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because these products lack specific cu-acting sequencesrequired for the stte-
specific cleavage of the concatemeric DNA and its encapstdation mto vn-us par-
ticles, they are not further processed or packaged. It is now known that the
essential sequencesreside wtthm the viral a sequence,an approx 400-bp element
that occurs as direct repeats at the genomic termini and m mverted orientation at
the junction between the L and S segments.The insertion of the a sequence into
the origin-containing plasmtd allows the concatemertc rephcatton products to be
cleaved and encapsidated when transfected cells are supermfected with wild-
type helper vtrus (25). In contrast to nonencapsidated DNA, the packaged mol-
ecules are resistant to the action of exogenously added DNase and can be further
propagated as defective genomes when virus progeny from the transfected and
superinfected cells are used to Infect fresh cell monolayers. These properties
provide the basis of convenient assaysfor packaging of HSV DNA that have
proved useful for definmg the encapsidation signals and should have application
in elucidating and characterizing the viral gene products that are required.

3.41. Assay for Encapsidated (DNase-resistant) DNA
1 Transfect BHK cells with a plasmid containing a known HSV origin and
sequences to be tested for packagmg activity, supermfect with wt HSV-1 and
process as described in Section 3 1 , steps 1-6.
Transient Assays 223

2. Twenty-four hours pi, remove medium from monolayers and scrape the cells into
1 mL RSB.
3. Transfer to a 5-mL plastic tube and add 50 pL 10% NP40 to lyse the cells fol-
lowed by 5 pL of a IO-mg/mL stock solution of DNase I (see Note 7).
4 Incubate for 2 h at 37™C
5 Add 1 mL 2X CLB contaming 1 mg/mL proteinase and incubate for a further 2-6 h
at 37™C
6 Extract the DNA sequentially with phenol and chloroform and analyze for the
presence of replicated plasmid sequences as described m Section 3.1. steps 7-9
The presence of a DpnI resistant fragment m DNase I treated samples that
comigrates with the marker DNA is indicative that the test plasmid has been both
replicated and encapsidated mto virus particles (see Note 8)
3.4.2. Assay for Propagation
of Plasmid Molecules as Defective Genomes
1 Carry out transfection and superinfection of BHK cell monolayers as described
in Section 3.4.1,) step 1.
2 Twenty-four hours PI, scrape the cells into the growth medium and somcate ex-
tensively to prepare a vuus stock from the transfected cells
3 To determine whether amplified plasmid sequences have been encapsidated as
defective genomes, this stock is further passaged at high MOI. Use 0.5 mL of the
stock to infect a fresh monolayer of BHK cells in a 35-mm Petri dish. Allow virus
to adsorb for 1 h at 37°C remove moculum, and add 2 mL EC5 to the cells
4. Incubate at 37°C for 24 h and prepare and analyze total cell DNA for the pres-
ence of replicated plasmid sequences as described in Section 3.1. Sequences cor-
responding to the test plasmid will be detected only if it IS both replicated and
packaged into vnus particles m the original transfected cells (see Note 9)

4. Notes
1. HSV-1 oris and orit share considerable sequence homology and appear to be
essentially equivalent m transient replication assays. Although both origins con-
tam palindromic sequence elements, the palindrome m oriL is sigmficantly longer
and is susceptible to deletions when cloned m most E coli strains. In contrast, the
oris sequence is stable on clonmg and plasmrds contammg it therefore are more
frequently employed m the replication assays (I-3).
2 The efficiency of the calcium phosphate transfection procedure is influenced by
several factors, including the concentrations of plasmid and carrier DNAs and
the pH of HeBS, and these should be optimized. A convenient method for evalu-
ating and comparing transfection efficiencies is to use a plasmid that expresses
the E coEzZacZ gene from a strong constitutively expressed promoter (e.g., the
HCMV major IE promoter; see Section 3.2.) and to stain and count cells express-
mg the P-galactostdase product (26). The incluston of such a plasmid can also
provide a valuable internal control to confirm that the transfectron efficiency does
not vary significantly between different plates withm an experiment, Alternative
224 stow

methods for mtroducmg plasmid DNAs mto cells (e.g , hpofection, electro-
poration, and various modifications to the calcmm phosphate procedure) are prob-
ably suitable for transient rephcation assays, with the optimal procedure being
influenced by the cell type used.
3. In some experiments it may be desirable to isolate DNA from the nuclei of trans-
fected cells. A simple method of preparmg nuclei ts to lyse the cells m RSB
containmg 0 5% NP40 and to recover the nuclei by centrifugation at 6008 for 2
min. The nuclei are resuspended and DNA isolated followmg digestion with pro-
tease in CLB as described for total cellular DNA (27).
4. Although we use probes labeled m vitro with 32P by either nick translation or
random priming, nonradioactive probes should, in prmciple, be equally suitable.
5 In our hands the introduction of multiple plasmid species mto cells seems to be
much more effective using the calcium phosphate precipitation method than
hpofection, but the reason for this is not clear.
6 Sf cells respond much better to lipofection than to calcium phosphate transfec-
tion that has a cytotoxic effect and causes significant disruption of cell mono-
layers A variety of reagents for hposome-mediated transfectron are available
commercially, and the lipid composition of homemade preparations can be var-
ied to optimize transfection efficiency.
7. Initially sonication rather than exposure to NP40 was used to break open the cells
prior to DNase I treatment (25), but m practice the latter method (28) proved to
be simpler and more reproducible.
8. DNA fragments reststant to DpnI cleavage, but smaller than the input plasmtd,
also will be detectable at lower abundance. These represent the terminal frag-
ments of packaged molecules generated by specific cleavage of concatemers at
the a sequence during the encapsidation process (25,29)
9. Although the use of virus supermfection to provide helper functions for replica-
tion and encapsrdation represents a convenient and reproducible method to ana-
lyze DNA encapudatlon, this is not the best method to generate stocks containing
the maximum proportion of defective genomes. If this is the objective it is better
to provide the helper functions by cotransfection with intact infectious HSV-I
DNA (6˜7).

References
1 Challberg, M D (199 1) Herpes simplex virus DNA replication Semin Vzrol 2,
247-256.
2. Weller, S. K. (199 1) Genetic analysis of HSV genes required for genome rephca-
tion, m Herpewrus Transcrrption and Its Regulation (Wagner, E K , ed.), CRC,
Boca Raton, FL, pp 105-135.
3. Olivo, P. D. and Challberg, M. D. (1991) Functional analysis of the herpes stm-
plex vuus gene products involved in DNA replication, m Herpesvzrus Trunscrzp-
tzon and Its Regulatzon (Wagner, E. K., ed.), CRC, Boca Raton, FL, pp. 137-150
4 Frenkel, N., Locker, H., and Vlazny, D. A. (1980) Studies of defective herpes
simplex viruses. Ann NY Acac SCL 354, 347-370.
Transient Assays 225
5. Cuifo, D. M. and Hayward, G. S. (1981) Tandem repeat defective DNA from the
L segment of the HSV genome, in Herpesvwus DNA (Becker, Y., ed ), Martmus
Nijhoff, The Netherlands, pp 107-128.
6. Vlazny, D. A. and Frenkel, N. (198 1) Replication of herpes stmplex vtrus DNA:
localization of replication recogmtion signals within defective vu-us genomes.
Proc Nat1 Acad. Sci USA 78,742-746.
7. Spaete, R. R. and Frenkel, N. (1982) The herpes simplex virus amphcon* a new
eucaryotic defective-vuus cloning-amplifying vector. Cell 30,295-304.
8. Stow, N. D (1982) Localization of an ongin of DNA replication wtthin the TRs/
IRS repeated region of the herpes simplex virus type 1 genome. EMBO J 1,863-867.
9. MacPherson, I. and Stoker, M (1962) Polyoma transformatton of hamster cell
clones-an investigation of genetic factors affecting cell competence. Virology
16,147-151.
10. Kitts, P A., Ayres, M. D., and Possee, R. D. (1990) Lineartsation of baculovnus
DNA enhances the recovery of recombinant vuus expression vectors Nucleic
Acids Res. 18,5667-5672
11. Rose, J K., Buonocore, L., and Whitt, M. A. (1991) A new cattomc hposome
reagent mediating nearly quantitative transfectton of animal cells Biotechnzques
10,52@-525.
12 Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H W., Wenz, M.,
Northrop, J. P., Ringold, G. M., and Danielsen, M. (1987) Lipofectton: a highly
efficient lipid-mediated DNA transfection procedure. Proc Nat1 Acad. SCL USA
84,7413-7417.
13 Sambrook, J., Frttsch, E. F , and Mamatts, T. (eds ) (1989) Molecular Cloning A
Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
14 Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D , Setdman, J. G., Smith,
J. A., and Struhl, K. (eds.) (1987) Current Protocols in Molecular Bzology,
Wiley, NY
15. Twigg, A. J. and Sherratt, D. (1980) Tram-complementable copy number mutants
of plasmid ColEI Nature 283,2 16-2 18
16. Vieira, J. and Messing, J. (1982) The pUC plasmids, an M 13mp7-derived system
for insertion mutagenesis and sequencing with synthetic universal primers. Gene
19,259--268
17. Mead, D. A., Szczesna-Skorupa, E., and Kemper, B. (1986) Single stranded DNA
“blue” T7 promoter plasmids. a versatile tandem promoter system for cloning and
protein engmeermg. Prot Eng 1,67-74.
18. Challberg, M. D. (1986) A method for identifying the viral genes required for
herpesvirus DNA replication. Proc. Nat1 Acad Scz. USA 83,9094-9098.
19. Wu, C. A , Nelson, N. J., McGeoch, D. J., and Challberg, M D. (1988) Identtfica-
tion of herpes simplex vuus type 1 genes required for origin-dependent DNA
syntheses. J Vwol. 62,435-443,
20. Heilbronn, R. and zur Hausen, H. (1989) A subset of herpes simplex virus rephca-
tion genes induces DNA amplification within the host cell genome. J. Vlrol 63,
3683-3692.
stow
226

21. Stow, N D., Hammarsten, O., Arbuckle, M. I., and Elias, P (1993) Inhibition of
herpes simplex vu-us type 1 DNA replication by mutant forms of the origm-bmd-
mg protein I/iroZogy 196,413-418
22. Martinez, R., Shao, L., and Weller, S. K (1992) The conserved hehcase motifs of
the herpes simplex virus type 1 origin-bmdmg protein UL9 are important for func-
tion. J Vwol 66,6735-6746
23. Zhu, L. and Weller, S K (1992) The six conserved heltcase motifs of the UL5
gene product, a component of the herpes simplex vnus type 1 hellcase-primase,
are essential for its function. J Vzrol 66,469-479
24. Stow, N D. (1992) Herpes simplex virus origin-dependent DNA replication m
insect cells using recombinant baculovnuses. J Gen Vu-01 73,3 13-32 1
25 Stow, N. D , McMonagle, E. C , and Davison, A J. (1983) Fragments from both
termmi of the herpes stmplex vnus type 1 genome contain signals required for the
encapsidatlon of viral DNA. Nucleic Acids Res. 11, 8205-8220.
26 Ho, D Y. and Mocarski, E S. (1988) P-galactosldase as a marker in the perlph-
era1 and neural tissues of the herpes simplex virus-infected mouse. VzroZogy 167,
279-283.
27 Stow, N D. and Hay, R. T (1993) Viral DNA replication, m Molecular Vzrology
A Practical Approach (Davison, A J and Elliott, R. M , eds.), IRL, Oxford, pp
75-107
28 Stow, N. D and Stow, E C. (1986) Isolation and characterization of a herpes
simplex vnus type 1 mutant containing a deletion within the gene encoding the
Immediate-early polypeptide Vmw I 10 J. Gen Vu-01 67,257 l-2585.
29 Stow, N D., Murray, M D., and Stow, E C. (1986) Cls-acting signals Involved m
the replication and packaging of herpes simplex VU-US type 1 DNA, m Cancer
Cells 4 DNA Tumor Vrruses, Control of Gene Expression and Repllcatlon
(Botchan, M , Grodzlcker, T , and Sharp, P., eds.), Cold Sprmg Harbor Labora-
tory, Cold Spring Harbor, NY, pp. 497-507
HSV Amplicons in Gene Therapy
Niza Frenkel and Ronit Sarid


1. Introduction
Herpes simplex vn-us (HSV) amplicons are defecttve vnus vectors capable of
introducmg amplified foreign genes into vartable types of eukaryottc cells, such as
fibroblasts, macrophages, gha, and neurons in dtfferent organisms including ro-
dents, monkeys, and human (refs. 1-3; reviewed tn ref. 4). The defective vnuses
follow their nondefective counterparts in the ability to infect mitotic, as well as
postmitottc cells. This makes them potentially useful vectors for use m nondtvid-
mg cells, such as in nerve cells. Available retrovn-us vectors employed to date for
gene therapy require cell division and therefore cannot be used to target neurons.
HSV resides latently in the host, from which it is reactivated, producmg recur-
rent infections (5-7). HSV latency takesplace in ganglia, with the vnus traveling
through nerve cells moving from the sites of replication to the sites of latency.
Being a neurotropic vector, HSV is used to localize nerve cell connections syn-
aptrcally between peripheral sites of entry and the central nervous system and
mterbrain conections (reviewd in refs. 8 and 9). During latency, the vnus enters
a benign stable state that does not alter electrophystological properties of the
cells (IO, 12). Recently, Farkas and colleagues have examined the electrophysi-
ological properties of cholmergrc and dopammergtc neurons after mfectton with
WV-l-derived vectors for a few days and have found no alterations (12). These
neurotroptc properties of the virus have made tt an attractive vector for
experimantatton in nerve cells in vivo. Actually, constructed amplicon-type vec-
tors were shown to be expressed in neuronal cells in vitro (13,14), as well as in
nondrvtdmg neurons m the adult animal in vivo (15-18)
Since the amplicons are defective vnus vectors, they depend on the helper
virus that supplies in tram DNA replication functions needed for the replica-
tion of the amplicon DNA sequences, as well as trans-acting vtrton polypep-
From Methods m Molecular Medwne, Vol IO Herpes Sfmplex Vtrus Protocols
Edlted by S M Brown and A R MacLean Humana Press Inc , Totowa, NJ

227
228 Frenkel and Sarid
tides and functions required for the packaging of the replicated amplicon DNA
(19). In addition, the helper virus must contain specialized functions, essential
for gene expression, within the target cells and tissues. Cotransfection of cells
with the seed amphcon along with helper virus DNA, or alternatively, trans-
fection of cells with amplicon DNA and then superinfection of the cells with
the helper virus result m virus stocks that typically constst of two components:
(1) Defective genomes containing multtple identical head-to-tail repeats of the
HSV seed amplicon, most likely arising by rolling circle replication. The
concatomeric genomes are cleaved to unit-length genomes at the cleavage/
packaging signals (pat-l-pat-2 signals), which they contain. The full size of
packaged defective virus particles is close to 150 kbp, which is the size of the
HSV-I genome (2&22) Therefore, defective genomes and standard genomes
are not separable by then DNA sizes m the vn-us stocks. (2) Helper viruses:
The resultant virus stocks can be routinely propagated at high multiplicity of
infection (MOI) for several passagesto amplify defective-to-helper virus ratios.
The state of the input amplicons m resultant defective virus stocks is analyzed,
e.g., by restrtction enzyme analyzes of viral DNAs, by using marker genes to
quantify the ratios of helper and defective viruses in Southern blots, or employ-
mg P-galactosidase as an expressed marker gene (23,24).
Virus propagation at high MO1 results m typical fluctuations m infectious
virus yield, reflecting the fact that the cells are dually infected with the helper/
standard vnus and the defective genomes. After efficient replication of defective
genomes, above the threshold level, the stocks with very high ratios of defective
genomes were found to interfere with the replication of the standard vn-us, even-
tually reducing the production of the helper vnus to minimal infectious virus/
cell, eliminating altogether the standard replicating vnuses in the majority of the
cells. At that point in the cycle, in the absence of helper virus, the defective
viruses cannot be replicated, the ratio of helper-to-defective virus was found to
be reversed, interference was reduced, the series recovered, and repeatedly so.
The amplicon-derived genomes replicate efficiently and have advantages
over the helper virus DNA. The 152 kbp of HSV- 1 DNA contain three origins
of replication. By comparison, the vectors contain one origin of replication in
each repeat unit sizes smaller than 15 kbp (reviewed in refs. 7 and 19).
Inasmuch as the amplicon depends on the helper vnus for its preparation
and large-scale preparation of the amplicon stocks, they are potentially hazard-
ous owing to the lytic properties of HSV. This will be dealt with later in the
text, where amplicon and helper combmation will be dealt with specifically.
Once generated, virus stocks containing mixtures of helper vnus and ample
constructed amplicons can be potentially used for experimental studies aimed
at future development of gene therapy.The stocks containing defined ratios of
helper virus and amplicon can be repeatedly reproduced.
HSV Amp/icons 229
The defective viruses cannot propagate and spread m the recipient cells in
the absence of then helper virus counterparts, as documented m vitro using
wild-type (19,25), as well as mutant helper viruses (13,26,27).
1.1. Amp/icon Plasmid Properties
The amphcon contams three kmds of genetic elements:
1. Sequences that allow Its propagation in bacteria, including the Eschenchza coli

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