4. Determine the EDso concentration by interpolation of the graphed data.
This experiment should be performed in duplicate and on several different
occasions to test the reliability of the ED,, obtained. The EDSodrug concentra-
tion determined for one cell lme may differ from that obtained with another if
the uptake of the drug was different. A therapeutic index, derived from the
IC50/ED50 ratio can be computed. Promising antiviral agents are usually those
with a therapeutic index of > 10.
3.2.2. infectious Virus Meld Drug Dose-Response Curves
Plotting the dimmishmg infectious vu-us yields from HSV-infected cell cul-
tures treated with mcreasmg concentrations of drug provides another useful
way of measuring antiviral activity. Drug dose-response curves allow the
kinetics of antiviral activrty to be studied, yreldmg more mformatron than can
be obtained from the EDs0 value alone.
1. Infect setsof cell monolayerswith HSV at an MO1of 5 PFU/cell, and allow 1 h
at 37В°C to absorb the vuus Remove unbound vn-usby washing the cell layers
three times wtth PBS + 5% calf serum
2. Overlay the infected cells with drug-free tissue-culture medium or medium con-
taining increasing concentrations of drug, and incubated at 37В°C for 24 h The
drug concentrations selected for antiviral studies should have been demonstrated
(from Section 3 1 1 ) to be nontoxic for the cell lme used
3. Harvest the total infectious virus yield by scraping the infected cells into the
4. Sonicatethe infected-cell suspension a few minutes,to break the cell mem-
branes and release the mtracellular vu-us Care must be taken to keep the sonibath
cool, since HSV is not stable at temperatures >4OвЂќC Alternatively, release the
intracellular HSV by three cycles of freeze-thaw In this procedure, the harvest
from step 3 1s rapidly frozen by plunging mto a dry-ice bath (dry-ice granules
bathed in ethanol andallowed to equilibrate) andthen quickly thawedby unmer-
sion in a 37В°C water bath Care should be taken when handling dry-ice/ethanol
5. Titratethevnusyieldsfromeachculture, determine infectivity by plaqueassay
6. Plot the virus/drug dose-response curve The dose-response kinetics are character-
ized by: the concentration that gives the first reduction in infectious virus yield; the
rate at which infectivtty is lost with increasing drug concentration (I e , the slope of
the curve); the greatestreduction in vnus infectivity attained,andwhether or not
the curve plateaus. These important features assist selection of a single (or stan-
dard) dose of the drug, whtch may be used in subsequent experiments
If the compound is suspected to be active at a very early stage m the virus
replication cycle (e.g., virus entry or uncoating--see Section 3.4.), it will be
necessary to pretreat the cells with the drug before infection and to ensure that
the drug is present throughout the vn-us replication cycle, including the vtrus
The infectious virus yield/dose-response experiment should be repeated sev-
eral times to confirm the kinetics of the curves. The dose-response kinetics
may differ with different cell lmes, cell types, or if exponentially growing or
resting cell cultures (see Note 2) were used.
Separation of the vuus yields mto cell-associated and cell-released fractions
(see Note 3) could provide additional information indicating whether drug
treatment affects the release of mfectious progeny vu-ions (28,31) Estimation
Anti-HSV Activity of Antiviral Agents 395
of the number of virus particles present in the cell-associated and cell-released
fractions by negative-stam electron microscopy allows particle/PFU ratios to
be calculated, providing additional information relevant to maturation and
release of virus particles produced m the presence of the drug.
3.3. investigating the Genetic Target of the Drug
The major goal in the investigatton of the antiviral mechanism is to identify
the vuus gene product that is the target of the drug.
3.3.1. Single-Round Selection of Drug-Resistant Vms
To identify the gene that encodes the protein target of the drug, it is first
necessary to isolate drug-resistant virus mutants. This should be possible if the
target is the product of a single HSV gene, but may be more difficult, if not
impossible, if overlappmg genes are involved. There are two ways m which
such mutants might be isolated: first, by multiple cycles of selection, where
HSV is initially passaged at a low (semipermissive) concentration of drug,
which is gradually increased at each subsequent passage, second, by a single
round of selection in the presence of a concentration of drug that inhibits plaque
formation by about 99.999%.
Multiple-cycle selection will show, relatively quickly, whether drug resistance
is possible and indicates that at least a component of the antiviral activity is vnus-
specific. However, the contmuous selective pressure placed on the vnus to increase
drug resistance with each successive passage is likely to result in an accumulation
of point mutations, at best within the target gene and, at worst, within a number of
different genes whose products interact with each other. In the latter case, it may be
difficult, if not impossible, to map and identify the genes involved.
Single-round selection has the advantage that the stock of drug-resistant
virus so generated is likely to differ from the wild-type virus by only a single
mutational event and so is suitable for further genetic analysis.
1. Infect 100-200 cell monolayers (on 50-mm tissue-culture dishes) with HSV at a
multpihcity of infection (MOI) of lOOO/PFU/dish After the absorption period,
wash the monolayers to remove unbound vnus, and then overlay with culture
medium containing, 1 25% methylcellulose, and a concentration of drag suffi-
cient to inhibit plaque formation by about 99 999%. Incubate the cultures at 37OC
for 48 h or until plaques develop.
2. Pick virus plaques, that are well separated from each other, into 0 5 mL PBS +
5% calf serum and sonicate to release the intracellular V˜I.K
3. Titrate the virus yield from the plaque, and determine the infectivity nr the
absence and presence of the drug (concentration as before). Select and pick
second-generation plaques from the drug-free cultures of those plaques that have
an approximately equal titer in the absence and presence of the drag.
4 Continue this process for at least three generations, always ptckmg from the drng-
free cultures and monitormg drug resistance with the drug-treated culture. From
time to time, it may be necessary to boost the titers of the plaque-produced virus
by a passage m the absence of the drug. Take care to ensure that the final stocks
of drug-resrstant vn-uses produced are not clonally related (1.e , they were not
picked from the sameplate).
3.3.2. Mapping the Drug Resistance Gene
Once drug-resistant virus stocks have been produced, tt becomes possible to
identify the genetic locus specifying drug resrstance by standard marker-rescue
techniques (see Chapter 3). The salient points are given below.
1. Purify DNA from a preparation of the drug-resistant virus particles (see DNA
extraction protocol; Chapter 3)
2. Cotransfect intact wild-type HSV-DNA and individual purified DNA fragments,
derrved from the drug-resrstant vn-us, mto duplicate cell monolayers (see vn-us
DNA transfections protocols; Chapter 3). Then overlay one set of monolayers
with drug-free tissue-culture medium and the other with medium containing the
concentratton of drug used m the selectton of the drug-resistant vnus.
3 Virus plaques that grow on the drug-treated cultures arise from recombinant
viruses, which have incorporated the drug resistance marker contained within the
cotransfected DNA fragment. These plaques should yield approxtmately equal
titers m the presence and absence of the drug.
4 Several controls are required. drug-free transfected cultures to indicate whether
transfection has been successful, wild-type HSV-DNA alone to exclude drug
resistance m the wild-type DNA stock used, intact drug-resistant vnus DNA to
confirm the drug resistant phenotype, and each purified DNA fragment alone to
detect uncleaved drug-resistant genomes contammatmg the stocks of purified
5. Confirm the locatton of the drug resistance gene within the identified fragment,
by repeating the experiment m mirror image that IS cotransfecting cell cultures
with uncleaved DNA from drug-resistant vnus and purified fragments of wild-
type DNA The fragment containmg the drug resistance marker will now be tden-
ttfied by HSV recombmant vnuses having the drug-sensttlve phenotype
To fine-map the genetic locus containing the drug-resistant marker, the above
procedure must be repeated, using progressively smaller restrrctron fragments.
3.3.3. identification of Drug Resistance Mutation by DNA Sequencing
Restriction enzyme mapping should positron the drug reststance locus within
a few kilobases of DNA. To identify the virus gene mvolved and to pmpomt
the mutation conferring drug resistance, this DNA fragment and the approprr-
ate parental wild-type DNA fragment must be sequenced (see DNA sequenc-
ing protocols; Chapter 4). If the gene product is one that has already been
Ant!-HSV Activity of Antiviral Agents 397
as the target of other antiviral compounds (e.g., the DNA poly-
merase), then it will be useful to check for drug crossresistance,smce this could
yield Insights mto the molecular basis for drug inhibition of protein function.
3.4. Investigation of the Antiviral Mechanism
If no drug-resistant variant can be isolated or if the function of the identified
drug resistance gene cannot be deduced from its DNA sequence, it will be
necessary to mvestlgate the point in the virus replication cycle at which the
drug-sensltlve function operates m order to focus further mvestigatlons. Cheml-
tally Induced inhibition of virus replication can be separated into virucldal and
antiviral actlvltles. Vu-ucldal actlvlty ehmmates mfectlvlty when vn-us particles
are mixed with the drug m solution, whereas antlvlral activity inhlblts the syn-
thesis of mature infectious progeny virlons in infected cells.
Virucidal activity may be caused by disintegration of the entire HSV par-
ticle, solubilizatlon of the virus envelope, or the chemical modification, degra-
dation, or masking of some essential envelope proteins. HSV particles treated
with a vn-ucidal agent are blocked at adsorption and/or penetration, the earliest
stage of the virus replication cycle.
3.4.7. Virucidal Activity
1. Prepare a set of HSV aliquots (1 x lo6 PFU in 2 mL) in drug-free tissue-culture medium
or medium contammg increasing concentrations of drug, and incubate at 37В°C
2. Withdraw duplicate O.l-mL samples for titration of residual mfectlvlty at the
time of drug addition and at various times thereafter. Titrate each sample without
delay or storage, lest the infectivity continue to decrease. The titer In the vu-us
aliquot must be sufficiently high to allow dilution, during tltratlon, of the viru-
tidal agent below the concentration at which cytotoxlclty is apparent on the mdi-
cator cells. This type of experiment is frequently performed at a range of
temperatures, e g., 4вЂќC, room temperature, and 37В°C.
3.42. investigating the Effect of Drugs on HSV Adsorption
Two techniques have been used to investigate adsorption of HSV to target
cells. The first technique provides an indirect measure of vu-us adsorption,
quantifying the decrease m infectivity, with time, m the moculum applied to
cell monolayers (20,321. Because noninfectious particles might also adsorb to
the cells, use vu-us stocks with low particle/PFU ratios for these experiments
(i.e., <50:1). The second technique quantitates HSV particles bound to cell-
surfaces by measuring the radioactivity associatedwith 35S-methlonme-labeled
HSV partrcles, accumulating with time and resistant to removal by washmg of
the cell layer. Both techniques should be performed at 4вЂќC, since at this tem-
perature, HSV binds to, but does not penetrate cells.
1 Prepare radlolabeled HSV by mfectlon of roller bottle cultures of cells (-2 x 1OS/
roller) with HSV at an MO1 of 1 PFU/300 cells in growth medium containing I/S
normal concentration of methlomne. Incubate for 24 h at 3 lвЂќC, then add 25 pCi/
mL 35S-methlonine, and Incubate at 3 1В°C until complete CPE 1sevident.
2. Harvest the extracellular HSV yield, and purify the vu-us particles by density
gradient centnfugatlon (see Chapter 1)
3 Cool confluent monolayers of cells (on 50-mm tissue-culture dishes) to 4В°C
4. Prepare a series of radiolabeled HSV inocula ( IO6 PFU/mL) using culture medium
precooled to 4вЂќC, and contammg either no drug or increasing concentrations of drug.
5. Immediately after preparing the virus mocula, decant the growth medium from the
cell monolayers, and infect duplicate cultures with 1 mL of virus/drug inoculum
6. At the time of infection and at increasing times thereafter (e.g., 15,30,45,60,90,
120, 180, and 240 mm), remove the moculum from the appropriate culture dishes,
and remove unbound virus by three washes with PBS.
7 Prepare cell extracts from the washed cell layers by adding 300 pL of SDS-PAGE
8 Estimate the amount of radloactlvlty m each sample by spotting 20 pL of cell extract
onto a Whatman no 1 filter paper disk (there 1sno need to TCA precipitate this
matenal), place m scmtlllatlon vial with 5 mL of scmtlllatlon fluid, and measure the
amount of radioactivity counts/mm m each sample using a scmtlllatlon counter Plot
the percentage vn-us adsorption with time (100% is the cpm obtained with the drug-
free control at the maximum time allowed for virus adsorption).
The Important features of the virus adsorptlon curve are the time at which
bmdmg of virus to the cells is first detected, the rate at which virus particles
bind to the cells, the time at which the curve plateaus, and the maximum amount
of virus binding to the cells. The proportion of the total moculum binding to
cells at any time may be computed by comparing total counts/mm in the cell
extract with total counts/min in the inoculum at 0 min.
3 4.3. hvestigating the Effect of Drugs on HSV Penetration
The effect of drug treatment on the rate of virus entry mto cells may be
monitored by measuring the mfecttvity that becomes resistant to low pH inac-
tivation with time (20,32). This experiment is only valid for compounds that
can readily be washed from the intracellular compartments of cells. The HSV
penetration assay depends on the observation that HSV binds to cells at 4вЂќC,
but does not penetrate until the temperature 1s raised.
1. Seed cells m several sets of 24-well plastic tissue-culture trays (one tray for each
time-point), and allow to grow overnight at 37В°C
2. Prepare a series of vu-us inocula (100-200 PFU/cell layer) m drug-free tissue-
culture medium and cool to 4В°C
3. Cool the cell cultures to 4В°C
4. Decant the tissue-culture medium from the monolayers and infect with 100-200
Anti-HSV Actiwty of AntivIral Agents 399
PFU HSV/monolayer m a 200 pL inoculum. Incubate at 4В°C for 2 h to allow
maximum adsorptron of the virus.
5 Wash the monolayers with ice-cold PBS to remove unbound vnus, overlay with
drug-free medium or medium contaming increasing concentrations of drug, and
then shift to 37В°C to allow vnus penetration.
6. At various times (0, 15, 30, 45, 60, 75, and 90 min) after temperature upshift,
remove the appropriate 24-well tray from the incubator and inactivate vnus par-
ticles that have not yet penetrated the cells by treating the infected cell cultures as
a. Wash once with 1 mL saline.
b Wash once, for 1 min only, with acidic glycine
c Wash once with tissue-culture medmm to neutralize the acid.
d. Fmally overlay the infected cells with drug-free medium contammg 1.25%
7 Incubate the infected cell cultures for 2 d at 37В°C to allow virus plaques to
develop. Fix/stain the cultures with Giemsa, count the numbers of plaques on
each culture, and plot the data.
The important features of the virus penetration curve kmetrcs are: the time
at whrch the first virus particles enter the cells; the rate of vrrus penetration; the
maximum number of virus particles entering the cells; and the time at whtch
the curves plateau.
3.4.4. lnvestigating the Stage of the HSV Replication Cycle
Blocked by the Drug
If the drug Inhibits virus replication at a point subsequent to vrrion entry, the
stage at which the replication cycle is blocked may be deduced from electron
microscope studies (see Note 4) or from single-step HSV growth experiments
in which a single (standard) antiviral concentratron of drug (determined from
Section 3.2.2.) is added to infected cell cultures at progressively later times
throughout the vn-us replication cycle.
1 Infect a series of cell monolayers with HSV at an MO1 of 5 PFU/cell. Allow the
cells 1 h at 37В°C to absorb the virus, then wash three times with PBS 5% calf
serum to remove unbound vn-us, and overlay with drug-free culture medium.
2. At various times after infection (e.g., 2,4,6,8, 10, 12, 16, and 20 h post infectton),
add the drug to the culture medmm of the appropriate set of Infected monolayers.
3 Similarly, at various times after infection (e g , 0,2,4,6,8, 10, 12, 16,20, and 24
h pi), harvest selected cell cultures (as described m Section 3 2.2 )
4. Determine the total infectious virus yields by titration, and plot the data.
If the protein target for the drug specifies an early functron, late addrtion of
the drug will have no inhibitory effect on the infectious virus yield. Drug-
induced inhibition of a protein function that is required late, or throughout the
replication cycle, will result in loss of infectivity whenever the drug is present.
The converse experiment where drug is removed from infected cell cultures at
progressively later times can also give useful data, as can experiments in which
pulses of drug are used. However, such experiments can be dtfficult to interpret
due to the dtffculty of ensurmg efficient removal of drug from intracellular sites.