or fungal orrgin; second, use of molecular knowledge of a partrcular antrviral
target to design specific inhibitors, e.g., short peptides to inhibrt essential pro-
tein-protein interactions (1,2), or antrsense RNAs to mhrbrt virus gene
expression selectively (J-5).
The aim of this chapter 1sto assistworkers new to the field of antiviral research
by providing a series of experimental procedures through which the anti-HSV
activity of candidate antiviral compounds may be evaluated in vitro, the potency
of the compound assessed,and the antiviral mechanism investigated. Because
antiviral research draws heavily on a wide range of techniques that are generally
applied m other fields of virology, some experimental protocols mentioned, but
not grven, here are crossreferenced to other chapters of this book.
The most potent antiviral agents currently available for the treatment and
management of herpesvrrus mfecttons are nucleoside analogs. The drscovery
of acycloguanosine (Acyclovir; ACV), was an Important milestone m the hrs-
From Methods in Molecular Medrone, Vol 10 Herpes Simplex Virus Protocols
E&ted by S M Brown and A R MacLean Humana Press Inc , Totowa, NJ
tory of antiviral chemotherapy, since rt demonstrated that selective antrvn-al
activity was obtainable. Acyclovn has now become the benchmark compound
against which candidate antiherpes agents are evaluated. The highly selective
antiviral activrty of ACV derrves from the fact that ACV IS activated only in
virus (HSV, or varrcella zoster virus [VZV]) infected cells; there is little
appreciable actrvatron of ACV m uninfected cells. The HSV (or VZV) spect-
fled thymidme kinase (TK) activates ACV by phosphorylation to ACV
monophosphate. Thereafter, cellular enzymes complete the processmg to ACV
trrphosphate, in which form the drug IS incorporated mto growing DNA strands
resulting m vnus DNA chain termination, and freezing of the virus DNA poly-
merase molecule to the terminated DNA strand (6; see ref 7 for revrew).
Much has been (and contmues to be) done to develop further the ACV fam-
ily of compounds by rmprovmg the btoavarlabrhty (e.g., Valacyclovn and
Famcrclovir/Penciclovtr; refs. 8-10) or extending the antiviral range (e.g.,
Gangciclovn for the treatment of human cytomegalovirus; ref. II). However,
HSV mutants resrstant to ACV have been isolated from both in vitro and in
vivo infections. The commonly perceived threat to health management, espe-
cially of the tmmunocompromrsed patient, represented by the spread of such
ACV-resistant HSV ensures that there IS likely to be an endurmg need for new
anti-HSV compounds, partrcularly those directed against targets other than the
viral DNA polymerase.
Figure 1 shows the replicatron cycle of HSV and identifies stages at which
virus replication might be blocked by an antrvnal compound. Anttvn-al targets
will usually be virus-encoded proteins or nucleic acrd molecules, but tt IS
concervable that some cellular function, more critically required for viral than
cellular growth, could possibly also serve as an antiviral target, e.g., posttrans-
lational processing of a viral protein
Treatment of vn-us particles with a vn-ucrdal compound results m failure of
the virus to infect cells. Entry of HSV into cells is a complex, two-step process
of adsorptron, followed by penetration and mvolvmg interactions between dif-
ferent glycoproteins on the virus envelope (2%14) with cell-surface
proteoglycans that serve as the receptors for the virus (15,16) Vn-ucrdal com-
pounds may block, or render nonfunctional, one or more of the essential vnus
glycoproteins involved m entry. Compounds that render the cellular receptors
for the virus inoperative can also be expected to prevent infection of the cells.
Amanttdine and Rimantidine (see ref. 17 for revrew), which inhrbrt
uncoating of Influenza type A (18,19), have no activity against HSV, and, as
yet, compounds specifically inhibiting HSV uncoating have not been reported.
Following uncoating of the vn-us particle, the HSV genome IS delivered to
the nucleus of the infected cell, and transcription and rephcation of the HSV
genome then begin. Transcrrptron of the HSV genome is organized mto three
Anti-HSV Activity of Antiviral Agents 389
1 Adsorption and penetration
4. HSVDNA synthesis
8 Egress of virions from the cell
Fig. 1. Stages the HSV replication cycle that might be blocked by antiviral agents.
phases, immediate early (IE), early (E), and late (L) (see Chapter X). HSV
transcription has recently been demonstrated to be an accessible anti-HSV tar-
get by the finding that antisense RNAs can specifically block HSV- 1 IE1, IE4,
and IE5 gene transcription (5) and by the observation that the anti-HSV effect
of the biflavone (Ginkgetin) operates through a strong inhibition of IE tran-
HSV-DNA synthesis represents the most commonly used anti-HSV target:
Several compounds licensed for use in humans (e.g., ACV and derivatives,
Vidarabine, triflouorothimidine, Foscarnet) operate by blocking viral DNA
synthesis. Using a plasmid-based system, Wu et al. (21) have identified seven
HSV genes that are necessary, and sufficient, for HSV-DNA origin-dependent
DNA synthesis; these include HSV- 1 genes UL9 (the origin binding protein),
UL5, 8, and 52 (a protein complex having DNA helicase/primase activity),
UL30, and 42 (the DNA polymerase complex) (see ref. 22 for review) Clearly,
compounds that interfere with the expression of one or more of these genes or
the function of their protein products (mcludmg protein-protein and protem-
DNA interactions) can be expected to have anti-HSV activity.
HSV-DNA synthesesgenerates long head-to-tail concatemers of DNA mol-
ecules, which must be cut to genome length for packaging mto nucleocapsids.
Although HSV- 1mutants unable to package the vnus DNA have been described
(23-27), no antiherpes agent specifically targeting DNA packaging is yet known.
Translocatron of the HSV vn-us particle to the cytoplasm of infected cells
depends on genome encapsidation. Although coating of the nucleocapsid by
tegument proteins 1srequired for vnion maturation, and presumably full infec-
tivity, the mechanism of tegument assembly is not yet fully understood, nor
has the site at which tegumentation occurs been unambiguously identified
Envelopment of the particle occurs at the inner nuclear membrane and/or at a
cytoplasmtc site. The particle may lose the nuclear membrane-derived enve-
lope and acquire another derived from Golgi apparatus or post-Golgi vesicles.
Interaction with the Golgi apparatus or post-Golgi vesicles IS required for com-
plete processing of the HSV envelope glycoproteins. Various inhibitors of gly-
coprotem processmg (tumcamycm, castanospermme) or of Golgi apparatus
function (e.g., monensm and brefeldm A) impair the mfectivity of the HSV
yield and some also inhibit the release of HSV from infected cells. These com-
pounds, however, are considered too toxic for in VIVOuse.
1. Phosphate-buffered salme (PBS) 170 mA4NaCl,3 4 mA4KC1, 10 mA4Na2HP0,,
2 ml4 KH,PO, Store at 4Â°C
2 TrypsmlEDTA (20% [v/v] trypsm/EDTA) 0 25% w/v Difco trypsm dissolved m
phosphate-buffered salme (PBS) 0 6 mA4EDTA dissolved m PBS Store trypsm
at -20Â°C and EDTA at 4Â°C.
Saline* 0.14M NaCl (sterile filter), and store at room temperature
Acidic glycme-(HCl): O.lMglycme m salme, pH 3.0 (sterile filter). Storeat room
Trypan blue dye* 0.5% (w/v) trypan blue (Stgma, St Low., MO) prepared m PBS
and passed through a 0 22-ym filter Store at room temperature. During long-term
storage (>l mo), a precipitate may form, and the solutton should be refiltered before
use Trypan blue dye IS a suspected carcmogen and must be handled carefully
6 Neutral red dye* 0 4% (w/v) neutral red (Sigma) prepared in PBS and passed
through a 0.22+&I filter. Store m the dark at room temperature. Neutral red dye
1s a hazardous chemical and should be handled carefully
Anti-HSV Activity of Antiviral Agents 391
7 Tris-buffered saline. 25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 5 mA4 KCl, 5.5
mM glucose, 0.7 mJ4 Na,HPO,.
8. Reticulocyte standard buffer 10 mA4Tns-HCl, pH 7.5, 10 mMKC1, 1.5 mMMgC1,.
9 Cell lysis buffer: 20 mM Tris-HCI, pH 7.5,2 mMEDTA, 1 2% sodium dodecyl
sulfate (SDS) (w/v).
10. Protemase K: 1 mg/mL in water.
11. 20X SSC 3M NaCl, 300 mM sodium curate.
12 Denaturing soluttonâ€™ 1.5 A4 NaCl, 500 mM NaOH.
13. Neutrahzation buffer 3MNaC1, 500 mM Trts-HCl, pH 7 2
14 Prehybridization buffer 5X SSC, 0.5% SDS, 5X Denbardtâ€™s buffer, contauung 100 pg/
mL calf thymus DNA. The calf thymus DNA must be botled immedrately before use.
15. Hybrtdrzatton buffer: Prepare as for prehybrtdtzation buffer except that the HSV
DNA (100 pg/mL) probe IS also included (both calf thymus and HSV probe DNAs
should be boiled immediately before use).
16. 50X Denhardtâ€™s buffer, 1% Ftcoll400, 1% bovine serum albumin, 1% Polyvmyl
pyrrohdone prepared m water
17. Wash solution 1. 2X SSC, 0.5% SDS
18 Wash solution 2. 1X SSC, 0.1% SDS.
3.1. Cytotoxicity Testing
In order to study the anttvnal activity of a new drug, rt is Important to determine
whether antivnal actrvity can be uncoupled from the confoundmg effect of cellular
toxicity. Cytotoxictty tests define the upper-limit drug concentratron, which can be
used in subsequent anttviral studies. The vital-staining techniques, whereby cells
are treated with trypan blue or neutral red dyes, are among the simplest cytotoxic-
rty tests to perform, need little equipment, and give reliable results (28-30). Trypan
blue IS excluded by hve cells, but stains dead cells blue. In contrast, neutral red ts
taken up by live cells, stainmg them a brownish-red color, whereas dead cells
remam colorless. Cell vtabthty, as determined by vital-staining tests, should be
confirmed by additional experiments, e.g., measuring incorporation of radiolabeled
amino acids into proteins and/or 3H thymidine into cellular DNA. Absence of cyto-
toxicity in in vitro tests does not necessarily exclude toxicity m vtvo. Similarly, a
moderate level of cytotoxicrty m in vitro tests may not necessarily exclude m viva
use of the compound, perhaps as a topical treatment.
3. I. I. lnves tiga ting the Effect of Drugs
on Cell-Culture Growth and Cell Viability
1 Seed cells sparsely on tissue-culture dishes (e g., 5 x lo5 BHK cells on a 50-mm
dish), and allow to settle overnight.
2 Replace the growth medmm with either drug-free medium or medium containing
increasing concentrattons of drug (see Note 1)
3 At 0, 1,2,3, and so forth, days after drug addition, decant the overlay medium
into separate centrifuge tubes, and then remove the cells from the culture dishes
by two washes with 20% trypsm/versene.
4 Pool the trypsm/versene washes with the overlay medium from each cell culture,
and centrifuge at low speed (1OOOgfor 10 min at 4Â°C) Resuspend the cell pellet in
1 mL of tissue-culture medium Return the resuspended cell pellet to the appropriate
culture, and use to resuspend the trypsnnzed monolayer Thus ensures that cells that
may have become detached from the monolayer are not lost to the experiment
5. Mix 0 1 mL of cell suspension with 0.1 mL of a stock solution of trypan blue or
neutral red dye.
6. After allowing 5 min for staining, count the number of stained cells and the total
number of cells in each sample (in duplicate) using a hemocytometer. Cells mixed
with neutral red should be kept m the dark during the staining period. Most cell
lines can be kept for up to 30 min in the presence of trypan blue without affecting
7. Graph the results, and interpolate from the curves the concentration of drug that
kills 50% of the cells; this is the 50% inhibitory concentration (IC,,)
8. Compare the total numbers of cells (live and dead) in drug-treated and drug-free
cultures to determine the effect of the drug on cell-culture growth.
It should be appreciated that the same drug might give different results when
experiments are performed using exponentially growing or resting cell cul-
tures, different cell types from the same species, primary or established cell
lines, or similar cell cultures derrved from the same organ, but from different
animal species. The cell hne eventually selected for antiviral activity assays
will, in many cases,be a compromise between drug tolerance and the abihty of
the test virus to grow adequately in the selected cell line.
3.1.2. Reversibility of Drug-Induced Impairment
of Cell-Culture Growth
If the window between antiviral activity and cytotoxicity is narrow, it will be
important to investigate whether Impairment of cell-culture growth 1sreversible
and to determine the period of time that a particular concentration of drug can be
left in contact with cells without affecting their subsequentgrowth (28,29). Drug-
removal experiments (Fig. 2) provide the answer to these questions.
1. Separate sparsely seeded cell cultures into sets (Fig 2), and overlay with either
drug-free medium or medium containing increasing concentrations of drug.
2. At 0, 1, 2, 3, and so on, days after drug addition, harvest a control culture and a
culture treated with each concentration of drug to determine the percentage cell
viability and total cell counts, as described m Section 3 1.1
3. At each time-point, remove the drug from designated sets of cell cultures, by
three washes with drug-free medium, and then overlay with drug-free medium
and reincubate the cultures at 37â€™C.
Anti-HSV Activity of Antiviral Agents
Daysafterdrug removal (cell count/ cell vtabdlty)
012345 Recovery period
removal 0 1 2 3 4
III 1 3 (cellcount/cell viablhty)
Fig 2 Schematic protocol: reversibihty of drug-induced impairment of cell-cul-
4 To measure the subsequent growth of cell cultures after drug removal (recovery
from drug treatment), either harvest (as described in Section 3.1.) sets of cultures
dally to monitor recovery, or harvest after a preselected number of days follow-
mg drug removal
5. To determine the maximum period of time that cell cultures will tolerate contact
with a particular concentration of drug, compare the total number of cells in the
drug-free and drug-treated cultures followmg the recovery period
3.2. Measuring Antiviral Activity
3.2.1. Measuring the EDso Concentration
The most commonly used measure of antiviral potency is the EDSo,that is,
the concentration of drug that eliminates 50% of the virus infectivity.
1. Infect sets of cell monolayers on 50-mm tissue-culture dishes with HSV at lOO-
200 PFU/dish. After a 1 h absorption period at 37Â°C remove unbound virus by
washing three times with PBS containing 5% calf serum
2 Overlay the infected monolayers with either normal tissue-culture medmm con-
taining 1.25% methyl cellulose (to mhlbit satellite plaque formation) or with the
same medium containing increasing concentrations of drug.
3. Incubate the infected cell layers at 37Â°C for 48 h or until virus plaques are visible