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proteins form a trimeric complex that exhibits both DNA hehcaseand DNA pmnase
acnvnies (for excellentreviews on HSV-DNA replication,seerefs. 1-3). Recent stud-
ies on the enzymatic activities of theseproteins hasbeen greatly facthtated by then
overexpression using a variety of heterologous expression systems,most notably
the baculovirus Autographa cal$&zzca nuclear polyhedrosts virus (AcNPV).
The transient assaysfor HSV- 1 DNA replication are based on earlier obser-
vations of the structure of HSV-1 defective genomes that arise on serial high
multiplicity passage of virus stocks and were shown to consist of tandem
duplications of small segments of the viral genome (4,.5). Since the tandemly
repeated structure was regenerated when monomeric umts produced by restrrc-
tton endonuclease cleavage were transfected into permissive cells in the pres-
ence of wild-type HSV-1 DNA as helper, tt was deduced that each unit must
contam a functronal vrral replrcatton origin and that repllcatlon probably
occurred by a rolling crrcle mechamsm (˜6,7). It was reasoned, therefore,
that rf cells were transfected with a circular plasmtd mto which a functional
HSV origin had been cloned and subsequently supermfected with wt HSV- 1,
the repltcatton proteins produced by the helper vu-us would be able to rec-
ognize and act on the plasmid-borne HSV-1 origm, resulting m amplification
of the whole plasmtd. Such specific amplificatton then could be detected by
hybridization with a probe contammg the vector sequencesalone (8). The pro-
tocols presented in the following sections descrrbe how this approach can be
utilized to assay for the abrlity of viral sequences to function as replication
ongins, of weld-type and mutated forms of the rephcatron proteins to partlcr-
pate m DNA synthesis, and of replicated molecules to be packaged into vu-us
particles. However, because these methods rely on transfection of tissue cul-
ture cells, it should remembered that, m contrast to the cell free assays avail-
able for other vnuses, they are unable to provide mformatlon on the role of
host proteins in these processes.
2. Materials
2.1. Cells and Media
1 Any cell line permissive for HSV infection that can be transfectedat reasonably
high efficiency should be appropriate for theseassays. have extensively used
We
baby hamster kidney (BHK) 21, clone 13 cells (9) grown in Eagle™s medium
supplementedwith 10% calf serum, 10% tryptose phosphatebroth, 100 U/mL
pemcillm, and 100 pg/mL streptomycin(ETC,O)at 37°C in an atmospherecon-
taming 5% (v/v) carbon dioxide. Cell monolayersin plastic Petri dishes(approx
2 x lo6 cells/35mm plate) arepreparedthe day before use, and followmg mfec-
non or transfectronthey are maintained m Eagle™s medium contaming 5% new-
born calf serum plus antibrotrcs (EC5). Eagle™s medium supplemented with
antrbiottcsISused for the washmgof cell monolayers,unlessspecified.
Transient Assays 217
2. The modified assay in insect cells (see Section 3.3.) uses monolayers of Spodopteru
fmgiperdu (St) cells (stram IPLB-SF-2 1; ref. 20). These are maintained at 28°C with-
out carbon dioxide m TC 100 medmm supplemented with 5% fetal calf serum, 100 U/
mL pemcillin and 100 pg/mL streptomycm (TC 1OOC).Monolayers of approx 4 x lo5
cells in 2 cm* plastic Linbro wells are suitable for the replication assaysand TC 100
without serum or antibiotics can be used m the lipofection of these cells.
2.2. Other Reagents
1. Trts-buffered saline (TBS): 137 mA4NaC1, 5 mM KCl, 0.7 mMNa2HP04, 5.5
mM glucose, 25 mA4 Tris-HCl, pH 7.4.
2. TE: 10 mA4Tris-HCl, pH 7.5, 1 mA4EDTA
3. 20 mg/mL protease: Sigma grade XIV protease dtssolved m TE, predigested for
1 h at 37“C and stored at -2O™C.
4 Cell lysis buffer (CLB): 10 mA4Tris-HCl, pH 7 5,1 mMEDTA, 0 6% (w/v) SDS.
5 200X RNase mix: 1 mg/mL RNase A, 10,000 U/mL RNase Tl in TE (stored
at 4°C)
6 Rettculocyte standard buffer (RSB): 10 mM Tris-HCl, pH 7.5, 10 mM KCl, 1.5
n-d4 MgClz
7. 10% (v/v) NP40 m HzO.
8. Hepes-buffered salme (HeBS): 137 mMNaC1,5 mMKCl,O.7 mMNa2HP04, 5.5
mM glucose, 2 1 mM HEPES-NaOH, pH 7.05; autoclaved or filter sterilized and
stored at room temperature
9 2 mg/mL Double-stranded calf thymus DNA (Sigma, St. Louis, MO) m H,O
(stored at -2OOC)
10 2M CaCl,: autoclaved or filter sterilized and stored at room temperature
11. 25% (v/v) Dimethyl sulfoxide (DMSO) in HeBS, made up mrmedtately before use.
12 Liposomes prepared as described (11,12) from dimethyl dtoctadecylammonium
bromide (DDAB) and dioleoyl phosphatidyl ethanolamine (DOPE). A solutton
contammg 4 mg DDAB and 10 mg DOPE in chloroform is evaporated to dryness
under vacuum, the lipids resuspended in 10 mL sterile H,O, and sonicated using
a Dawe Soniprobe at approx 100 W until no further decrease in turbidity occurs.
Store at 4°C. (Can be used for at least 6 mo )
13. Detailed descripttons of the reagents and methods used for restriction enzyme
digestion, agarose gel electrophoresis, Southern blot transfer, preparation of
[32P]-labeled probes by mck translation or random priming, and hybridization,
which are beyond the scope of this chapter, can be found in standard manuals of
molecular biological techmques (13,14)

3. Methods
3.1. Assay for the Presence
of a Functional Origin of HSV-1 DNA Synthesis
In order to determine whether a particular fragment of HSV-1 DNA con-
tains a functional origin sequence, or if a known origin remains functional fol-
stow
218

lowing mutagenesis, the most convenient procedure is to transfect a bacterial
plasmid containing the fragment into mammalian cells and supermfect with
wild-type HSV-1 to provide helper functions. The majority of our work has
utilized common plasmrds such as pAT153 (15), pUC derivatives (26) or the
pTZ series (17) as vectors for the viral origin-containing fragments, and these
are propagated in dam+ E. colz strains such as DH5 or XLl-blue. The DNA is
mtroduced into monolayers of BHK cells m 35-mm Petri dishes by the calcium
phosphate technique. Followmg supermfectron, total cellular DNA is prepared
and examined for the presence of amplified input plasmrd DNA sequences by
a combmation of restriction enzyme drgestron, Southern blot analysis, and
hybridization to a labeled probe prepared usmg the DNA of the initial plas-
mrd vector. The DNA 1susually digested with DpnI in the presence of a second
enzyme that cuts the input DNA once and consequently can convert the high
molecular weight concatemertc replication products to monomerrc units that
are more readily transferred from the gel and more easily detected since they
represent a discrete, uniformly sized species. The use of DpnI allows reph-
cated plasmid sequences to be distinguished from unreplicated input DNA
since the latter contains methylated A residues within the DpnI recognmon
sequence and therefore is susceptible to DpnI digestion Following amplifica-
tion of these sequences m mammalian cells these sites are no longer methy-
lated and the DNA becomes resistant to cleavage by DpnI. Digestion with the
two enzymes therefore should yield a species that comigrates with linearized
DNA of the input plasmid and represents DNA that has been replicated subse-
quent to transfection, and several smaller species that represent the DpnI cleav-
age products of unreplicated mput DNA (an example of this type of analysts 1s
shown m Fig. 1).
1. Preparethe calcium phosphate-DNA coprecipitatefor transfection(1 mL of pre-
cipitateis sufficient for transfectionof two cell monolayersin 35-rnm Petri dishes,
one of which should be superinfectedwith the other serving as a mock-infected
control) To 1mL of HeBS add20 pg calf thymuscarrier DNA and 0 5 ng of the
test plasmid. Mix gently, then add 70 uL 2M CaC& and mix rapidly Allow the
precipitateto form for approx 5 min at room temperature. amount of test plas-
This
mtd is suitable for a plasmid of approx 3.5 kbp. Equtmolar amounts of other
plasmids should be used (see Note 2).
2. Remove growth medium from the monolayers and add 0.4 ml/plate of the tine
precipitate.
3. Incubate at 37°C for 45 min then add 2 mL EWplate.
Continue incubation for a further 3 h 15 min at 37°C
4.
5. Pour off medium and wash the cells once with Eagle™s medium Then add 1 mL
25% DMSO m HeBS for 4 mm, pour off, wash once more, and add 2 mL EC5
6. Incubate for a further 2 h at 37°C and then mock-infect or supermfect the cells
with wt HSV-1 at an MO1 of 5 PFU/cell Vnus should be adsorbed at 37°C for 45
219
Transient Assays

M
Ml Z 7R -5 -8 -9 -29-30 -42-52




Fig. 1. An example of a transient DNA replication assay in Sf cells (Section 3.3.).
Sf cells were transfected with plasmid pSTl9, which contains a functional copy of
HSV-I ori, and either mock-infected (MI) or superinfected with the parental
baculovirus, AcRP23lacZ (Z), a mixture of AcNPV recombinants specifying all seven
HSV- 1 DNA replication proteins (7R) or similar mixes from which one recombinant
in turn had been omitted (-5 indicates the mixture from which the UL5 expressing
virus had been omitted, and so on). Total cellular DNA was cleaved with EcoRI (which
cleaves pST19 once to yield a 2.9 kbp fragment) and DpnI, the fragments separated by
agarose gel electrophoresis and transferred to a nylon membrane. Replicated pST19
DNA was detected by hybridization to pTZ 19U (the vector used in the construction of
pST19) DNA, which had been 32P-labeled in vitro by nick translation. Size markers
of 3.8 and 2.8 kbp are shown in lane M. Small fragments resulting from DpnI cleavage
of unreplicated input pSTi9 DNA can be seen in all lanes (except M). A product
corresponding in size to linearized PST 19 DNA is present in lane 7R (arrowhead) and
is indicative of PST 19 replication in the presence of all 7 HSV- 1 DNA replication
proteins. The absence of this band from tracks -5, -8, -9, -29, -30, -42, and -52
confirms that all seven HSV-I proteins are required for efficient DNA synthesis. The
larger band present only in lane Z results from homology between the pTZ 19U probe
and sequences present in AcRP23lacZ but not the other recombinant baculoviruses.
Reproduced from ref. 24 with permission.

min and the monolayers then overlayed with 2 mL EC5 prior to continuation of
incubation at 37™C.
220 stow

7 Twenty-four hours after Initial transfectton, prepare total cellular DNA (see Note
3). Remove medium from cells and wash with TBS Add 2 mL CLB containing
0.5 mg/mL protease to the drained cell monolayer and incubate at 37°C for 2-6 h.
Transfer the cell lysate to suitable tubes, extract sequentially with phenol, then
chloroform and precrprtate nucleic acids with ethanol. Recover the nucleic acids
by centrrfugatlon and redissolve m 100-200 pL TE containing 1X RNase mtx.
8. Drgest a sample of the DNA corresponding to that recovered from approx 2 x 10™
cells with DpnI and an appropriate second enzyme (3 h m a 40-pL final volume in
the presence of 3-5 U of each enzyme 1sadequate usually) and separate the frag-
ments by agarose gel electrophoresis. An appropriate marker IS approx 5 ng of
the transfected plasmid cut with the second enzyme alone. Blot the gel and
hybrrdize to an appropriate labeled probe (see Note 4)
9. After hybridization, wash the filter and detect any signal by autoradiography.
Rephcated plasmtd DNA in the total cellular DNA samples is detected as a band
comtgratmg wtth the marker DNA
3.2. Assay for HSV-1 DNA Replication Gene Function
The assay for HSV-1 orrgm function described earlier was modified to pro-
vide a convenient method for determining the requirement for specific HSV- I-
encoded proteins in vrral orrgm-dependent DNA synthesis (28,19). In thts
approach, cells were transfected wrth a known HSV-1 origin-contaimng plasmid;
and viral helper functions, rather than being provided by a superinfecting vn-us,
were specified by a series of cotransfected recombinant plasmids. With thus
assay Challberg and his colleagues were able to rdentrlj the set of seven HSV- 1
products that are both necessary and sufficient for viral DNA synthesis. In the
initial experiments these genes were all expressed from their own promoters
and two additional plasmids encoding the HSV- 1 transactrvators Vmwl 10 and
Vmw 175 were also requned to be present to actrvate their transcription. Sub-
sequently, It was shown that the requirement for Vmw 175 and Vmw 110 could
be circumvented if expression of the seven replication genes was driven by the
human cytomegalovn-us (HCMV) major IE promoter that allowed constitutive
high level expression (20). This assay provides a convenient method to deter-
mine whether mutations in any of the seven replication proteins affect their
ability to partictpate in viral DNA synthesis. Cells are cotransfected with an
origin plasmrd, a plasmrd encoding the mutated protein and stx other plas-
mids specifying the wt versions of the remaining rephcatron proteins (21).
1. Make up a mix of the DNAs in HeBS. One millihter should contam 1 pg of each
of seven plasmlds encoding the essential HSV-1 DNA rephcatton proteins plus
0.5 pg of plasmtd containing a functional ortgin of replication and 12 pg calf
thymus carrier DNA (see Note 5)
2. Form precrprtate and add to cells (0.4 mL/35 mm dish) as described in Section
3 1 (steps 1 and 2)
Transient Assays 221
3. Feed cells with 2 mL EC5 after 45 min and treat with DMSO 4 h posttransfectron
as described in Section 3.1. (step 5).
4. After DMSO treatment incubate for a further 48 h at 37°C.
5. Prepare total cellular DNA and analyze for replication of the HSV ortgin-
containing plasmid as described in Section 3.1. (steps 7-9).
Weller and colleagues have described a transient replication complementa-
tion (TRC) assaythat combines several of the features of the described proce-
dures (see Sections, 3.1. and 3.2.) and provides an alternative method of
determining whether mutations affect the functions of cloned HSV replication
genes. In this approach the replication gene to be tested is cotransfected with a
plasmid containing a known functional HSV origin, and the cells subsequently
superinfected with a mutant helper virus containing a deletion of the gene of
interest. Amplification of the origin-containing plasmid indicates that the prod-
uct of the transfected gene IS able to particrpate m viral DNA synthesis (22,23).
3.3. Assay for the Ability of Baculovirus Expressed HSV-1 DNA
Replication Proteins to Participate in Viral DNA Synthesis
The high level expression of HSV- 1 DNA replication proteins by recombi-
nant baculoviruses provides an attractive system for the study of the biochemi-
cal activities of wild-type and mutated forms of the proteins. However, it was
also desirable to demonstrate that the heterologously expressed proteins
retained their abtlity to carry out HSV-1 DNA synthesis. The absence of a cell-
free system for origin-dependent HSV-1 replication therefore prompted the
development of a suitable transient assayusing baculovirus recombmants in Sf
insect cells. It was shown that transfection of Sf cells with a plasmid contain-
ing a known functional HSV-1 origin and superinfection with a mixture of
seven recombinants expressing the HSV replication proteins resulted in
amplification of the origin containing plasmid (24). The amplification was
dependent on the presence of all seven HSV-1 DNA replication proteins
and exhibited the important characteristics of viral DNA synthesis in per-
missive mammalian cells. This assay therefore provides a convenient method
by which a single recombinant baculovirus can be used to evaluate the ability
of a mutated replication protein to participate in viral DNA synthesis and
to produce amounts of the mutated protein sufficient for biochemical
investigations.
1, Set up monolayers of Sf cells in Linbro wells the day before required (final cell
number to be approx 4 x 10s cells/2 cm2 well in 1 mL TC 1OOCmedmm).
2 The HSV origin containing plasmrdis introduced by a liposome-mediatedproce-
dure. Separately dilute 0.4 pg plasmid DNA with 0.5 mL TClOO (without serum
or antibiotics), and 15 pL liposomes with 0.5 mL TC 100. MIX the diluted DNA
and liposomesand stand 10 mm at room temperatureprior to application to the
cells (see Note 6) Remove the growth medium from cell monolayers and wash
once with TC 100
3 Add 0.3 mL DNA-liposome mix per well and incubate for 4 h at 28°C.
4. Prepare a mixture of recombmant baculoviruses m TC 1OOCsuch that a final vol-
ume of 150 uL contains 2 x IO6 PFU of each vuus Remove DNA/liposome mix
from wells and add 150 yL/well of the vnus mix (5 PFU/cell of each virus)
5 Incubate for 1 h and add 1 mL TClOOC per well
6 Continue mcubatton at 28°C for a further 48-72 h prior to the preparation of total
cellular DNA. This is done essentially as described m Section 3.1 except that the
cells are more conveniently harvested by gentle pipetmg mto the supernatant
followed by centrifugation for 15 s m a microfuge. Resuspend the cell pellet m
0.4 mL CLB contammg 0 5 mg/mL protemase and Incubate at 37°C for 2-6 h
Extract sequentially with phenol then chloroform and precipitate nucleic acids
with ethanol Recover the nucletc acids by centrifugation and redissolve in 60 pL
TE containing 1X RNase mix
7 Analyze the DNA for the presence of replicated on-contammg plasmtd molecules
as described m Section 3.1. (steps 8 and 9).

3.4. Assays for Encapsidation of HSV-1 DNA
In the described assays,replication of a plasmid contaming an HSV-1 ortgm
m the presence of HSV-DNA repltcation proteins ytelds concatemertc molecules
composed of tandem head-to-tail repeats of the original plasmid. However,

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