Graham and van der Eb (46). Although the purity of the
transfected DNA is of obvious importance, it is not necessary to purify it by CsCl
gradients, as long as the contaminating RNA is removed by polyethylene glycol pre-
cipitation In order for this transfection/mfection technique to work, it is necessary
that the UL26 gene be driven by an a promoter; we recommend the promoter of a4.
At >l O”C, HSV-1 bmds but does not penetrate cells For consistent results, it IS
14
very important that one works fast when removing the inoculum and placing the
flasks in the water bath so as not to allow the expression of post a genes The
temperature of the water during must be very precisely regulated
Any covalently closed circular DNA (e.g., plasmid DNA) may be used, but care
15.
must be taken during the purification to insure that it is maintained as form I;
CsCl gradients are recommended. Since the amount of viral DNase ˜111 be high,
the nuclear extracts should be diluted m reaction buffer and several different
diluttons should be tested. To prepare DNA specifically labeled in uractls, 1 mCi
of [3H]uridme is simply added to a 200-mL bacterial culture m log phase, which
is then used to prepare the plasmid DNA

References
1. Ejercito, P. M., Kieff, E. D., and Roizman, B. (1968) Characterization of herpes
simplex V˜I.IS strains differing in their effect on the social behavior of infected
cells. J, Gen Virol 2,357-364.
2. Braun, D. K., Roizman, B., and Pereira, L (1984) Characterizatton of post-trans-
lational products of herpes simplex virus gene 35 bmdmg to the surfaces of full
capsids but not empty capsids. J Vzrol 49, 142-153
3. Pereira, L., Wolff, M. H., Fenwick, M , and Roizman, B (1977) Regulation of
herpesvn-us macromolecular synthesis. V. Properties of a polypeptides made in
HSV- 1 and HSV-2 infected cells. J. Viral. 77, 492-50 1
254 Blah0 and Roizman
4. Blaho, J. A and Roizman, B (1991) ICP4, the major regulatory protein of herpes
simplex vu-us, shares features common to GTP-binding proteins and is adenylated
and guanylated J Vwol 65,3759-3X9
5 Wtlcox, K W., Kohn, A , Sklyanskaya, E., and Roizman, B. (1980) Herpes sim-
plex virus phosphoprotems I Phosphate cycles on and off some viral polypep-
tides and can alter their affinity for DNA. J. Vwol 33, 167-182.
6 Blaho, J. A , Michael, N , Kang, V , Aboul-Ela, N , Smulson, M E., Jacobson, M
K , and Roizman, B. (1992) Differences m the poly(ADP-ribosyl)ation patterns of
ICP4, the herpes simplex vuus maJor regulatory protem, m Infected cells and m
isolated nuclei J Vv-ol 66,6398-6407
7. Bames, J D. and Roizman, B (1993) The U,lO gene of herpes simplex vuus 1
encodes a novel viral glycoprotem, gM, which IS present m the virion and m the
plasma membrane of Infected cells J Vzrof. 67, 1441-1452
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Flume, G (1994) The herpes simplex vnus UL20 protein compensates for the drfferen-
tral disruption of exocytosis of vinons and viral membrane glycoprotems associated
with the fragmentation of the golgi apparatus J. Vzrol. 68,7397-7405
9 Erickson, J S and Kaplan, A S. (1973) Synthesis of protems m cells Infected
with herpesvirus IX Sulfated protems. Vzrology 55, 94-102
10 Maclean, C A., Clark, B , and McGeoch, D J. (1989) Gene U,l 1 of herpes sim-
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3147-3157.
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Myristylation and polylysine-mediated activation of the protem kmase domain of
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Vwology 179, 168-l 78.
12. Blaho, J A., Mitchell, C., and Roizman, B. (1994) An ammo acid sequence shared
by the herpes simplex vm.ts 1 CLregulatory protems 0, 4, 22, and 27 predicts the
nucleotidylylation of the UL21, UL3 1, UL47, and UL49 gene products. J Bzof
Chem 269, 17,401-17,410.
13. Blaho, J. A., Mitchell, C., and Roizman, B. (1993) Guanylylation and adenylyla-
tion of the a regulatory proteins of herpes simplex virus require a viral p ory function
J. Vwol. 67, 3891-3900
14. Gottheb, J , Marcy, A I , Coen, D M , and Challberg, M. D (1990) The herpes
simplex virus type 1 UL42 gene product. a subunit of DNA polymerase that func-
tions to increase processivity. J Vv-ol 64,5976--5987
15. Powell, K L and Purifoy, D. J M. (1977) Nonstructural proteins of herpes sim-
plex virus I. purificatron of the induced DNA polymerase. J, Vzrol. 24,61 E-626.
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expression from those conferrmg a gene recognition wrthin the regulatory domains
of herpes simplex wrus 1 c1 genes Proc Nat1 Acad Scz USA 81,4056-4069
17 Mavromara-Nazos, P., and Roizman, B (1987) Actrvatton of herpes simplex virus
1 x 2 by viral DNA replication Vzrology 161, 593-598.
18 Lui, F. and Roizman, B. (1991) The promoter, transcriptional unit, and coding
Analyses of HSV Proteins 255
sequence of herpes simplex virus 1 family 35 proteins are contained within and m
frame with the II,26 open reading frame. J Vzrol 65,20&2 12.
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tease also contains its coding domain the gene encoding the more abundant sub-
strate. J Vzrol. 65, 5 149-5 156.
20. Lm, F. and Roizman, B. (1992) Differentiation of multiple domams m the herpes
simplex virus 1 protease encoded by the I-I,26 gene Proc Nat1 Acad. Scl USA

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