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3.4.5. lnvesfigating the Effect of Drugs on HSV-DNA Synthesis
In the past, HSV-DNA synthesis has been studied by separation of 3H-
thymidme labeled infected-cell DNA into viral and cellular fractions by cesium
chloride gradient centrlfugatton (33). More recently, however, this technique
has been superseded by a dot-blot, Southern blotting method. The protocol
described here uses a radtolabeled probe to detect HSV-DNA bound to a nylon
filter, but a nonradioactive detection system might also be used. To reduce the
background HSV-DNA signal, caused by residual maculating HSV particles,
it is advisable to extract DNA from isolated nuclei (34).
3.4 5 1 PREPARATION DNA FROM
OF NUCLEI
1. Infect monolayers of cells (-2 x 1O6 cells) on 35mm tissue-culture dishes wtth
HSV at an MO1 of 5 PFU/cell
2. Allow the cells 1 h at 37°C to absorb the virus, and then remove unbound virus
by three washes with PBS + 5% calf serum
3 Overlay the infected cell cultures with either drug-free medium or medium con-
tammg mcreasmg concentrations of drug, then incubate at 37™C for 24 h.
4 Scrape the infected cells into the medium, and pellet by low-speed centrifugation
(1OOOg for 10 mm). Carefully remove the supernatant, wash the cells with Tris-
buffered salme, and then repellet the cells (1OOOgfor 10 mm).
5. Raise the cell pellet in 1 mL of reticulocyte standard buffer containmg 0 5%
NP40, and mcubate on ice for 10 mm to lyse the cells.
6 Pellet the nuclet by low-speed centrifugation (1OOOg for 10 min)
7. Carefully ptpet off the cytoplasmic fraction (supernatant), and resuspend the
pelleted nuclei by vortexmg m 1 mL of water to which 1 mL of lysis buffer is
then added. Incubate at 37°C for 6 h.
8 Dilute the sample wtth 20X SSC to give a final concentration of 6X SSC
3.4 5 2 DOT-BLOT/DNA HYBRIDIZATION
1. Prepare a serves of fivefold dilutions (of 300 pL total volume) of each nuclear
extract m 6X SSC
2 Boll for 10 min, and then cool on ice
3. Assemble the dot-blot apparatus according to the manufacturer™s mstructions,
e g , place two sheets of Whatman 2MM chromatography paper presoaked m 2X
SSC on the base of the apparatus, and overlay these with a sheet of Hybond N
(Amersham, Little Chalfont, Buckmghamshire, UK) nylon membrane presoaked
in 2X SSC, then close the apparatus
4. Apply the vacuum to the dot-blot apparatus, and load 200 yL of each sample into
401
Anti-HSV Activity of Antiviral Agents
the approprtate well of the apparatus.
5 When all the sample has passed through the membrane filter, break the vacuum
and disassemble the apparatus. Lay the nylon membrane (DNA side up) on top of
a stack of three Whatman 3MM chromatography paper sheets soaked by parttal
nmnersron m denaturmg buffer. Incubate at room temperature for 10 mm.
6 Transfer the nylon membrane (DNA side up) to a stack of three 3MM chroma-
tography paper sheets soaked by partial immersion in neutrahzation buffer, and
incubate at room temperature for 5 min
7. Allow the membrane to air-dry, then munobilize the DNA on the membrane by
baking at 80°C or by UV crosslmking on a transdlummator (as described m the
manufacturer™s instructions).
8 Incubate the membrane at 65°C for 2 h with prehybridization buffer
9. Prepare the radiolabeled HSV-DNA hybridization probe as described m the ran-
dom-priming or nick-translation protocols (see Chapter 4)
10. Incubate the membrane at 65°C overnight m hybridization buffer containing the
radiolabeled HSV-DNA probe.
11. Carefully remove the radioactive hybridtzatton buffer, and then wash the mem-
brane twice in wash solution 1 (1 min at 65™C), twice in wash solution 1 (15 min
at 65”C), and finally twice in wash solution 2 (15 mm at room temperature)
12. Seal the membrane m a polythene bag, and expose to X-ray film to obtain an
autoradiograph
By using a densitometer to measure and comparing the optical absorbance
of the dots on the autoradiograph, the effect of the drug on HSV-DNA synthe-
sts can be computed. Alternatively, if HSV-DNA standards of known concen-
tration are included on the same membrane with the test samples, it will be
possible to quantitate the amounts of HSV-DNA synthesized.
3.4.6. investigating the Effect of Drugs
on Viral Protein Synthesis and Posttranslational Processing
The HSV-infected cell polypeptide patterns obtamed on SDS-PAGE gels
are well characterized. Similarly, the temporal classification of most HSV pro-
teins is known from SDS-PAGE studies using temperature-sensitive mutants
or from studies with drugs that block HSV replication at defined stages m the
virus replication cycle (35).
SDS-PAGE can be used to investigate the effect of drugs on HSV polypep-
tide synthesis. Drug-induced inhibition of HSV replication is usually reflected
in changes in the quantities, or the apparent molecular weights, of HSV-speci-
fied polypeptide bands on SDS-PAGE gels. Reduction in the quantity of a par-
ticular protein can have a number of possible explanations, including inhibition
of the transcription of the affected gene, inhibition of translation of the mRNA,
or reduction in the stability (half-life) of the mRNA or the protein product (see
Chapter 13 for transcription and mRNA stability protocols). If the protein
Dargan
402

affected by the drug specifies a regulatory function, the drug may indirectly
affect the synthesis of other proteins under rts control. Drug-induced changes
in the apparent molecular weights of protem bands on SDS-PAGE gels argue
for alteration m the normal pattern of posttranslational processmg of the
affected protein, resulting m accumulation of unprocessed precursor forms,
intermediately processed forms, or breakdown products of the affected pro-
teins. The protocols for Investigating HSV-infected cell proteins by SDS-
PAGE are given m Chapter 7.
Posttranslational processmg of HSV proteins can be of several types, mclud-
ing glycosylation, phosphorylatlon, mynstllatlon, and sulfation (see Chapter 7
for labeling protocols).

4. Notes
1 The test compound should be soluble in either aqueous solution, tissue-culture
medium, distilled water, and so forth, or organic solvents, dlmethylsulfoxlde
(DMSO), ethanol, and so forth Drug solutions should be sterihzed by ultrafiltra-
tion (0.22 p), or if heat-stable, by autoclavmg before use Where organic solvents
are used, additlonal controls for the effect of the solvent must be included m
experiments If the molecular weight of the test compound IS known, then molar
amounts should be used; otherwise, weight/volume doses may be used
2. To prepare resting cell cultures, the cells are sparsely seeded (1 x lo5 cellGO-
mm dish) in tissue-culture medium contammg 0.5% serum and incubated at 37OC
m a well-humidified incubator After about 7 d, growth of the culture should
have stopped. The decrease m cellular metabolism can be momtored by measur-
ing the mcorporatlon of 3H-thymldme mto cellular DNA during an overnight
labeling period. The growth-factor-depleted (conditioned) medium from resting
cell cultures must be retained at the time of mfectlon, so that it can be reused,
with drug added as appropriate, to overlay the infected cells
3. Before harvestmg the mfectlous vu-us yields at step 3 of Se&on 3 2 2 , there IS an
optlon to harvest the total vnus yield or to separatethe virus yield mto cell-
associated and cell-released components; the latter can provide information on
whether the drug impalrs the release of vlrions from the cell or whether the drug
has a strong virucidal effect To obtain the cell-released yield, remove the super-
natant tissue-culture medium from infected cells and clarify by low-speed cen-
trlfugatlon (lOOOg/lO mm/4”C) To obtain the cell-associated yield, raise the cell
pellet (generated by the centrifugation step above) m fresh culture medium, and
use this to harvest the cells, whxh remain attachedto the tissue-culturedish, by
scraping them into the medium The cell-associated and cell-released volumes
are brought to the same volume with fresh medium, and the cell suspension IS
then somcated to generate the cell-associated VIIUS fraction
4. The herpesvirus replication cycle has been extensively studied by electron
microscopy, and the temporal sequence of morphologrcal events occurring dur-
ing HSV replication has been examined by time-course experiments (36), experi-
403
Anti-HSV Activity of Antiviral Agents
ments m which virus replication 1s blocked by drugs (37), and experiments m
which virus rephcatlon 1sarrested by various temperature-sensitive mutations in
the mfectmg vn-us (38-41)
HSV-infected cells treated with drugs that mhlblt IE gene expression show
little morphological evidence of infection, except that the nucleus of the infected
cell exhibits condensation and marginatlon of the cellular chromatin. HSV
Infected cells treated with drugs that block HSV-DNA synthesis exhlblt marg-
matlon of chromatin and mtranuclear accumulation of large numbers of imma-
ture nucleocapsids (I e., both A- and B-type capslds [42]). Infected cells treated
with drugs that block HSV rephcation at a stage after viral DNA replication and
packaging exhlblt, m addition, mature nucleocapslds having an electron-dense
DNA-containmg core (C-type capsids [42])

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