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using NAD as substrate. The modification that occurs tn nucler may differ from
that which occurs in Intact cells (6) and may result from either the elongation
of pre-existing chains or addition at novel sites on the proteins.
1. Infected cell nuclei are Isolated as described m Section 3.7.
2 Nuclei are resuspended m 25 mM Tris-HCl, pH 8 0,3 mM MgC&, 20 mM KCI,
0 2 mM PMSF, 4 mM 2-mercaptoethanol containing 5 pLM (100 @I) [32P]NAD
3. The nuclei are incubated at 25°C for 30 mm, pelleted, and the label IS removed
and dtscarded
Analyses of HSV Protems 243
4 The nuclei are then washed three times with 200 pL of reaction buffer minus NAD.
5. The nuclear proteins are then extracted as described in Sectlon 3 7. and analyzed
by using denaturing gels (see Section 3.1 ), followed by autoradiography
3.9. Nucleotidylylation in Nuclei
At least eight HSV proteins are nucleotidylylated (22). This conclusion is
based on the fact that viral proteins are labeled m isolated nuclei by either
[cx˜˜P]ATP, [cx˜˜P]GTP, or [2-3H]ATP, where the tntlum atom IS m the purme
base.
1 Infected cell nuclei are isolated as described in Section 3.7
2. Nuclei are resuspended m 50 pL of 50 mi14 Tns-HCl, pH 7.5, 5 mM MgCl,, 30
mM of [IX˜˜P]ATP or [a32P]GTP (3000 Ci/mmol) plus 150 pM ATP or GTP,
respectively. The specific activity of [2-3H]ATP IS approx 25 Ci/mmol.
3 The nuclei are incubated at 15°C for 30 mm and nuclear proteins are then
extracted as descrrbed rn Section 3.7
4. Visuahzatlon of the proteins is done conveniently by denaturing gel electrophore-
SIS, followed by etther autoradiography or fluorography (see Note 9).
5. As a result of the extremely long fluorographlc times required to observe proteins
labeled with trltmm (>4 mo), we have developed a simple mtrocellulose filter bind-
mg assay (see Section 3.12 ) m order to measure the nucleotldylylatlon of proteins
3.10. Phosphorylation in Nuclei
Phosphorylatlon of viral nuclear proteins can be done exactly as described
for nucleotidylylatlon in Section 3.9. simply by substituting [y3™P]ATP for the
labeled nucleotide. To differentiate between phosphorylation and nucleo-
tldylylatlon, the unlabeled ATP or GTP 1s substituted for nonhydrolyzable
nucleotide analogs, such as GTPyS or GDPpS (guanosine S-O-[2-thiodi-
phosphate]) (23),
3.11. Induction and Purification
of HSV ProteinGST Fusion Proteins
In general, herpesvirus proteins are not tolerated well when expressed m
bacteria (see Note 1O), presumably owing to either the high G-C content of the
viral DNA or the high prolme-rich content of the proteins. The following pro-
tocol reproducibly generates highly purified, intact fusion protein to yields as
high as 8 mg/mL of bacterial culture.
1. Fresh single colonies of E coli BL2 1 cells containing glutathione-S-transferase
(GST) fusion protein expressing plasmids are picked and incubated m 3 mL of
Lurla broth (L-broth) for no more than 6 h.
2. This 3-mL culture is used to inoculate a 50-mL overnight culture that IS mcu-
bated no longer than 12 h.
3. This 50-mL overnight culture 1sused to inoculate 500 mL of L-broth, which IS
Blaho and Roizman
244
grown to anOD,,, of -0.446, at which point 60 pL of fresh 20% IPTG 1sadded
and the cells are incubated for an addttional 1 h All bacterial growth IS at 37°C
under ampicillin selection (100 pg/mL)
4. All of the following techmques are done at 4°C (wet ice). Pelleted cells are resus-
pended in 5 mL PBS, briefly somcated, and 500 pL of Triton X-100 is added,
prior to pelleting the cellular debris at SOOOgin a Sorvall SS34 rotor
5. The supernatant fluid is mixed with 2.5 mL of a 50% slurry (v:v m PBSA) of
glutathione-agarose (Sigma) and rotated for 30 mm
6. The agarose beads are pelleted and then rmsed:
a Two times m 10 mL of PBS;
b. Two times m 10 mL of 0 02% Tween-20 in PBS (VW), and
c. Two times in 10 mL of 50 mMTns-HCl, pH 8.3.
7. Fusion protein bound to the glutathione beads is purified followmg two sequen-
tial 1.5 mL elutions with 5 mA4 glutathione-50 mM Tris-HCl, pH 8.3.
8 Fusion protein are either used immediately or dialyzed into 50 mMTris-HCl,*pH
8.3, allquoted, stored at -70°C and thawed once prior to use.
9. The vtral fusion polypeptides generated in this fashion are frequently suitable sub-
strates for protein modification reactions. To test the usefulness of fusion proteins m
nucleotidylylation reactions, the protocols described earher for reactions m nuclei
was done with nuclear extracts (see Section 3 7 ) by simply mixing the extract with
an equal volume of fusion protein (-10 ug) m a 2X reaction cocktail (this brings the
NaCl to 0.2A4). The modified fusion proteins are then analyzed by either denaturing
gels (see Section 3 1.) or by mtrocellulose filter bmdmg (see Section 3.12 )
3.12. Nitroceiiuiose Filter Binding
of Posttranslationally Modified Proteins
The methodsfor specifically modtfymg viral proteins describedabove ultimately
yield soluble radiolabeled polypepttdes.The extent of protein labeling can be easily
quantified m a nitrocellulose filter bindtng assay.This technique ISsensitive and the
method of choice for the analysisof trtttatedpolypeptides (seeNote 11).
1 Nttrocellulose membranes (Schleicher & Scheull, BA83) are first wet m phos-
phate-buffered salme.
2. Mixtures contammg radiolabeled polypeptides are then passed through the
nitrocellulose under vacuum using a “dot-blot” manifold (Gibco-BRL,
Gaithersburg, MD).
3. The filters are washed by sequentially passing 0 5 mL of phosphate-buffered
saline through the membranes three times
4. The membranes are then dried for 30 min in an oven (13O™C)
5. The radioactivity retained is measured by liquid scmtillation using a Beckman
scmttllation counter To do this, the membranes are first dissolved in 1 mL tet-
rahydrofuran (Sigma) prior to adding 5 mL BCS-NA scintillant (Amersham)
6 The radioactivity of of 32P-labeled proteins bound to membranes may also be
quantified using a p-counter (Betagen)
Analyses of HSV Proteins 245

3.13. DNA Polymerase
The DNA polymerase of HSV-1, the product of UL30 gene, forms a com-
plex with the product of the UL42 gene. The UL42 protem Increases the
processivity of the complex (13). The polymerase activity is measured by using
purified enzyme by measuring the formation of acid-precipitable counts start-
ing with radiolabeled nucleotides (15).
1 Infected cells are harvested 18 h postinfection and are lysed m 20 mMTrrs-HCI,
pH 7.5, 0.5 mMDTT by sonication.
2. The salt concentrations are adjusted to 1 7M KCI, 5 mM EDTA, 500 ug/mL
bovine serum albumin (BSA) and the mixture is incubated on ice for 30 mm,
pnor to pelletmg the cellular debrrs and dralysrs m 50 mMTris-HCI, pH 7 5, 0.5
nu!4 DTT, 0 2% NP40, and 20% glycerol,
3™ The extract IS passed over a DEAE-cellulose column and the polypeptides
retained are eluted using a O-O 3MKCl linear gradient. (Additional steps of purr-
fication may be performed m the same manner using sequential phosphocellulose
and DNA-cellulose chromatography.)
4. Fractions are assayed for acid-precipitable radioactivrty as follows Fifty micro-
liters of sample 1s added to mixtures (200 pL) contammg 6 6 mM Trts-HCl, pH
7.5, 3 mA4 MgC12, 2 mM P-mercaptoethanol, 100 pg of denatured salmon sperm
DNA, 5 pM [3H]TTP (50 C˜/mrnol), and 0.3 mUeach ofdATP, dCTP, and dGTP
and incubated at 37°C for 10 min.
5. The mixture is immediately spotted onto glass-fiber filters (Whatman GUF) and
washed three trmes m ice-cold trichloroacetic acid (lo%), and then two times m
ice-cold ethanol (95%), prior to drymg
6. The incorporation of radioactive precursors mto acid precipitable oligodeoxy-
nucleotides IS measured by liquid scmtillation counting (see Section 3.12.).
3.14. Thymidine Kinase
Thymidine kinase (t& the product of the HSV- 1 UL23 gene, has been used
as a highly sensitive reporter gene product (16, I 7) m studres of viral gene regu-
lation. In general, chtmeric genes containing the coding sequences of the &
gene are either transfected mto cells, which are then infected with a a- virus,
or the chimeric gene itself resides within the genome of the virus that is used to
infect cells. In both cases, cells deficient in the cellular enzyme homolog are
used (murine Ltk-, hamster BHKtk-, or human 143tk- cells). Thymtdme kinase
activity is measured in infected cell extracts and quantitated as cpm of
[3H]thymtdine converted to thymidylate per pg of total cellular protein,
Thymidylate formation IS assayed by its retention on DEAE filter membranes.
1. The medium from a 25 cm2 dish of infected cells (-4 x 106) IS aspirated off and
the cells are washed two trmes wrth 5 mL phosphate-buffered sahne
2. Cellular proteins are extracted by adding 400 uL of 10 mM Tns-HCl, pH 7.5, 10
Blaho and Rolzman
246
mMKC1, 1 mMMgCl,, 1 rnA4/3-mercaptoethanol, 50 pM thymtdme, 0 5% NP40
and mcubatmg at 25°C for 5 mm
3 KC1 IS added to 0 15M by the addmon of 10 pL of 3M KCl, and the mtxture
(contaming cellular debris) 1sshaken off of the plate mto a mtcrocentrifuge tube,
which 1s then spun m a Brmkman mtcrofuge for 2 mm
4. Ftfty mtcrohters of the supernatant 1s then mtxed wtth 10 pL of [3H]thymtdine
(71 Ci/mmol), 5 pL cold thymtdine (1 mg/mL) and 25 pL of 0 76MTris-HCl, pH
7 5,7 6 mM ATP, 7.6 mM MgCl*, 12 mJ4 phosphocreatine, 40 mA4 DTT, and 40
mMNaF, and 10 pL of creatme kmase (33 U/mL)
5 The mixture IS reacted at 37™C for 30 mm and IS termmated by borlmg m 0 1%
SDS for 1 mm
6 Duplicates of 50 pL of each reactron are spotted onto DEAE filter paper, which IS
then drted using a heat lamp
7 The filters are sequentrally washed using the following solutions 10 mM ammo-
mum formate for 15 mm, 10 mM ammonium formate for 15 mm, dtstllled water
for 10 mm, and ethanol (95%) for 5 mm (see Note 12)
8 The filters are finally dried and the trmated thymtdylate bound is measured by
llqutd scmtlllatton usmg a toluene-based fluor (e g., POPOP/PPO)


The HSV protease is encoded by the UL26 gene. Its substrate 1s the protease
precursor molecule itself that is cleaved twice, and ICP35, the more abundant
product of the UL26.5 gene cleaved once. U,26 and U,26.5 are 3™ coterrninal
(l&19). ICP3 5 forms the scaffolding of the capsid; during packaging of DNA,
ICP35 1s removed from the capstd. Two assays for the protease activity have
evolved in the past 5 yr. The first assay employs the intact, transfected or
Infected cell. The second assay is based on in vitro cleavage of the substrates
(the protease precursor and ICP35).
3.15.1. Protease Cleavage Following Transfectlon
The UL26 proteolyttc activity 1sassayed conventently by transfectmg a copy
of the UL26 gene (which inherently contains U,26.5) that is driven by the viral
a4 promoter, infecting the transfected cells with HSV- 1(F) at 39.5”C, and then
analyzing the ICP35 polypepttdes by immunoblotting. Because HSV- 1(F) con-
tains a ts lesion in the a4 gene, rt does not express its own UL26 protease or its
own ICP35 protein at 39.5”C.
1 Approximately 4 x lo6 BHKtk- cells m a 25-cm2 screw-capped culture flask are
transfected (1.5) with 10 pg of a plasmtd containmg the l-J,26 gene driven by the
HSV-I a4 promoter (see Note 13) and incubated at 37°C for 20 h
2. The cells are then cooled to 10°C for at least 30 mm, prtor to exposmg them to 10
PFU of HSV- l(F) for 2 h at 10°C
3. Followmg absorptton, the viral moculum 1s quickly removed and replaced with
Analyses of HSV Protems 247

cold (4°C) medium The flask immediately is immersed m a circulated water
bath preeqmhbrated at 395°C and incubated for 20 h (see Note 14)
4. The infected cells are washed twice m phosphate-buffered saline and harvested
by scraping them off the dish After pelleting, the infected cell proteins are
extracted by resuspension m 50 mA4Tris-HCl, pH 7 0, 8 5% sucrose, 5% /3-mer-
captoethanol, 2% SDS, sonication on ice, and boiling for 1 mm
5. The polypeptides are separated in denaturing gels (see Section 3 1 ), electro-
phoretically transferred to mtrocellulose, and the probed for ICP35 usmg monoclonal
antibody H725 (see Note 7).
6 In order to control for the synthesis of the U,26.5 gene product without pro-
teolytic cleavage, it IS necessary to repeat this procedure using plasmid that con-
tams only UL26.5 driven by a4 but not the ammo portion of Ut,26 (18)
3.15.2. Protease Cleavage in Cell-Free Extracts
The protease used m this assay is derived from the 635-ammo acid open
reading frame of UL26 and it is either transcribed and translated in vitro (20) or
expressed m bacteria as a fusion protein (21). The induction and purrficatron of
bacterial fusion proteins was descrtbed in Section 3.11. The technique
described below is for the m vitro expression of the protease from a recombt-
nant plasmid. The cleavage measured 1s autoproteolysts and it is assayed by
denaturing gel electrophoresrs. Cleavage of the ICP35 substrate is performed
easily by adding exogenous substrate to the reaction. The substrate may be
either pure ICP35 protein, an ICP35 fusion protein, or a synthetic peptide that
contams the core cleavage site Leu-Val-Asn-Ala/Ser (22).
1 Linear DNA containing the Ui,26 gene in the polylmker of a pGEM plasmid
(Promega) is transcribed in the presence of the cap analog GppG (New England
Biolabs) with RNA polymerase from either bacteriophage T7 or SP6 (Promega).
2. The UL26 RNA is added to a mixture (50 uL) containing nuclease-treated rabbit reticu-
locyte lysate (Promega) and 10 uCt [35S]methtonme and reacted for 10 mm at 4°C
3. Translation is mhtbtted by the addttton of cycloheximide to 100 pg/mL.
4. The mixtures are then incubated for up to 6 h m order to allow the proteolysts to
go to completton. At this time, the reactions are terminated by the addition of
SDS to 0.1%.
5. The cleavage products are visualized following denaturing gel electrophoresis
and autoradiography (see Section 3 1.).
6. Since the Ur,26 protease IS a serine protease, m vitro synthesis and mcubation in
the presence of 10-25 mMphenylmethylsulfony1 floride (PMSF) enables the for-
mation of full length, noncleaved precursor protem.

3.76. Protein Kinase Us3
The Us3 gene product was predicted to encode a protem kmase based on its
sequence (23); this prediction later was confirmed experimentally (24,25). The
248 Blaho and Roizman

assay for the Us3 kmase 1s essentially identical to that previously described for
the viral kmase of pseudorabies virus, Inasmuch as both enzymes phosphory-
late basic peptlde substrates in vitro (26,27) in order to differentiate Us3 activ-
ity from that of cellular kinases it is necessary to partially purify the kmase
from infected cells by anion exchange chromatography (27).
1. Infected cells are lysed by Dounce homogenization m 10 mM Tris-HCl, pH 7.5,
10 mM KCl, 1 5 mM magnesium acetate, cellular debris is pelleted, the supema-
tant IS removed, and dialyzed m 20 mMTris-HCI, pH 7 5, 1 mMEDTA, 10 mM
P-mercaptoethanol, 10% glycerol
2. The crude fraction IS chromatographed on a DEAE-cellulose column and the
bound polypeptldes are eluted using a linear gradlent from 0-0.4MKCI and tested
for activity
3 Partially purified Us3 1sadded to a mixture (100 pL) containing 20 mM Tns-HCl,
pH 7.4,50 mMKC1, 10 mMMgC12, 10 mMP-mercaptoethanol, 0.1 mM[y3*P]ATP
(0.5 pCi>, 0 8 mg protamme sulfate/ml, and incubated at 30°C for 30 mm
4 The reactlons are terminated by spotting them onto Whatman 3MM paper disks
and washing them sequentially for 15 min each m the following: two times m
20% TCA, four times m 10% TCA; one ttme m ethanol (95%)
5 The disks are then dried and the amount of radiolabeled protamme remammg IS
measured by hquld scmtlllatlon (see Section 3 12 )

3.17. U, 13 Kinase
Evidence that the product of the ULl 3 gene IS associated with protein kinase
activity has been published (28,29). Thus, in cells infected with UL1 3-mutants,
ICP22, ICP47, and VP22 are not processed and the accumulation ICPO and the
Us1 1, UL26, and UL26.5 gene products ts decreased (27-31).
The gene product is a component of the vmon (32,33). However, since the
protein itself has not been purified, evidence has not been presented that it has
enzymatic activity.
3.18. Alkaline Exonuclease
The HSV-1 alkaline exonuclease is encoded by the UL12 gene. The enzyme
plays a role in viral growth and DNA synthesis The DNase activity can be
easily assayedusing agarose gel electrophoresis and measurmg the conversion
of form I DNA to form III as a function of the concentration of DNase (34)
1 Infected cell nuclear extracts are prepared as described m Section 3 7
2. Covalently closed circular DNA (0.5 pg) 1s mixed with the infected cell nuclear
extract (5 pL) in reactions (25 pL) containing 50 mM Tns-HCl, pH 8.0, 4 mM
P-mercaptoethanol, 10 mA4MgC12 and incubated at 37°C for 30 mm (see Note 15)
3. The reaction is stopped by the addltlon of 5 pL of 5% SDS, 0 15M EDTA, 0.1%
bromophenol blue, and 30% glycerol.
4 The DNA is then resolved m an agarose gel using 40 mM Tns-acetate, 2 mM
Analyses of HSV Proteins 249
EDTA buffer (pH 8.3) followed by ethidmm bromide (0.5 pg/mL) stammg and
visualization by fluorescence enhancement.
3.19. Helicase/Primase
The HSV hellcase-primase consists of three polypeptides that are the prod-
ucts of the UL5, UL8, and U,52 genes (35). Also, the HSV origin binding pro-
tein, encoded by the UL9 gene, was shown to contain both a helicase acttvlty

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