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to 37OC,to allow penetration to begm, and at vartous trmes the monolayers are
treated with low-pH buffer to tnacttvate vu-us that has not penetrated the cell
Vnus penetration ISmeasuredin termsof subsequent plaqueformatton andexpressed
as a percentage of the number of plaques formed on control monolayers
To mmimtze penetration during the 4°C adsorptton stage, steps l-4 below are
carried out m a 4°C cold room Warm clothing and gloves are strongly advtsedt
1. Remove the mednun from 90-100% confluent cell monolayers m six-well trays,
replace with cold (4°C) medtum and Incubate at 4°C for 1S-30 mm
2 Briefly somcate vnus stocks (3G60 s), and dilute m precooled ETC,n, to 150-200
PFU/200 yL (see Note 7).
3 Remove the medmm from the wells, and dram thoroughly Add 200 pL virus/well,
and mcubate at 4°C for 60 mm (see Note 8) Note that for each vu-us, one six-well
tray will represent one time-point
4 Remove the vn-us, and wash the cells twice with cold PBS
5 To start penetration, add 2 mL prewarmed ETClo (37”C), and transfer the cells to
a 37™C incubator This represents 0 time Agam, add the overlay to all viruses for
each time-point together the first set added should represent the last time-pomt
handled, whereas the last set added should be the first time-point harvested (see
Note 9)
6. At the relevant time-pomt, remove the medium from the trays, and add 1 mL PBS
to three wells (control), and 1 mL citrate buffer, pH 3 0, to the remammg 3 wells
of each tray Incubate for 3 mm at room temperature with gentle shaking It IS
important to include a set of PBS control wells for each time-pomt, since abso-
lute plaque numbers do vary from tray to tray (see ref 13)
7. Remove the buffer and wash the cells twice with PBS, shaking 5-l 0 s each wash
8 Dram the wells thoroughly, then add 2 5 mL MC5, and incubate at 37°C until
plaques are clearly visible
9 Stain and count as described above (Section 3.2 )
10 Penetration is measured as the percentage of acid-resistant vu-us with time
Each time-point = (avg PFU on low pH-treated wells/
(3)
avg PFU on PBS-treated wells) x 100
HSV Entry and Spread 15

3.5. Virus Release
To measure the effictency of release of vu-us particles during infection, the
percentage of total mfecttous progeny virus that 1s present within the extracellu-
lar medium is measured with time, following rnfectton at either high or low
multtphcity. Infection at high multiphctty (5-20 PFU/cell) follows mfectton
through one infectious cycle. Infection at low multtplictty (0.001 PFU/cell) allows
multiple cycles and may amphfy small dtfferences m overall virus growth
1 Briefly sonicate vtrus stocks (30-60 s), and drlute to either 5 x 1O7PFU/mL (for
mfectron at 5 PFU/cell, assuming 5 x 1O6 ceils/60-mm Petri dish), or 1 x lo4
PFU/mL (for mfectton at 0 001 PFU/cell) (see Notes 10 and 11)
2. Remove the medium from 90-100% confluent cell monolayers m 60-mm Petri
dishes Add 0 5 mL vn-us/plate, and incubate at 37°C for 1 h.
3. Remove the virus moculum Add 1 mL citrate buffer, pH 3 0, per plate, and mcu-
bate at room temperature for 2-3 min (see Note 12)
4 Remove the buffer, wash the cells twice with PBS, and dram the monolayers
thoroughly.
5. Add 2 mL ECs/plate (EC, If the monolayers are very confluent), and incubate the
cells at 37™C
6 At the relevant time-points, remove the medium to a glass biJou bottle (or alter-
native vial suitable for sonicanon), and measure the volume Store on ice. This
represents the released or supernatant, vnus (SV)
7 Wash the monolayer gently twice wrth PBS-If any cells detach, recover these by
centrtfugatton (e g., Fisons coolspm, 2000 rpm, 5-10 mm). Add 2 mL EC5 to the
monolayer, scrape the cells mto the medium (e g , using a commercial cell
scraper, or a plunger from a sterile syringe), and transfer to a glass bijou Pool
any cells recovered from the washes. Store on Ice This represents the cell-asso-
ciated (CA) virus.
8. Sonicate the SV stocks briefly (30-60 s), and the CA stocks until clarified (around
3 x 60 s) Store the SV and CA virus stocks at -70°C until they can be titrated.
9. Quantttate the yields of infectious vnus by titration, This IS basically the method
described m Chapter 1 with some minor modrficattons Prepare serial lo-fold
dilutions of virus in a total volume of 1 mL ETC,, m 5-mL bijou bottles, mtxmg
the dtlutrons by swuhng Because of the large number of tttrattons bemg handled,
it is relatively easy to contaminate your hands with hqutd from the hds when
opening and closing the vials-swirling IS less hkely than vortexmg to result m
virus contammatton on the lids of the vials Virus should then be plated m duph-
cate on 60-mm Petri dishes, using 200 pL/plate and adsorbed for 1 h at 37°C. To
minimize secondary spread of virus, the moculum is removed before overlaying
with MCS. Note that MCs contains I .5%, not l%, carboxymethylcellulose
10. Calculate the yield of virus m the CA and SV samples, since well as the percent-
age of progeny vnus that IS released at the different time-pomts.
Virus yield = titer x volume
(4)
%SV = [SV yield/total vtrus yield (SV + CA yield)] x 100
16 MacLean

3.6. Cell-to-Cell Spread
Cell-to-cell spread can be assayedby infecting cells at low multiphclty and
allowmg the virus to grow under condltrons that limit the extracellular spread
of virus. We standardly use commercial pooled human serum, which contams
high levels of neutralizing anttbody to HSV m the overlay medium Neutrahz-
ing monoclonal antIbodIes (MAb) could also be used.
1 Brlefly somcate the virus stocks (30-60 s), and dilute to 1 x lo4 PFU/mL (0 001
PFU/cell) (see Notes 13 and 14)
2. Remove the medmm from 90-100% confluent cell monolayers m 60-mm Petri
dishes Add 0 5 mL virus/plate, and incubate at 37°C for 1 h
3 Remove the virus moculum, and add 1 mL citrate buffer, pH 3 0, per plate, and
Incubate at room temperature for 2-3 mm
4 Remove the buffer, wash the cells twice with PBS, and dram the monolayers
thoroughly
5 Add either 2 mL ETCIO or 2 mL Ehu/plate, and incubate the cells at 37°C
6 At the relevant time-points, remove the medium If desired, the supernatant from
the ETClo-treated plates can be kept, and treated as m Sectton 3 5 It 1s also a good
Idea to titrate one or two of the later EHu supernatants to monitor neutrahsatlon
7 Wash the monolayers gently twice with PBS-If any cells detach, recover these
by centrifugatlon (e.g., Fisons coolspm, 2000 rpm, 5-10 mm) Add 2 mL EC, to
the monolayer, scrape the cells mto the medmm, and transfer to a glass bijou
Pool any cells recovered from the washes. Store on Ice
8. Somcate the SV stocks briefly (3MO s), and the CA stocks until clarified (around
3-60 s) Store at -70°C
9 Quantltate the yields of mfectlous virus by titration (see Section 3 5 , step 9)
Vu-us yield = titer x volume (5)
Calculate the ratlo of CA yields under human serum compared to normal medmm,
for each virus

4. Notes
1 If preferred, radiolabeled virlons can be prepared following mfectlon at high
multlphclty (low multlpllclty of mfectlon is simply more economical with virus
stocks) In this case, infection 1s carried out at 37°C Use 5 PFU/cell, Infect m
Emet/SC$, add the radlolabel at 4 h pi, and harvest around 24 h pi
2. Large pellets after F˜oll gradient centrifugatlon can suggest either the presence
of cell debris or overheating of the Flcoll during preparation of the solutions
This can conslderably reduce the yield of purified vlrlons F˜coll solutions can be
prepared by leaving overnight in the refrigerator to dissolve, and only minor
warming with stirring 1s then required to get the Flcoll mto solution Solutions
are cooled to 4°C before gradients are prepared
3 Petri dishes (35-mm) can be used instead of six-well trays We use the latter,
smce they are easier to handle for the washing stages
17
HSV Entry and Spread
4, The mam problem wtth thts assay 1sm handling the samples quickly enough It 1s
therefore important to be well-orgamzed before startmg, e g., have all the trays
and scmtillatton vials labeled, and have all medta, buffers, pipets, and so forth,
available. For I7syn+, a reasonably detailed time-course would be 0, 5, 10, 15,
20, 30, 45, 60, 90, and 120 mm postvnus addrtton We find that, with practice,
one person can handle 5-mm time gaps for two viruses
5 No blocking step IS mcluded in this procedure.
6 Virus stocks are titrated shortly before use, adsorbmg the virus at 37°C for 1 h
under condtttons rdenttcal to those used m the adsorptron studres. Best results are
obtained If the peak or final plaque counts are between 100 and 250 For 35-mm
wells, counts above 300 are usually inaccurate The actual number of PFU added
to the wells can often differ by up to threefold from that expected. This IS prob-
ably owing at least partly to dilution error To mmimrze variatton, we keep a
stock vial of each vnus specifically for these expertments and regularly recheck
titers. We find errors are less if at least 50 uL of sonicated virus stock are used to
prepare serial 1O-fold diluttons, and these then used to generate the final dilution
requtred. It IS also sensrble to test regularly the accuracy of the ptpets used to
prepare the dtluttonst
To calculate the amount of vtrus to use in these experiments, titrate vtrus stocks
at 4°C on 35-mm Petrt dtshes/srx-well trays under condmons tdenttcal to those to
be used for adsorption m the penetratton assay
In practtce, mcubatton at 4°C does allow some penetratton. Although some labo-
ratories cool cells on Ice, we find that BHK Cl3 cells do not survive well at this
temperature. Monolayers become loose after even short pertods at 0°C and are
often lost If the base level of penetratton IS too high, vnus can be Incubated at
4°C for shorter pertods (10-15 mm) before shiftmg to 37°C
Again, the mam problem with this assay IS m handling the time-pomts qurckly
enough. Be well orgamzed before startmg-have all buffers, ptpets, and so forth,
ready, and all six-well dishes correctly labeled For 17syn+, penetratton has usu-
ally reached 80-l 00% by 20-30 mm, and so a reasonable time-course would be
0, 5, 10, 15, 20, 30, 45, and 60 min after temperature shift. For more detail, we
occasronally use 3-mm trme-pomts up to 21 mm. Handlmg these time-points 1s
sigmficantly easrer If two people work together.
10 Ideally, vnuses should have been recently trtrated on the same batch of cells used
m the growth experiments. The input dtlutions should also be tttrated, etther
nnmedtately after use, or followmg storage at -70°C.
11. A reasonably detatled ttme-course would be. 0, 2,4, 6, 8, 10, 12, 16, 20, 24, and
32 h PI, following Infection at high multtphcity; and 0,4,8, 12,24,36,48,60,72,
and 96 h pt, following Infection at low multtphctty 17syn+ usually reaches a
plateau for total virus yield around 16 h pi, and for SV yields around 24-32 h pr
(high MOI), or around 36-48 h PI, and 60-96 h PI, respecttvely (low MOI)
Because of the numbers of samples (and titrations) Involved, and because results
can depend very much on the health of the cells, we tend to use only a smgle plate
per ttme-point per vnus, repeating the experiment a further l-2 ttmes
MacLean
18
12 The addrtton of citrate buffer pH 3.0 serves to mactivate any remammg,
nonpenetrated virus, ensuring a synchronous mfectron and givmg more accurate
values for progeny vtrus titers at early ttmes. This step IS particularly important
following a high multtphcity mfection
13. Ideally, viruses should have been recently titrated on the same batch of cells used
m the growth experiments The input dilutions should also be tttrated, either
tmmedtately after use or followmg storage at -7O™C
14. Reasonable time-points are 0, 12,24,36,48, 60, 72, and 96 h pi Assuming only
two or three vtruses were being compared, these time-points would usually be
carried out m duplicate for each vuus

References
1. Spear, P G (1993a) Membrane fusion induced by herpes simplex vnus, m Vzrul
Fusion Mechanwns (Ben& J , ed ), CRC, Boca Raton, FL, pp 201-232
2 Spear, P G (1993b) Entry of alphaherpesvnuses mto cells. Sem Vwologv 4, 167-180
3, Sztlagi, J F and Cunningham C (199 1) Identtflcatton and charactertzatton of a novel
noninfectious herpes simplex virus-related particle J Gen Vzrology 72, 66 l-668
4 Campadelh-Flume, G , Arsenakis, M., Farabegoli, F , and Rotzman, B (1988) Entry
of herpes simplex virus 1 m BJ cells that constitutively express viral glycoprotem D
is by endocytosts and results m degradation of the vnus J Vzrol 62, 159-l 67
5 Johnson, R M and Spear, P G (1989) Herpes simplex vnus glycoprotem D
mediates Interference wtth herpes simplex virus mfection J Vwol 63,8 19-827
6 Shteh, M -T , WuDunn, D , Montgomery, R I, Esko, J D , and Spear, P G
(1992) Cell surface receptors for herpes simplex virus are heparan sulfate
proteoglycans J Cell Bzol 116, 1273-1281
7 Gruenhetd, S , Gatzke, L., Meadows, H , and Tufaro, F (1993) Herpes simplex
vtrus infection and propagation m a mouse L cell mutant lacking heparan sulfate
proteoglycans J Vwol 67, 93-100
8 Banfield, B W., Leduc, Y , Esford, L., Schubert, K , and Tufaro, F (1995)
Sequenttal tsolatton of proteoglycan synthesis mutants by usmg herpes stmplex
virus as a selective agent. evidence for a proteoglycan-independent virus entry
pathway. J Vwol 69, 3290-3298
9 Herold, B C , WuDunn, D , Soltys, N., and Spear, P G (1991) Glycoprotem C of
herpes simplex vnus type 1 plays a prmcipal role m the adsorptton of virus to cells
and in mfecttvity. J Vzrol 65, 109&1098
10 Karger, A and Mettenleiter, T C ( 1993) Glycoprotems gII1 and gp50 play domi-
nant roles in the btphasic attachment of pseudorabies virus Vwology 194,654-664.
11. Huang, A. and Wagner, R (1964) Penetration of herpes simplex vnus mto human
eptdermotd cells Proc Sot Exp Btol Med. 116,863-869
12. Highlander, S. L., Sutherland, S L , Gage, P. J , Johnson, D C , Levine, M., and
Glortoso, J C (1987) Neurtrahzing monoclonal antibodies specific for herpes
simplex virus glycoprotem D inhibit vnus penetratton J Viral 61, 33563364
13. Mettenletter, T. C. (1989) Glycoprotem gII1 deletton mutants of pseudorabtes VI-
rus are impaired m vtrus entry Vvology 171, 623-625
3
Preparation of HSV-DNA
and Production of Infectious Virus
Ala&air R. MacLean


1. Introduction
This chapter deals with (1) the preparation of herpes simplex vn-us (HSV)
virlon DNA of a quality and purity suitable to be used for the generatlon of
infectious vn-us, and (2) its use m the preparation of infectious vn-us. An Import-
ant development m the understanding of virus genetics and gene products has
been the ability to carry out reverse genetics. This is dependent on the ability to
manipulate the genome m vitro and reconstitute mfectlous virus. Our under-
standing of DNA viruses and positive stranded RNA viruses (where DNA and
RNA/cDNA, respectively, are generally mfectlous) ISconsiderably greater than
for negative stranded RNA viruses, where until recently, it had been Impossl-
ble to generate vm.˜s from either RNA or cDNA. Wlthm the herpesvmdae,
knowledge of the function of HSV gene products is one of the more advanced
owmg to the relatively straightforward techniques required to generate vn-us
from HSV-DNA, and to introduce desired mutations by cloning small parts of
the genome, manipulating them, and then remtroducmg the mutations by a
process of cotransfectlon and m VIVOrecombmatlon with Intact vn-us DNA.
Other a-herpesviruses, such as EHV- 1 and PRV, are equally amenable to such
mampulatlon, and knowledge of their gene products is also well advanced. In
contrast, this technology IS only now, and with much less success,being applied
to other members of the family, such as EBV, HCMV, and VZV, and knowl-
edge of their genetics IS much less advanced. The use of cosmlds to reconstl-
tute intact virus will aid in the advance of knowledge for these viruses. For
examples of uses of recombinant DNA technology, the reader IS referred to
other chapters (especially those on cloning and mutagenesis). I will concen-
trate on the techniques currently m use in my laboratory, but will also mention
From Methods m Molecular Medune, Vol 10 Herpes Smplex Vws Protocols
Edlted by S M Brown and A R MacLean Humana Press Inc , Totowa, NJ

19
20 MacLean

other techmques m use elsewhere that may be more appropriate m certain
cell types.
2. Purification of HSV-DNA
This chapter deals only with the purification of vlrlon HSV-DNA for use m
transfection procedures, although such DNA can also be used for analysis of
genome structure by restrlctlon enzyme digestion and Southern blotting. More
rapid small-scale procedures for the purification of infected cell DNA for
Southern blotting, are described elsewhere m thrs book. The integrity of HSV-
DNA 1s absolutely crucial for Its mfectlvlty. Because of Its high molecular
weight (150 kbp), HSV-DNA 1s easily fragmented, and must therefore be
handled gently with no vortexing, vigorous shaking, or plpetmg. It 1simportant
that there IS no contammatmg nuclear DNA m the preparation, since cellular
and nonmfectlous concatemerlc HSV-DNA will mhlblt the transfectlon eff-
clency by lowering the ratio of mfectlous to nonmfectlous DNA.
2.1. Protoco/
Our standard cell line for HSV growth 1sBHK 2 l/C 13 cells (I), but any cell
lme permissive for HSV growth (such as Vero or CVl cells) can be used.
1 Ninety percent confluent BHK 21/C13 cells are infected at a multiplicity of mfec-
tlon (MOI) of 0 003 plaque-formmg units (PFU)/cell m a minimal volume of

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