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A single-cycle growth experiment involves infecting every cell m a mono-
layer and monitormg the growth during one round of repllcatlon. To do this,
cells are inoculated with an MO1 of 5 or 10 PFU/cell to ensure that every cell is
infected and the progress of the InfectIon 1s normally monltored durmg 24 h.
A multlcycle growth experiment amplifies the effect of any small Impair-
ment during several rounds of repllcatlon. In this case, cells are infected at a
low MOJ (usually 0.01-O 1 PFU/cell), and the mfectlon 1smonitored over 72 h.
The method for both 1sthe same with only the virus moculum and the pomts
of harvest varying.
1. Count a BHK 2 l/Cl 3 cell suspension and seed 35mm plates with 2 x 10” cells/
dish m 2 mL of ETClo Seed a single plate per time-point for each virus being
assayed Especially for large experiments where several viruses are being com-
pared, It 1s advisable to label the plates at this stage, smce it saves time when
mocularmg with vu-us
2 Incubate overmght at 37°C
3 Pour off the growth medium
4. Inoculate with virus, e.g ,2 x lo6 cells infected at a MO1 of 5 PFU/cell means an
moculum of 1 x 10™ PFU/plate. Therefore, it is necessary to dilute the virus to
1 x lo* PFU/mL and add 100 pL/plate Make sufficient diluted virus for all ofthe
time-points, so that the inocula gomg onto a series of plates IS fom a single virus
solution
5 Incubate at 37°C for 1 h to allow the virus to absorb.
6. Wash the plates with 2 mL of PBS/calf serum to remove any unabsorbed virus
7 Overlay the plates with 2 mL of ETC,,, (accuracy here 1svery important) This 1s
0 h on the time scale
8. Incubate at the appropriate temperature (normally 37°C)
9. Harvest the virus samples at the designated time-points by scrapmg the cell mono-
layer into the medium and transferring the suspension to a clearly labeled sterile
bottle that IS suitable for somcatlon (black cap vial)
Time-points for harvesting are arbitrary, but for a high MO1 (single-cycle)
Harland and Brown
8
experiment, 0-, 2-, 4-, 6-, 12-, and 24-h points are usual, and m some cases, 8-
and 16-h pomts may also be reqmred For a low-MO1 (multlcycle) experiment, 0-,
4-, 8-, 12-, 24-, 48-, and 72-h samples are usual.
10 Sonicate the samples thoroughly m a sombath to disrupt the cells, and release the
virus mto the medium. Store the samples at -70°C until they can be titrated.
11 Titrate the virus as described above, and calculate the titers at each time-pomt
Smce virus from 2 x IO6 cells IS harvested into 2 mL, the final titer per mllllllter
IS equivalent to Its titer per IO6 cells
12 Plot out the titers on a log graph scale with PFU/106 cells (log,,) on the y-axls
and time (m hours) on the x-axis

References
It ˜111 be obvious that we have not Included any references Since the step-
by-step procedures explained m this chapter are fundamental and have been m
operation for many years, we are assummg that it will be unnecessary for the
reader to requu-e more detalled mformatlon. Most of the basic references are m
papers published over 20 years ago!
2
HSV Entry and Spread
Christine A. MacLean


1. Introduction
This chapter deals with assays commonly used to follow herpes simplex
vtrus type 1 (HSV-1) entry mto and spread between cells m tissue culture
These are complex processes,known to involve several of the 20 or more HSV-
encoded membrane proteins (see refs. I and 2 for recent reviews). HSV entry
is mediated by a number of proteins on the surface of the virus particle. Recog-
nition of and bmding to target cells are known to involve at least three glyco-
protems-gB, gC, and gD. gC mediates the mittal mteractton with cells,
recognizing heparan sulfate proteoglycans on the cell surface. gB also interacts
with heparan sulfate proteoglycans, and can substitute for gC in gC negative
viruses. This initial, heparm-sensitive attachment to cells is relatively weak,
and is followed by a more stable attachment to cells, apparently mediated by
gD. Followmg attachment, the virus particle fuses with the cell membrane to
mediate entry. Fusion is known to require gB and gH/gL, and possibly also gD,
but their precise functions are uncertain. The roles of other virus-encoded mem-
brane proteins in entry are unclear, but it is possible that different protems may
be required for entry mto different cell types.
Following infection, spread of progeny virus m tissue culture occurs via
both the release of mature mfectious virus particles mto the extracellular
medium and the direct cell-to-cell spread of vn-us UL20 plays a role m mem-
brane trafficking events involved m the maturation and egress of vnus particles,
whereas several virus membrane proteins are probably involved m the mem-
brane fusion event required for cell-to-cell spread, mcludmg gB, gD, gE/gI, gH/
gL, and gK.
This chapter will describe assaysthat address vtrus entry, m terms of the
initial attachment of vnus to cells (adsorptron) and the subsequent fusion between
From Methods In Molecular M&one, Vol IO Herpes 8mplex Vwus Protocols
Edlted by S M Brown and A R MacLean Humana Press Inc , Totowa, NJ

9
10 A&Lean
the virus and cell membranes (penetration), and virus spread, m terms of both
mtracellular and extracellular vtrus yields (virus release) and vuus growth
under conditions that ltmtt extracellular spread of vu-us (cell-to-cell spread).
Detailed methodology IS provided for the assays used m our laboratory,
although some attempt will be made to refer to procedures used by others. The
assaysdescrtbed here involve the use of tissue-culture cells, the growth of virus
stocks, and extensive virus tttratton. The reader should therefore be familiar
wtth the procedures described m Chapter 1.

2. Materials
1 Cells We standardly use baby hamsterkidney 21 clone 13 (BHK C13) cells,
although any cell line permrsslve for HSV mfectlon should be suitable BHK
Cl3 cells are grown m ETClo (see step2), at 37™C m a humrdlfied atmosphere
contammg 5% (v/v) carbon droxtde Cell monolayers are seeded at 1 x 1O6cells/
35-mm Petrr dash or a well of a srx-well tray, or 2 x lo6 cells&O-mm Petrr dish,
20-24 h before use Cell monolayers are used when about 8690% confluent,
and we assume approx 2 x IO6 tells/35-mm dish
2 Media ETC,, Eagle™s medium supplemented with 10% newborn calf serum,
5% tryptose phosphate broth, 100 U/mL pemctllm, and 100 mg/mL streptomycm
EC,/EC, Eagle™s medium supplemented with antrbtotlcs, and either 5 or 2%
newborn calf serum, respecttvely
Emet/SC, Eagle™s medium contammg one-fifth the normal concentration of
methronme and 2% newborn calf serum
MC,/MC, Eagle™s medium supplemented with antibiotics, 1 5% carboxy-
methylcellulose, and either 5 or 2% newborn calf :rum, respectively
EHu. Eagle™s medium supplemented with antrbiotrcs and 10% pooled human
serum
3 PBS 170 mA4 NaCl, 3 4 mA4 KCI, 10 mM Na2HP0,, 1 8 mA4 KH,PO,, supple-
mented with 6 8 mA4 CaCl, and 4.9 mA4 MgCl,
4 Citrate buffer. 40 mA4 citric acid, 135 n-u!4 NaCl, 10 mA4 KCl, pH 3 0

3. Methods
The methods described here are based on the use of HSV- 1 strain 17syn+ m
BHK C 13 cells. It 1simportant to remember that growth charactertsttcs and/or
kinetics of entry may differ when using different strains of HSV-1 or different
cell types
Before undertaking these procedures, consult local regulations for the safe
handling of HSV. We generally work with the vu-us on the bench or m a class
II biological safety cabinet, and inactivate all waste either by steeping over-
night in a 1% solution of vircon or chloros, or by autoclavmg. The most obvt-
ous risks from HSV are from splashesto the eye, or contact with areas of broken
skin (e.g., cuts, eczema). Because of the large numbers of infected monolayers
11
HSV Entry and Spread

that may need to be handled at once m some of the procedures below, rt IS
rather easy to be a little careless.
Unless otherwise stated, mampulatrons are carried out at room temperature,
as raprdly as posstble. Seed stocks of vn-us or cells are generally handled m a
biological safety cabinet using sterile technique, but all other procedures are
conducted on the bench using good microbiologtcal practice.
3.1. Adsorption of Radiolabeled Virions to Cells
This assay measures the proportton of total virus particles that bmd to cells
with time. Purified radrolabeled virus particles are allowed to bmd to cells for
given periods of time, the cells washed extensively to remove unbound virus,
and the cells then lysed and the amount of bound radiolabel measured. The
radrolabeled virrons used m these experiments should be free from contamr-
nating cell membranes or debris. We generally use 35S-methronme-labeled vu+
ons, purified by Ficoll gradtent centrrfugation (3), but other radiolabels (e g.,
3H-thymidme) and/or different gradient purification procedures are also suit-
able (e.g., see refs. 4-6). We generally find that around 15-20% of radrola-
beled virrons bind to cells wrthm 60-90 mm at 37°C.
3 1.7. Step I: Preparation of Radiolabeled Virlons
1 Infect N-90% confluent cell monolayers In 80-02 roller bottles (assume around
2 x I O8BHK C 13 cells/roller bottle) at 0 00 1 PFU/cell m EC, at 3 1“C (see Note 1).
2 Once plaques become vlsrble (12-24 h pi), remove the medium and wash the
cells twice with, and subsequently mamtam them m, Emet/SC,. Approximately
2-4 h later, add 35S-methlonme (Amersham, SA >lOOO Ct/mmol) to a final con-
centratron IO-20 mCr/mL. A total volume of 20 mL 1s usually sufficient, but
ensure that the cells do not dry out.
3 Once all the cells appear rounded, but still attached to the roller bottles (3-4 d pi),
carefully remove the culture supernatant (avotd detachmg the cells), and pellet
the cell debrts by centrifuging the supernatant m a Fisons coolspm centrifuge (or
equivalent) at 2000 rpm for 30 mm at 4°C. Care should be taken to avotd ceil
debris in virus stocks membrane fragments can copurtfy wrth vnions on Ficoll
gradients, and excessive cell debris can trap/sequester vnus m large aggregates,
resulting m low yields of purified vmons
4. Again, carefully remove the supematant, and then pellet the virus by centrrfuga-
tion at 12,000 t-pm for 2 h at 4°C m a Sorvall GSA rotor. Remove all the superna-
tant, add 1 mL of Eagle™s medmm wrthout phenol red, and then very gently scrape
the vtrus pellet mto the medmm and allow the vuus to resuspend overnight at
4°C Vnions should be handled very gently at all stages to avoid damage to the
virus envelopes.
5. Prepare 35-mL contmuous 5-15% Ftcoll gradtents (Ftcoll400, m Eagle™s medium
without phenol red) in transparent centrtfuge tubes that can be easily pterced by a
syrmge needle, and cool on ice or at 4°C. We generally use Beckman Ultra-
12 Maciean
clearTM centrtfuge tubes Gently prpet the vrrton suspension until homogeneous,
layer It onto the Ficoll gradtents, and centrifuge for 2 h at 12,000 rpm, m a Sorvall
AH629 rotor at 4°C The number of roller bottles of virus loaded per gradient
will depend on the yield of vnus expected For 17syn+ wild-type, vrrus from 2-5
roller bottles would normally be loaded onto a single 35-mL Ftcoll gradient
(approx l-5 x lo9 PFU at the end of step 4)
6 Vlsuahze the vrrron band under a light beam (see ref 3 and Note 2) Carefully
remove the vmon band by side puncture, using a 5-mL syringe and broad (18/19G)
gage needle, dtlute the vnus m Eagle™s medium without phenol red, and then
recover the vnus by centrrfugatron at 2 1,000 r-pm for 16 h in a Sorvall AH629
rotor at 4°C.
7 The virus pellet should appear as an opaque halo at the base of the centrifuge tube
Remove the supernatant carefully, and dry the tube with a tissue to remove excess
lrqurd (avoid drsruptmg the pellet) Add 500 mL ETC,s, gently scrape the vu-us
mto the medium, and allow rt to resuspend overnight at 4™C
8 Very gently, resuspend the vtrus preparation until homogeneous, using an
Eppendorf prpet, and then determine
a. The quality of the preparation, by electron mtcroscopy,
b Particle numbers (parttcles/mL), by electron microscopy,
c The virus titer (PFU/mL), and
d Radtoacttvrty (counts/mm/mL), by liquid scmttllatton counting
9 Vrrrons can be stored at -70°C until use

3.7.2. Step 2: Adsorption of Radrolabeled Viaons to Cells
1 Remove the medium from 90-100% confluent monolayers m six-well trays (see
Note 3), and add ETCIO contammg 1% BSA for 30-60 min at 37°C This step IS
Included to reduce nonspecific bmdmg of vrrrons, although in practice, we find
ltttle dtfference If this step 1somitted
2. Dilute the radtolabeled vu-tons m prewarmed ETCIo. The amount of virus added
will vary m terms of counts. When comparmg different vrruses, we sun to use
comparable particle numbers, while trymg to keep the counts wlthm the range
l&l000 cpm/pL (20-200,000 cpm/well). This is usually lo™-lo3 particles/cell,
and does not reach saturation bmdmg (see refs 7 and 8)
3. Remove the blocking medium Smce volume influences adsorptton rates, all wells
should be dramed thoroughly
4 Add 200 pL prewarmed vnus/well, m trrplrcate for each time-pomt, plating drffer-
ent time-points on separate trays (since shaking will affect the rate of adsorptron)
Plate all vu-uses for each time-point together. the first set added should be the last
time-point harvested, whereas the last set added should be the first time-point har-
vested (see Note 4) Transfer monolayers to 37°C; this represents 0 time
5 At the relevant time-points, remove the vnus supernatant using an Eppendorf
ptpet, and transfer to a scmtrllation vial
6 Wash the cells three times with 1 mL PBS, shaking the trays for 5-10 s each time,
and transfer each wash to a scmtrllatron vial
HSV Entry and Spread 13

7 Harvest the cells (and bound virus) by scrapmg into 300 pL PBS/l% (v/v) SDS,
and transfer this to a scmtlllatlon vial.
8 Add 4 mL EcoscintTM-A (National Diagnostics, Atlanta, GA) to each vial, vortex
briefly, and count each sample m a liquid scmtlllation counter for 1 min
9. The percentage of bound virus at each time-point is calculated from*
(cpm bound/total recoverable counts) x 100 (1)
where cpm bound = cpm m cell harvest and total recoverable counts = (cpm m
virus supernatant + cpm in washesl/2/3 + cpm bound).
3.2. Adsorption of Infectious Virus to Cells
In this assay, Virus is allowed to attach to cells for given periods of ttme, the
cells washed extensively to remove unbound virus, and the amount of bound
wus then measured in terms of subsequent plaque formation. Either crude
vn-us preparations or gradient purified vtrlons can be used as input virus.
1. Remove the medmm from 90-l 00% confluent monolayers m six-well trays, and
drain all wells thoroughly
2 Briefly somcate vu-us stocks before use (30-60 s), and dilute the virus m
prewarmed ETClo to 150-200 PFU/200 pL (see Notes 5 and 6).
3. Add 200 pL vnus/well, m trlphcate for each time-point, plating different time-points
on separate trays Plate all vuuses for each time-point together the first set added
should represent the last time-point handled, whereas the last set added should be the
first time-point harvested. Transfer monolayers to 37”C, this represents 0 tune.
4 At relevant time-pomts, remove the vu-us using an Eppendorf pipet, and discard
5. Wash the cells three times with 2 mL PBS, shaking the trays for S-10 s each time
6 Dram all wells thoroughly Overlay the monolayers with 2 5 mL MC5 (or MC2 if
the cells are very confluent), and incubate at 37°C until plaques are clearly vls-
ible (usually 2 d pi).
7 Stain the cells by adding l-2 mL Glemsa stain, leaving the cells at room tem-
perature for 2-24 h before washmg.
8 Count the plaques under an Inverted microscope.
9. The percentage of infectious vu-us binding to cells at a given time 1s calculated
from
(avg. no. of PFU at given time/avg. no. of PFU at peak or final time-point) x 100 (2)

3.3. Modifications of the Adsorption Assays
Sections 3.1. and 3.2. describe adsorption of vu-us at 37”C, under standard
conditions. It is obviously possible to modify these procedures m a number of
ways: e.g., to wash m the presence of reagents that may interfere with binding
(e.g., heparin) or to slow adsorption by incubating at 4°C (see refs. 9 and ZO).
To carry out these assays at 4”C, both cells and vn-us should be pre-
cooled before addltlon of vu-us to cells, and the experiments carried out in a
14 MacLean

4°C cold room. We do not cool the cells on ice, as described by others, since
we find that our BHK Cl3 cells do not survtve such treatment well. Time-
points are washed at 4”C, before transferring to room temperature for harvest-
mg (Section 3.1.) or addition of prewarmed MC5 (Section 3 2.). A reasonable
time course would be 0, 15, 30, 45, 60, 90, 120, 180, and 240 mm after vnus
addition. We find BHK C I3 cell monolayers do not survrve longer periods at 4°C
3.4. Penetration
Vnus penetration IS measured as the rate at which attached virus becomes
reststant to mactrvatron by low pH (Zi,ZZ). Vtrus 1s bound to cells at 4”C, a
temperature at which very little penetration should occur. Cells are then shifted

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