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28. McGeoch, D. J , Dolan, A , Donald, S., and Brauer, D H K. (1986) Complete
nucleotide sequence of the short repeat region m the genome of herpes simplex
virus type 1. J Gen Vzrol 71, 125-132
29 Igarashi, K , Fawl, R , Roller, R J , and Roizman, B (1993) Construction and
properties of a recombinant herpes simplex virus 1 lacking both S-component
origins of DNA synthesis. J. Vlrol. 67, 2 123-2 132
30. Stow, N. D. and McMonagle, E D. (1982) Propagation of foreign DNA sequences
linked to herpes simplex virus origin of replication, m Eukaryotlc Viral Vectors
(Gluzman, Y.), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York,
pp. 199-204
31 Glorioso, J C , Goms, W. F., Meaney, C. A., Fink, D J , and DeLuca, N. A
(1994) Gene transfer to brain using herpes simplex vnus vectors Ann Neural.
35, S28-S34.
32. Kwong, A. D. and Frenkel, N (1984) Herpes simplex vnus amphcon: effect of
size on replication of constructed defective genomes containing eukaryotic DNA
sequences J Virol 51, 595-603.
Watson, K., Stevens, J G , Cook, M. L , and Subak-Sharpe, J. H. ( 1980) Latency com-
33
petence of thirteen HSV-1 temperature-sensitive mutants. J Gen. Virol 49, 149-159.
34. Davison, M. J., Preston, V. G., and McGeoch, D. J. (1984) Determination of the
sequence alteration in the DNA of the herpes simplex virus type 1 temperature-
sensitive mutant ts K. J. Gen. Vzrol. 31,360-369.
Paterson, T. and Everett, R D. (1990). J. Gen Vu-o1 71, 1775-1783
35.
Matza, Y and Frenkel, N (1995). In preparation
36.
37. Graham, F. L. and van der Eb, A. J. (1973) A new technique for the assay of
mfectivity of human adenovirus 5 DNA Vzrologv 52,456-467
38. Gorman, C M , Merlmo, G. T., Willmgham, M. C., Pastan, I., and Howard, B. H.
(1982) The Rous sarcoma vnus long terminal repeat IS a strong promoter when
introduced into a variety of eukaryottc cells by DNA mediate transfection. Proc
Natl. Acad. Scz. USA 79,6777-6781.
Analyses of HSV Proteins
for Posttranslational Modifications
and Enzyme Functions
John A. Blaho and Bernard Roizman


1. Introduction
In order to Identify the nature of posttranslatlonal modlficatlons and enzyme
functions of herpes simplex vn-us 1 and 2 (HSV-1 and HSV-2) proteins, it is
necessary to apply both biochemical and genetic analyses. The experimental
methods described m this chapter have been applied to cells cultured in vitro
and infected with HSV-1 or to isolated nuclei of infected cells, to nuclear or
cytoplasmic fractions, and, in some instances, to purified extracts of infected
eukaryotic cells or of prokaryotic cells expressing a viral gene.

2. Materials
1 Chemicals and reagents: N,N™-dtallyltartardlamlde (DATD), endogylcosldase H,
galactosyltransferase, glutathione-agarose, tetrahydrofuran, phosphocreatme,
creatine kinase, NaF, Polygram CEL 300 PEI/UV plastic sheets, and DEAE-
cellulose were from Sigma Chemical Company (St Louis, MO) Neuramini-
dase are available from Boehringer Mannhelm (Indlanapohs, IN) Lectins and
the Vectastam kit are available from Vector Laboratories (Santa Cruz, CA)
Nitrocellulose membranes (type BA 83) and DEAE filters are available from
Schleicher and Scheull (Keene, NH). Pure poly(ADP-ribose) glycohydrolase
was a gift from Myron Jacobson (University of Kentucky). pGEM plasmids,
RNA polymerase, and rabbit reticulocyte lysate 1s available from Promega
(Madison, WI). Cap analog IS available from New England Biolabs (Beverly,
MA). Sephadex G-200 and poly(dT-dA) is available from Pharmacla (Uppsala,
Sweden). Glass fiber filters (type G/C F) are available from Whatman
(Maidstone, UK).
2. Radioactive materials. [32P]orthophosphate, [u˜˜P]ATP, [cx˜˜P]GTP, [8-3H]GTP,
From Methods fn Molecular Mechne, Vol 70 Herpes Sfmplex Vwus Protocols
Edtted by S M Brown and A R MacLean Humana Press Inc , Totowa, NJ

237
Blaho and Roizman
238

[2-3H]ATP, and C3H]CDP are avallable from Amersham (Arlington Heights, IL)
[35S]methlonme, [P32P]NAD, [3H]thymidme, [3H]UDP-galactose, [3H]TTP,
[3H]uracll, [3H]glucosamine, and [a32P]ATP are available from New England
Nuclear (Boston, MA)
Animal cells Human HEp-2, human HeLa, mouse Ltk-, and BHKtk- cells are ob-
tamed from the American Type Culture Collection (Bethesda, MD) All cell lines
are maintained m Dulbecco™s modified Eagle™s medium (DMEM) supplemented
with 5% newborn calf serum HSV-l(F) 1s a hmlted-passage clinical isolate of
HSV- 1 that IS temperature-sensitive and is used as the prototype HSV-1 strain (I)
Immunochemlcals* Rabbit prelmmune serum and polyclonal antisera against
specific viral polypeptides are produced by Josman Laboratories (Napa, CA)
Rabbit polyclonal antlbody to poly(ADP-nbose) 1s a gift from Mark Smulson
(Georgetown Umverslty) Monoclonal antibody H725 to ICP35 is a gift from
Lenore Perelra (UCSF) (2) Monoclonal antibody MAB30 to the HSV-2 large
subunit of rlbonucleotlde reductase should be requested from Laure Aurehan
(Johns Hopkins) Goat antirabbit lmmunoglobulm lmmunobeads are from Blo-
Rad (Duarte, CA) Protein A-sepharose CL4B 1s from Sigma

3. Methods
3.1. Denaturing (Sodium Dodecyl Sulfate [SDS])
DA TD Polyacrylamide Gels
Denaturing gel electrophoresls is one of the most convenient assays for the
detection of HSV protein modlficatlons and enzyme functions. The assay is based
on the observation that most modifications change the electrophoretlc mobility
of viral proteins in denaturing gels. In our laboratory, we exclusively use poly-
acrylamlde gels that are crosslinked with iV,N™-dlallyltartardlamide (DATD).
DATD has many advantages over his-acrylamlde m resolving glycosylated viral
protems (3). For example, these gels enable the resolution and detection of the
three posttranslatlonally modified forms of the ICP4 protein (4).
Use any standard vertical electrophoresis apparatus
Pour and set the followmg 9 3% separating gel using 0 5-mm spacers. 11 2 mL
water, 10 mL 0 14% ammonium persulfate (w:v), 5-mL 3M Tns-HCl (solution
A), pH 8 5, 13 mL DATD:acrylamlde (0 735% 28%, w.w.v) (solution C), 200
yL 20% SDS, 2 pL TEMED (see Note 1)
Pour and set the followmg stacking gel* 6 45 mL water, 12 mL 0 14% ammo-
mum persulfate, 3 mL solution B (1M Tns-HCl, pH 7 0, 20% SDS), 2.55 mL
solution C, 8 pL TEMED
To any solution of soluble protein (see Note 2), add l/3 vol disruption buffer (2
mL 55% sucrose, 4 mL 20% SDS, 2 mL IMTns-HCI, pH 7 0,2 mL 2-mercaptoe-
thanol, 25 mg bromophenol blue), boil for at least 60 s, load and run gel (at 0 5 mA/
cm for 16 h using 0.5 mm spacers) m the following running buffer: 6 g Tns-base,
28 8 glycme, 2 g SDS, and water up to 2 L (see Note 3).
239
Analyses of HSV Proteins
5 The most efficient electrical transfer of viral polypeptrdes from a 0 5-mm gel to mtro-
cellulose requires 120 V for 3 h at 4°C m running buffer containing 0.025% SDS
6 To do fluorography, the membranes are sprayed with En3Hance (NEN), dried,
and placed drrectly on X-ray film Exposures may take as long as 4 mo
3.2. Phosphoryla tion
Phosphate cycles on and off several viral protems during the course of pro-
ductive infection. Moreover, not all phosphoprotems are made at the same time
and, as a consequence, the identity of the labeled proteins may vary dependmg
on the time at which they are pulse labeled (5). The technique described in
the following was developed to label a proteins; it is included here because
these are the most difficult proteins to label (see Note 4). Labeling proteins
made later in infection is easily accomplished by delaying the time of addition
of the radioactive phosphate.
1. With few exceptions, HEp-2 cells are the cells of choice m our laboratory for
analyzing viral polypeptides (see Note 5).
2. Monolayer cultures contammg 4 x lo6 cells are mamtamed m Dulbecco™s Modi-
fied Eagle™s Minimal Essential Medium (DMEM) minus phosphate for 6 h.
3 Cells are then exposed to 10-20 PFU of HSV-l(F) per cell for 1 h
4 After absorption, the moculum is replaced with Dulbecco™s modified Eagle™s
minimal essential medium minus phosphate contammg 10 &I [32P,] (see Note 6)
and maintained for 6 h at 37°C.
5. The cells are harvested, washed (see Note 6) with phosphate-buffered saline
(PBS; 140 mMNaCl,3 mM KCI, 10 mMNa2HP04, 1.5 mMK2HP04 [pH 7.4]),
and either analyzed immediately (see Section 3.1.) or fractionated (see Section 3 6 )
6. Since the viral phosphoproteins are abundant and highly labeled, exposure of the
electrophoretically separated proteins to film for autoradtography is generally
relatively short
3.3. Poly(A DP-ribosyl)a tion
Poly(ADP-rtbosyl)atton is a posttranslational modification that takes place
in the nucleus. Modified proteins are thought to participate in DNA repair and
synthesis, as well as other cellular processes includmg differentiation, trans-
formation, and signal transductton. In vivo analysis of this modification has
relied on the use of specific antibodies and purified poly(ADP-ribose) glyco-
hydrolase, which specifically degrades the poly(ADP-ribose) chains (6).
Although interest in this modificatron is growing rapidly, these key reagents
remain commercrally unavailable and must be obtamed from researchers in
the field.
1 Infected cell nuclear extracts are prepared as described in Section 3 6
2. Nuclear extracts are denatured by boilmg for 60 s in 0.1% SDS.
240 Blaho and Roizman
3 After denaturation, SDS 1s removed by three serial acetone precipitations and
acidifications with 10% acetic acid To do this, 4-5 vol of cold (-2O™C) acetone
is added, the mixtures are stored at -70°C for 30 mm, prior to pelleting of the
proteins in a high-speed centrifuge After lyophkatlon, proteins are resuspended
m 1 vol of acid and the procedure is repeated
4 After the final lyophilization, the polypeptides are digested for 30 min at 37OC
with (0 1 U)-poly(ADP-nbose) glycohydrolase m 50 mMKP04, pH 7 5,50 mM
KCl, 0 1 mg/mL bovine serum albumin, and 10 mM P-mercaptoethanol (6)
5. The digestions are stopped by the addition of SDS to 0.1% and the protems are
electrophoretlcally separated m a denaturmg polyacrylamlde gel (see Section
3.1.), electrically transferred to mtrocellulose and reacted with antlbody to
poly(ADP-nbose) (see Note 7)
3.4. Glycosylation
The definitive proof that a viral gene product is glycosylated is to label the
protein with tritlated glucosamme (7). To do this, simply add 50 PCi of
[3H]glucosamme to the culture medium during infection and then harvest the
cells and analyze the proteins by fluorography, as described below (see Sec-
tion 3.4.1.). An alternative and more detailed approach to show glycosylation
is described in Section 3.4.1. This technique extends preexisting sugar chams
on proteins with labeled galactose using purified galacosyltransferase (G.C-F.,
personal commumcatlon); this enzyme ˜111 transfer galactose from UDP-
galactose to N-acetylglucosamme. Herpesvlral glycoproteins contain both
N-lmked and O-linked ohgosacchandes. Proteins containing N-linked sugars
are predicted to contain the sequence Asn-X-Thr/Ser. The technique for ldenti-
fying N-linked sugars is described in Section 3.4.2. and It mvolves cleaving the
sugar chains attached to proteins in cellular extracts with endoglycosidase H,
which degrades high mannose chains (7), and then testing for an altered elec-
trophoretic mobility of the protein of interest. The determination of O-linked
sugars utilizes the fact that N-acetylgalactosamme(GalNAc) is added to the
proteins very early in the processing (8). To do this, simply transfer infected
cell protein to nitrocellulose as described in Sections 3.1. and 3.3. and then
probe the blot with a lectin specific for GalNAc. We recommend the lectin
from Helixpomatza and the Vectastam system from Vector Labs as the devel-
oping reagents. Fmally, both N-linked and O-linked glycoproteins contain
slalic acid that may be removed by neuramimdase (8) This ts done exactly as
described m Section 3.4.2.,but substltutmgneuraminadase for endoglycosldase H.
3.4.7. Galactosyltransferase Activity
1. Prior to use, galactosyl transferase must be autogalactosylylated. In 500 pL, add 10
U of enzyme in 50 mA4 HEPES, pH 7.3,5 mMMnCl,, 1 mM P-mercaptoethanol,
1% aprotonin (v.v), and 0.4 mA4 UDP-galactose, and incubate at 37™C for 30 mm.
Analyses of HSV Proterns 241

2. Add 326 mg of ammonmm sulfate, incubate on ice 20 min, and precipitate the
enzyme by centrifugation (microfuge 30 mm at 4°C). The enzyme 1sresuspended
in 250 PL of 25 mM HEPES, pH 7 3,5 mA4 MnCl*, 50% glycerol, and stored at -
20°C in 50-I.˜L aliquots.
3 Infected cell extracts (5 pL), prepared as described in Section 3 4.1,) but without the
pulse-labeling, are added to reactions (50 uL) containmg 5 mM HEPES, pH 7.3, 7 5
mA4NaCI,2 5 mMMgCl,, O.l%NP40,2.5 mA4ATP, 5 mMgalactose, 1 uCi [3H]UDP-
galactose, 100 mU of autogalactosylylated transferase, and reacted at 37™C for 30 mm.
4 The reactions are terminated by boiling in 0 1% SDS prior to denaturing gel elec-
trophoresis (see Section 3.1.) and fluorography (see Note 8). To specifically tden-
tify viral proteins, tt is recommended that they be immunoprecipttated prior to
galactosylylation (see Note 9)
3.4.2. Endoglycosidase H Cleavage
1. Approximately 4 x lo6 infected cells are pulse-labeled with [3sS]methtonme as
follows At 5 h postmfectton, the media is replaced with DMEM contammg 50 uC1
[3sS]methionme, 1% newborn calf serum, and l/10 the normal amount methionme
and the cells are incubated for 2 h, prior to washing the cells three times with PBS
and replacing the media with DMEM containing 1% newborn calf serum.
2. At 16 h postmfection, the medium is removed, washed three times in PBS, and
the cells are lysed by the addmon of 300 uL PBS containing 1% NP40, 1% deoxy-
cholate, 10 PM tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK), and 10
yM a-tosyl-L-lysme chloromethyl ketone (TLCK) and briefly somcated. Thts
technique is standard for obtaining infected whole-cell extracts; for unlabeled
cells, simply omit the methionine label and use DMEM throughout
3. Cellular extract (10 pL) is reacted with endoglycosidase H (0.1 mu) m 50 uL of
100 mMsodium citrate (pH 5.5) and 100 mMP-mercaptoethanol at 37™C for 30 mm.
4. The reactions are terminated by boiling in 0.1% SDS prior to denaturing gel elec-
trophoresis (see Section 3 1 ). To specifically identify viral proteins, it is recom-
mended that they be immunoprecipitated prior to electrophoresis (see Note 9).
3.5. Sulfa tion
Some viral glycoproteins are sulfated (9). To analyze the sulfation of specific
viral gene products, add carrier-free H2[35S]0, (50 l.L%nL) directly to the infected
cell culture media at four hours postinfection. Cellular extracts are then made at 8
h postinfection and the labeled polypeptides are visuahzed in denaturing gels (see
Section 3.1.) following fluorography, as described m Section 3.4.
3.6. Myris tyiiza tion
At least two HSV-1 polypeptides have been shown to be myristylated, the
product of the Ut,l 1 gene (10) and the large subunit of HSV-2 ribonucleotide
reductase (II). Myristylated proteins contain a consensus modification
sequence, Gly-X-X-X-Ser/Thr, at their amino terminus. Identification of
Blaho and Roizman
242
myristylated viral gene products requires metabolic labeling of cells infected
with HSV. In general, either [3H]myristic acid or [3H]palmitic acid (125-250
l&t/mL) is simply added to the media of infected cells at 3 h postinfection and
the infected cells are labeled for as long as 20 h, prior to harvesting and analyz-
ing the viral polypepttdes in denaturing gels (see Section 3.1.) and by fluorog-
raphy (see Section 3.4.)
3.7. Isolation of Purified Infected Cell Nuclei
Since infected cell nuclei are extremely fragrle, care most be taken during
their tsolation. The followmg procedure uses HeLa cells (see Note 5) and pro-
vides a simple, htghly reproductble source of infected cell nuclei, as well as
nuclear extract proteins. However, this technique is not sufficient for the deter-
mmation of the mtracellular partitiomng of vu-al polypepttdes; more rigorous
techniques should be used for these cases.
1 Infected HeLa cells are harvested at 16-20 h post mfectton as follows The cells
are scraped mto the medrum, pelleted at 1000 rpm for 3 mm m a Beckman table-
top centrtfuge, washed once with 1 mL of-phosphate-buffered salme, resuspended
in 100 pL of 50 ti Trts-HCl, pH 7 5,5 mM MgCl,
2 The plasma membrane 1ssolubdtzed by the addition of 4 yL of 10% Nonidet P-40
and storage at 25°C for 5 mm.
3 The nuclei are separated from the cytoplasm by centrifugmg for 1 s m a Brmkman
microcentrifuge, washed m 100 pL 0 1% Nonidet P-40, 50 mM Trts-HCl (pH
7 5), 5 mA4MgC12, and centrifuged again for 1 s. The supernatant flutd from the
first pelleting may be saved and used as a crude cytoplasmtc extract
4. Nuclear protems are extracted followmg the addition of 100 pL of 20 mM Trts-
HCI, pH 8.0,420 mM NaCI, 1.5 mM MgCl,, 0 2 mM EDTA, 0.5 mA4 DTT, 0 5
mMPMSF, 25% glycerol, and incubation at 4°C for 30 mm, prior to pelleting of
the nuclear debrts and saving the supernatant fluid. The nuclear extract should be
stored m 20-yL ahquots at -70°C tf not assayed tmmedtately.
3.8. (ADP-ribosyljation in Nuclei
Section 3.3. describes a technique for detection of protein poly(ADP-
ribosyl)ated in infected cells. The addition of (ADP-ribose) to viral polypep-
tides in isolated nuclet is catalyzed by the cellular (ADP-rtbose) synthetase

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