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ETClo We carry this out in 20-mL roller bottles holding 1 x IO8 cells For HSV-
1 each roller bottle ˜111typically give a yield of 100 pg DNA and for HSV-2 l&
50 c(g DNA. We routmely prepare stocks of 5-10 roller bottles, but this can be
scaled down to only one roller bottle or less if neccessary.
2 The cells are incubated at 3 1°C until cytopathic effect (CPE) 1scomplete, usually
after 34 d.
3. Virus-infected cells are shaken mto the medium (glass beads can be used If drffi-
cultles are encountered m detaching the cells), and the medium decanted into
centrifuge tubes
4. Cells are pelleted by spinning at low speed (2K) m a benchtop coolspin for 15
mm at 4™C The supernatant is carefully decanted and stored at 4°C
5 Cytoplasmic vn-us IS extracted from the cells by lysing m RSB contammg 0 5% (v/v)
NP40, which lyses the plasma, but not the nuclear membrane. The cells are resus-
pended m 10 mL RSB/NP40 and Incubated on ice for 10 mm, the nuclei pelleted at
2K m acoolspmfor 10mm at 4”C, andthe supematant
carefully removedsoasnot to
disturb the pellet This supernatant 1sadded to the cell supernatant from stage 3
6 The nuclei are re-extracted with RSB/NP40, the supernatant again being added to
the cell supernatant and the nuclear pellet discarded
7 Virus from the cell supernatant and cytoplasm is pelleted by spinning m a GSA
rotor m a Sorvall superspeed centrifuge (or equivalent) for 2 h at 12K at 4°C The
supernatant is discarded, and the virus pellet resuspended m 10 mL NTE and trans-
21
Preparation of US V-DNA
ferred to a glass tube. Virus 1scompletely resuspended by somcation in a water bath.
8. Virus is lysed by the addition of SDS and EDTA to concentrations of 2 5% (w/v)
and 10 mM, respectively, followed by mcubation at 37°C for 5 mm From this
stage, the virus DNA is free and hence susceptible to shearing, and all manipula-
tions must be done carefully wtth gentle shaking and no vortexmg
9 The DNA IS phenol-extracted by adding an equal volume of NTE saturated phe-
nol, gently inverting, and allowing to stand for 10 min at room temperature. The
aqueous phase contammg the DNA is separated from the organic phase by cen-
trifugatton m a coolspm at room temperature for 10 mm at 2K. The upper aque-
ous phase IS carefully removed from the organic phase, takmg care not to disturb
the protemaceous interphase
10 The aqueous phase is re-extracted with phenol between one and three times, until
there IS no mterphase and the upper layer IS clear. If the volume of the aqueous
layer drops sigmficantly below 10 mL to mmimtze physical loss of DNA, the
volume should be increased back to 10 mL by the addition of NTE.
11. A chloroform:isoamyl alcohol (24: 1; v/v) extraction IS cart-ted out m a stmtlar
manner to the phenol extraction, except that the incubation and spin are for
only 5 min
12. The DNA IS prectpttated by the addition of 2 vol of ethanol and gentle mver-
sion. DNA should precipitate tmmedtately as fme strands No added salt or
-20°C mcubatton is requtred owing to the high molecular weight of HSV-
DNA. The DNA is pelleted by centrtfugatton m a coolspm at room tempera-
ture for 10 mm at 2K and washed with two-thirds of the tube volume of 70%
(v/v) ethanol.
13. The DNA should be an-dried m an inverted position for 15 mm before redissolvmg
in sterile distilled HZ0 (dH*O) containing 50 pg/mL RNase A It is Important not to
overdry the DNA, since this may cause difficulty tn redissolvmg. Typically, the
DNA from 10 roller bottles is resuspended m 1-2 mL The DNA is quantitated
either by OD at 280 nm with 1 OD unit equaling 40 l.tg DNA or runnmg on an
ethidim-bromide-strand agarose gel agamst a standard of known concentation.
RSB Buffer: 10 mM Tris-HCl, pH 7 5, 10 mM KCl, 1 5 mM MgCl,

2.2.Titration of HSV-DNA infectivity
Each preparation of HSV-DNA will vary n-r Its ability to generate virus.
Here its infectivity will be expressed as the number of PFU/pg DNA. The bet-
ter the quality of the DNA, the higher this figure wrll be and, in general, the
more efficient at generatmg recombinant VII-W.Each DNA preparation requires
to be titrated (usually in the range of 0.1-2 pg) DNA to establish Its PFU/pg
DNA, and an optimal figure is chosen. At low levels, there IS a linear increase
m the number of PFU as the DNA amount IS increased, but this plateaus and
then begins to fall owmg to inhibition at high levels of DNA. A point near the
top of the linear response should be chosen as the optrmal amount of DNA to
be used m transfecttons.
MacLean
22
2.3. Calcium Phosphate PrecipitatioWDMSO Boost
The standard method for introducing HSV-DNA into cells IS the calcmm
phosphate preclpltatlon/DMSO boost method (2,3) All buffers should be
stored and reactions carried out m sterile plastlcware, since the detergents used
to wash glassware may be mhibrtory if not properly rinsed.
1 HSV-DNA IS added to 400 pL HEBES buffer contammg 10 pg/mL carrier calf
thymus DNA
2 Calcmm phosphate is added to a final concentatlon of 130 mM, and the sample
gently mixed and allowed to sit at room temperature for 5 mm to allow the cal-
cium phosphate/DNA precipitate to form.
3 The DNA sample is added gently to tissue-culture cells m a 60-mm plate, from
which the medium has been removed. For maximum transfection efficiency, the
cells should be 50-70% confluent, actively growing, and should have been set up
the previous night from freshly typsnuzed cells, which had not been stored at 4°C
4 After 40 mm of incubation at 37”C, the cells are overlaid with 5 mL ETC,, and
mcubatlon allowed to proceed at 37°C for 4 h prior to boosting v&h DMSO This
1sthe optimal tlmmg for the DMSO boost, but it will be effective up to 7 h after
addition of DNA
5 The next stage is the DMSO boost. The DNA/medmm mixture is removed from
the cells, which are washed once with 5 mL ETC,O One mtllihter of 25% (v/v)
DMSO m HEBES IS added gently to the cells and incubated for exactly 4 mm at
room temperature. This time must not be exceeded owmg to the toxicity of
DMSO. The DMSO/HEBES 1spoured off, and the cells washed twice with, and
subsequently overlaid with 5 mL ETClo Speed 1s extremely important here
because of the need to dilute the DMSO as quickly as possible. Initially, it 1s
better not to handle more than 10 plates at a time at the DMSO boost stage and
even with experience never more than 20 plates at one time.
6 The plates are incubated at 37°C for 3 d or until CPE 1sextensive, at which point
the infected cells are harvested to recover VUUS.If mdlvldual plaques are required,
the ETC,O overlaying the transfection should be replaced after 16-24 h by a
mechum, such as ETMC 10% containing carboxymethylcellulose, to prevent sec-
ondary spread of cell released vn-us (see Chapter 1 on vu-us growth) and mcuba-
tlon continued for a further 48 h. Cells used for transfectlons m our laboratory are
BHK 2 l/C 13, but this procedure ˜111work with most established cell lines.
HEBES Buffer. 130 mA4NaCl,4.9 mMKC1, 1.6 mMNa,HPO,, 5.5 mMo-glucose,
21 WHEPES
2.4. Lipofection
We do not routinely use hpofectlon for the generation of infectious virus, since
we consistently find it lessefficient than calcium phosphate transfectlon. This con-
trasts with our results using plasmlds for transient expression assays where
lipofectlon has a much higher efficiency than calcium phosphatetransfection. How-
ever, for cell types refractile to calcium phosphate transfectlon, it is worth trying.
Preparation of HSV-DNA 23

1. The DNA (1-5 pg) IS added to 100 uL Optrmem serum-free medium (Life Tech-
nologtes) and IS mrxed wrth.
2 12-pL Liposomes made up no more than 1 mo ago are added to 100 pL Optrmem.
3 The DNA and liposomes are combined, mixed gently, and left to stand at room
temperature for 5-15 mm.
4 Cells m 35-mm plates (at lO--25% denstty, smce hpofection IS sigmficantly more
efficient in low-density cells) are washed twice with Optimem
5 DNA/liposomes are mixed with 800 pL Opttmem, added to the cells, and mcu-
bated for 6 h at 37°C
6. One mllhliter of ETC20 IS added, and incubation continued for 24 h at 37°C
7 The medmm is removed and replaced with fresh ETC,,,, and incubation contm-
ued at 37°C for 2 d or untrl CPE IS extensive, at whrch point the infected cells are
harvested to recover vnus
2.5. Preparation of Liposomes
1. Pipet 1 mL dtoleoyl L-a-phosphattdyl ethanolamme (DOPE) (10 mg/mL m
chloroform) (Sigma PO510) mto a glass universal, add 5 mg dtmethyldrocta-
decyl ammomum bromide solid (DDAB) (Sigma D2779), and dissolve by vortexmg
2 Evaporate chloroform using a stream of nitrogen Thts takes approx 5 mm
3. Lyophihze overnight m a freeze dryer
4. Resuspend dried lipids m 10 mL sterile dH,O either by vortexing or somcating m
sombath
5 Once suspended, somcate the lipids using a somprobe Sonicate at maximum
power, using bursts of 30 s, keeping the universal on ice in between, until the
suspension clears, mdtcatmg liposomes have been formed
6. Store the hposome preparation at 4°C. Prior to use, vortex bnefly Discard after 1 mo
2.6. Electropora tion
We do not carry out electroporatton of HSV-DNA mto eukaryotm cells, but
for cells that do not transfect/lipofect, such as primary neurons or lympho-
cytes, this is a possible way to introduce vnus DNA into cells.
3. Generation of Recombinant Virus
3.7. Marker Rescue
This procedure 1svery stmllar to that for transfection, except that m addition
to HSV-DNA, a HSV fragment (usually derived from a plasmid clone) con-
taming the genetm marker to be introduced into the genome is also added to the
transfection mix. This fragment should have flanking sequences of ideally
X500 bp on either side of the alteration, although recombmation at a somewhat
reduced frequency will still occur if only 200 bp flanking DNA are present: this
IS the mmimum amount that will allow detectable recombinatron to occur (4)
Flanking sequencesof >l kbp will not lead to an increase in recombmation fre-
quency. The fragment IS usually added at a range of molar ratios. A suggested
24 Maclean

range 1s l- to 50-fold molar excess.Maximum recombinatton frequency is usu-
ally reached at a IO-fold molar excess As the excessof plasmld and hence the
DNA present increases,the overall transfectlon efficiency will decrease, leading
to a subsequent decline m recombmatlon efficiency at high levels of plasmld. To
detect recombmants, the transfectlon mixture should be harvested, plated out at a
dilution appropriate to give single plaques, which should be isolated, and a DNA
stock grown from these for analysis of genome structure The recombination
frequency 1svariable between experiments, but 1sgenerally low, typically being
on the order of 0.1-l%. Thus, detection of recombmants by analysis of then
DNA profile is time consummg. If a detectable marker, such as the Escherichza
coli P-galactosidase gene 1sincluded m the rescuing fragment, recombinant vi-
rus can be detected by blue staining by adding X-gal (150 pg/mL) to the methyl-
cellulose overlay after the plaques have formed.
More recently, other methods of generating recombinant vu-us where the
frequency of recombmants 1s higher have been developed. One of these, the
use of cosmids to generate recombinant virus, IS described elsewhere in this
book. Two other methods are described below.
3.2. Ligation of a Fragment into a Unique Site
Rlxon and McLaughlan (5) have described the use of a vu-us with a unique
XbaI site m a nonessential site of the genome between genes US9 and 10
Digestion of this DNA to completion almost completely abolishes the ablllty
of the virus to generate VKUS. regenerate vnus, the two digested halves can
To
be ligated together. If a plasmld with sequences to be introduced, flanked by
XbaI sites 1sadded to the ligation mix, then recombinant virus 1sgenerated at a
high frequency (l-10%). This frequency 1s IO-fold higher than for the classl-
cal marker rescue technique. A virus with a unique restriction enzyme site 1s
generated in the desired location by standard marker rescue. This method does
not rely on the presence of flanking DNA for the generation of recombinant
virus, but is mainly useful for the insertion of extraneous DNA for expression
rather than for alteration or deletion m single genes. The transfectlon proce-
dure is as described above. Usually 0.5-2 pg of HSV-DNA and l-2 pg frag-
ment are used m the ligation reaction and subsequent transfectlon.
3.3. Recombination Across Digested HSV
Recently we have developed a method to generate nearly a 100% recom-
binants in any desired location of the HSV genome. This will be especially
useful for the introduction of multiple mutations mto one gene. First, a vuus
with a unique restriction enzyme site 1s generated m the desired location by
standard marker rescue. We have been using a PucI site, since there 1sno site
occurmg naturally m the HSV- 1 genome For ease of detection of the original
25
Preparation of US V-DNA
restrictlon enzyme site positive virus and recombmants, this ts usually done
with a fragment containing the J3-galactosldasegene as a marker, flanked by
is digested by PacI, and a fragment spanmng (at
PacI sites. The HSV-DNA
least 500 bp on each side) the dlgested DNA is added to the mixture. Transfec-
tlon takes place as described above. For viable vn-us to be generated, recombm-
atlon is required to take place across the two ends through the overlappmg
fragment; thus, almost all progeny will be recombmants. Nondigested parental
DNA containing the /3-galactosldase gene will produce blue-staimng plaques
in the presence of X-gal.
References
1 MacPherson, I. and Stoker, M G (1962) Polyoma transformatton of hamster cell
clones an mvestigatton of genetic factors affecting cell competence Vwology
190,22 l-232
2 Graham, F L and van der, E B (1973) A new technique for the assay of the
mfectlvtty of human adenovuus 5 DNA Vzrology 52, 456467.
3. Stow, N D and Wilkie, N M (1976) An improved techmque for obtammg enhanced
mfecttvity with herpes simplex vnus type 1 DNA J Gen Vzrol 33,447-458
4 Preston, V G (1981) Fme-structure mappmg of herpes stmplex vnus type 1 tem-
perature-sensitive mutations wrthm the short repeat region of the genome. J Vu-01
28, 150-161.
5 Rtxon, F J and Mclaughlrn, J (1990) Insertion of DNA sequences at a unique
restrtctton enzyme site engmeered for vector purposes mto the genome of herpes
simplex virus type 1 J Gen Vwol 71,2931-2939
4
HSV Mutagenesis
Robert S. Coffin


1. Introduction
Herpes genomes are large and complex, with many interactions among her-
pes encoded proterns, herpes DNA and RNA, and the host cell. These interac-
tions begin as the vnus enters the cell, and continue as the decision for latency
or Iyttc replication is made. Correctly regulated gene expression then allows
herpes genes to be expressed in a temporally regulated manner and to subvert
the host cell metabolism m favor of virus production. Finally, infectious prog-
eny virions are assembled and released. Explormg the function of the many
herpes-encoded proteins and the mechanisms to control then expressron dur-
mg these processes thus requires a fine dissection of the herpes genome, so as
to allow protein-coding regions to be linked wtth function and control DNA
regions to be identified.
Before modern molecular biological techniques, viruses were often studted
by the random generation of loss of function mutations giving an altered phe-
notype, either as they arose spontaneously during vnus propagation or m which
the natural rate of mutation was increased in some way. In a similar way, tem-
perature-sensitive mutations were identified in which mutations m an essential
gene allowed growth at a permissive temperature, but not at a htgher non-
permissive temperature, allowing the function of a particular gene to be probed.
These types of studies can nowadays most usefully be used to select for a vn-us
with a particular phenotype (especially if the locus likely to be responsible for
the phenotype is unknown), but cannot allow the finer details of protein or
DNA function to be understood. Molecular biology now allows targeted
changes to be made in the herpes genome (as long as the virus remains viable
or can be grown on a cell lme complementmg the mutation), which can be used
to unravel the details of the virus/host relationship. This chapter will concen-
From Methods tn Molecular MedIcme, Vol IO Herpes Bmplex Wrus Protocols
Edlted by S M Brown and A R MacLean Humana Press Inc, Totowa. NJ

27
Coffin
28

trate on the types of targeted mutatton, whrch can be made, what these muta-
tions can tell you, and how the mutant DNA sequences can be generated. The
methods by which sequences are introduced into the herpes genome (when
required) and recombinant plaques Isolated, purified, and the DNA structure
checked by Southern blotting, are covered elsewhere in this book.
1.7. Types of Mutation
Mutations can be made that either prevent the expression of a gene (by
its deletion or other disruptton), alter the protein product of a gene (by de-
letron or addition of translated sequences), affect the regulation of tran-
scription of a gene (by alteration of promoter regions), or whose primary
effect is at the RNA level (sequences affecting RNA processing and stabil-

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