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2.2. Detection of Proteins Present in Small Amounts
Herpesvirus proteins present in minute amounts may not be detected unless
they are tagged by insertion of an epitope recognized by a powerful mono-
clonal antibody. This technique was used successfully to identify the product
from gene UL37 (63), and to separate the otherwise indistinguishable proteins
Haarr and Lange/and
706
dertved from genes UL26 and UL26.5 (41). In both cases the commercially
available antibody was directed against a CMV epitope
A protein accumulating in a certain compartment of the cell, or in a struc-
tural component of the vnion may barely be detectable m extracts from whole
cells but quite evident m a particular cell fraction or m isolated virrons.
2.3. Mapping of Proteins to Their Encoding Genes
One of the first successful strategies to map HSV proteins used intertypic
recombinants between HSV-1 and HSV-2 and took advantage of the fact that
homologous proteins from the two types migrate slightly different m SDS-
PAGE gels (6465). Several [35S]-methionlne-labeled proteins, phosphopro-
tems and glycoproteins were thus assigned to restricted regions of the HSV
genome. Temperature sensitive mutants as well as other mutants are important
tools for more precise localization. Virus mutants deficient m essential genes,
like those encoding gB, gD, gH, and gL can only be propagated in cell lines
carrying the appropriate genes
Marker rescue experiments are cotransfections with specific DNA fragments
and genomic DNA carrying the defect under investigation. The rescuing fragment
contains the gene (66). Analysis with mtertypic recombmants and marker rescue
are still powerful methods for obtaining an initial and more or lesscrude mapping,
for example of genes encoding viral ligands mvolved in attachment (67,68).
The gene technology available today allows almost unlimited manipulations
with any of the HSV genes, and thus a detailed functional analysis of protein
domains. Transfection with plasmlds contammg the normal or modified HSV
gene of interest has been used successfully to study functional regions in gC
and gD (53,69), but gene expression from the plasmtd is transient. It is generally
accepted that the best way of studying a manipulated gene is to have it inserted
into the vn-us and then analyze the effects. The plasmid contammg the HSV
gene should have HSV-specific flanking sequences for recombmatron and
insertion at the correct position in the virus genome. This technique has been
useful for studies of gC (70).
Alternative methods for correlating a gene to its product include hybrid-
arrested or hybrid-selected in vitro translation, generation of antibodies to
ohgopepttdes synthesized according to predicted sequences in open reading
frames, and finally, sequencing of the ammo acids in isolated proteins.
When hybridizing a specific DNA fragment to a mixture of different RNA
molecules, those molecules bound to DNA will be inactive in m vitro transla-
tion, but reactivated by denaturing the hybrid The disappearance and reap-
pearance of a given protein can be detected then by SDS-PAGE or 2D gel
electrophoresis. Three different protein products from the thymidme kinase
gene were detected by this technique (71,72). The gene encoding the 65 kDa
Analysis of USV Polypeptides 107

DNA-binding protein was identified by in vitro translation of mRNA isolated
by hybrid selection using a specific DNA fragment (73).
Antibodies against synthetic peptides are directed against specific proteins
or protein regions, and can be used m search for previously unidentified pro-
teins. The products of gene UL47 were identified by this method (74). Alterna-
tively, the gene encoding a potential protein can be cloned into an expression
vector and the protein then isolated for immunization. To facilitate protein pu-
rtfication it may be useful to Introduce a tag of histidmes (75) or make a fusion
protein with glutathione-Stransferase (31,761 such that the expressedprotein can
bmd to columns of Nt2+-mtrtloacetic acid agarose or glutathione, respectively.
Sequencing of proteins isolated by 2D gel electrohorests was origmally
descrtbed by Vandekerchove et al. (77) and by Aebersold et al. (78). Proteins
are blotted onto membranes before bemg cut out for further analysis. Mem-
branes of polyvmylidene fluoride (PVDF), or a second generation thereof (˜79)
seem to be best. A problem with this type of microsequencmg is N-termmal
blocking that may require cleavage of the protein by CNBr (80) or with a pro-
tease such that internal fragments can be separated by HPLC and then
sequenced. At least some of the blockmg seems to occur during electrophore-
sis, fixation, or destaining of the gel owing to impurities in the reagents There-
fore it is recommended to use pure chemicals during all those steps.
2.4. Localization of Proteins
Within infected Cells and in the Virions
To localize a protein to a certain compartment in the infected cell or m the
virion the various components usually will have to be separated. Several meth-
ods are available for fractionation of cells. A simple procedure for separation
of cytoplasmtc and nuclear fractions 1sgiven in the followmg (Section 3.2.).
Both mature and immature virus capsids are isolated by gradient centrtfuga-
tion of material from nuclei of infected cells (81,82). Ltght particles consrstmg
of tegument and envelope, but devoid of nucleocapsid, are separated from
mature virions of HSV-1 by centrifugation m a Ficoll gradient (83). Distmc-
tion between localizatton m the tegument and in the envelope has to be done by
chemical stripping of the particle or by specific labeling of surface proteins, or
by a combination of these methods. Stripping is usually performed by incuba-
tion with 1% NP-40 in the presence of salt followed by centrlfugation in an
ultracentrlfuge to spin down the remaining particles. Complete solubtlization
of envelope and tegument by this treatment requn-es a high concentration of
sodium chloride (1M) Treatment for longer than 15 mm at 4°C under these
conditions may result m precipitation of already solubihzed proteins.
Iodination was used approx 10 yr ago to detect proteins on the surface of the
HSV-1 capsid (84). When studying envelope proteins the reagents should not
Haarr and Lange/and
108
penetrate the membrane. For this purpose the water msoluble compound
1,3,4,6-tetrachloro-3a-6a-diphenylglucolurrl (Iodo-gen) may be useful m com-
bmatton with todme (85). It reacts prtmartly wrth tyrosme residues. Iodo-gen
sticks to the inside of the test tube such that the reaction 1sstopped by transfer-
rmg the mixture to another tube. An alternattve method 1s btotmylatton with
sulfosuccmtmrdobtotm (sulfo-NHS-biotin), which is a water soluble reagent
unable to penetrate membranes. Btotmylated proteins are then detected by bmd-
mg of streptavldin ([˜251]-labeled or lmked to an enzyme). In our hands certain
proteins(e.g., gC) are more efficiently labeled by the former and others (e.g.,
gE) by the latter method.
The vtrton envelope is fragile and may rupture easily as a result of cen-
trtfugation, other mechamcal treatments and freezing and thawing (86) To
ensure a mmrmum of damaged parttcles we do the iodmatton on freshly har-
vested virus, before purification in a gradient. A result of such an experiment
1sshown m Fig. 6.
3. Methods
In the followmg section we describe only the procedures with which we
have some experience. Unless otherwise stated we use baby hamster kidney
(BHK) cells, Eagle™s mmtmum essential medium, newborn calf serum, and
35-mm plastic dishes for cell culture
3.1. Radioisotope Labeling
3.1. I. infected Cells
3.1 1 1 LABELING WITH [35S]-M˜˜˜˜˜˜˜˜˜
Almost confluent monolayers of cells m 35-mm dishes are incubated with
10-20 PFU/cell. Virus is removed after 1 h (zero time) and the infected cells
washed twice with medium containing one-fifth the normal concentratron of
methionme and supplemented with 2% serum. The mfectton then 1sallowed to
proceed for 0.5-l h to reduce synthesis of cellular proteins before addition of
[35S]-methionme to a final concentratton of W-250 pCt/mL, and incubation
continued for the appropriate period of time.
Labeling in pulse-chase experiments 1sperformed after washing and replac-
mg the medium with phosphate-buffered salme (PBS). At the end of the pulse
(1O-30 min), the label 1sremoved and the cells washed three times with medium.
Some cells are harvested (pulse) and others incubated further in medium for chase.
3.1.1.2. LABELING WITH [32P,]

Prior to labeling the cells are incubated 2-3 h in medium containing one-
tenth (10 pi™@ the normal concentratron of phosphate and serum dialyzed
109
Analysis of HSV Polypeptides




69 - 1




46 - /




30 - I




21.5- I




Fig. 6. Two-dimensional gel electrophoresis of [ ˜2SI]-1abeled surface proteins
of HSV-1 virions. HSV-1 virions were isolated and iodinated by the Iodo-gen
method as described in Section 3.4. before further purification in a Ficoll gradient
(see Section 3.3.). The slab gel contained 5-12.5% polyacrylamide crosslinked
with BIS. M, markers were run in a separate lane. Labeled proteins are indicated
by arrows.


against 0.9% NaCl. Phosphate starving often is started in uninfected cells, and
labels added after infection. For 2D gel electrophoresis, 50-l 50 pCi is used/ml.
3.1 .I .3. LABELINGWITH[3H]-M˜˜˜˜˜˜
In the procedure described by Hope et al. (87) the medium is supplemented
with 2% serum and 100 $i [3H] mannose/mL is used. The ethanol in which
the mannose is supplied is removed before use. Pulse-chase is performed as
for [35S]-methionine labeling.
3.1.2. Virions
3.1.2.1. LABELINGWITH[35S]-M˜˜˜˜˜˜˜˜˜
Subconfluent cells in roller bottles are infected with 0.02 PFUkell in a total
volume of 20 mL. One mCi of [35S]-methionine is then added per bottle, and 5-
mL portions of normal medium added at intervals such that the final volume
does not exceed 40 mL.
Haarr and Langeland
770
3 .1 .2 2 . LABELING WITH [32P-P,I
Cells in roller bottles areinfected asdescribed(seeSection3.1.2.1.)and incubated
for 5 h m medium with reduced phosphateand 10% dialyzed serum,as described m
Section3.1.1.2.One mCi [32P,] addedand ahquotesofphosphate-reduced medium
is
added at intervals suchthat the final volume does not exceed40 mL.
3.2. Preparation of Nuclear and Cytoplasmic Fractions
Infected cells are harvested by scraping with a rubber poltceman and centri-
fugation at 85Og for 5 mm. The pellet is resuspended and incubated for 5-10
min on ice m a solution containing 0 5% NP-40,0.25% sodium deoxycholate,
10 n-& Tris-HCl, pH 8.0, and 10 mM EDTA. After centrifugatton for 5 mm at
850-l OOOg nuclei are in the pellet. The centrtfugations were performed in
the
a Kubota 8700 centrifuge using the RS 3000/6 rotor.
3.3. Purification of Virions for Analysis of Structural Proteins
Harvesting starts when the cells are detached. Nuclei are spun down at 85Og
for 10 mm, m a Kubota centrifuge, and cellular debris by a subsequent cen-
trifugation m a Sorvall SS 34 rotor for 5 min at 14,500g. The supernatant IS
then centrifuged m the same rotor for 1 h and the pellet resuspended by leaving
it overnight m a small volume of medium without mechanical agitation.
Further purification m a Ftcoll gradient is describedby Sztlagyand Cunnmgham
(83). Briefly the resuspendedvnus pellet from four roller bottles is layered on a
preformed gradient of 5-l 5% Ftcoll400 dissolved m culture medmm without phe-
nol red, and centrifugatron performed for 2 h at 33,000g m a Beckman SW 41 TI
rotor. The upper band consistsof light particles devoid of nucleocapstds,and the
lower band contams the vnions. Alternattvely, a preformed gradient of 20-40%
Nycodenz dissolved m 1 mMphosphate buffer (pH 7 4) can be used, and cent&$
gation performed in a Beckman SW 4 1 TI rotor for 2 h at 68,000g. Light particles
and vmons are present in the first and second band, respectively, from the top.
In our hands the recovery of mfectious virus is approximately the same from
these gradients, and superior to that obtained from other gradients.
Virions and light particles are concentratedbefore electrophoresisby spmnmg m
rotor TLA 120.2 in a Beckman TLX table top ultracentrmtge for 15mm at 157,OOOg
or for 3 h at 279,OOOgrespectively. All proceduresare carrted out at 4°C.
3.4. lo&nation of Virion Proteins
The method 1s adopted from that described by Markwell and Fox (8.5).
Briefly, 2 mg Iodo-gen is dissolved in 2 mL chloroform. Allquotes of 25-75 l,,tL
are transferred to Eppendorf tubes, dried, and then stored at-20°C under nitro-
gen. [125I] (200 @i) is then added to each tube and subsequently 30-300 pL
vn-us preparation. Incubation with regular shaking 1sperformed for 10-l 5 min
Analysis of HSV Polypeptrdes 111

at O+C, and the reaction stopped by transferring the solution to another tube
without Iodo-gen. To restrict labeling as much as possible to surface molecules
the iodmation should be performed before loading virus on the gradient. (Some
vnus membranes may disrupt during gradient centifugation.)
3.5. Biotinylation of Virion Proteins
Virions are purified as described in Section 3.3. and selective labelmg of
surface proteins obtained as described above (see Section 3.4.). When labelmg
after gradient centrifugation it is recommended to concentrate the virus by add-
ing 1 vol of 100 mM Sorensens phosphate buffer pH 8.0, followed by centrifu-
gation in rotor TLA 120.2 in a Beckman TLX table top ultracentrifuge for 15
mm at 157,000g. The pellet is allowed to resuspend m 100 uL of the same
phosphate buffer.
To 100 PL of vnus preparation IS added an equal volume of sulfo-NHS-
biotin 0.4 mg/mL (final concentration 0.2 mg/mL). After incubation for 5 mm
the reaction is stopped by addition of 7 pL 1Mcitrrc acid, followed by 100 pL
67 mM Sorensens phosphate buffer pH 6.1. All steps are performed at @-4”C
Vnus is then spun down m the table top ultracentrifuge as described and sub-
jected to polyacrylamtde gel electrophoresis and Western blotting onto a nitro-
cellulose filter. The filter is mcubated overmght m PBS containing 0.05%
Tween-20, which is then removed and a new portion containing 0 3-0.4 pCi
[ 1251]-streptavidm/mL added (total volume approx 8 mL). Incubation 1scarried
out for 2 h at 37°C before removing the radioactive solution and extensive
washing for 8-12 h using a shaker at room temperature, PBS/Tween as above,
and changing the solution mmimally six times. The filter finally is rinsed m
HZO, dried, and exposed to film. Sulfo-NHS-biotin IS stored dry at 4°C under
nitrogen vapor, and the solution is made immediately before use.
3.6. In Vitro Translation and Hybrid Arrest
The protocol developed by C. M. Preston 1sused (88). Dried DNA is dis-
solved m a solution of 80 PL,formamide and 6 pL 1M PIPES (piperazine-N,N-
his-2-ethanesulfonic acid) (pH 7.4) and incubated at 90°C for 5 mm Then
follows sequential addition of 6 PL RNA (approx 6 ug) and 8 pL SMNaCl, and
mixing by pipeting up and down, before transferring the tube to 56°C and mcu-
bation for 1 h. The reaction mixture is then transferred to Ice and the nucleic
acids precipitated by addition of 1 mL ethanol and 0.4 mL isopropanol.
The nucleic acids are recovered by centrrfugation, the pellet washed with
ethanol and allowed to dry thoroughly before dissolving m 7 uL H20. An ali-
quot of 2.5 PL of the solution is stored on me, and is the hybridized
sample. The rest of the solution is heated at 100°C for 60 s to denature the
hybrid, transferred quickly to ice and an ahquot of 2.5 pL used for further
712 Haarr and Lange/and

reactions. 22.5 uL of an m vitro translation mixture contaming 20 PL
micrococcal nuclease-treated rabbtt reticulocyte lysate and 2.5 PL (2.5
$1) of [35S]-methionine are then added to each tube. After incubation at
30°C for 60 min the reaction is stopped by adding 25 yL of a solution
contammg 100 mM EDTA (pH 7.5) and 500 pg/mL of RNase A, and
incubation continued for 15 mm.
When translating mRNA directly, without hybridization and denaturatton,
approx 5 pg is used per tube.
3.7.20 Gel Electrophoresis

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