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sis: the tegument of the infecting virion contains the product of the UL41 gene,
the virion host shutoff (VHS) function, which degrades RNA molecules (4).

From Methods III Molecular Medmne, Vol 70 Herpes Smplex Vms Protocols
Edlted by S M Brown and A R MacLean Humana Press Inc , Totowa, NJ

193
Phelan and Clements
194
HSV- 1 mfectlon has wide-ranging effects on posttranscriptlonal processes.
Nuclear extracts from HSV-l-infected cells demonstrate an increase in the
efficiency of usage of certam poly (A) sites,predominantly sites that are mher-
ently weak, and the increase appears to be owing to an enhancement m the
binding of polyadenylatlon factors to their substrate RNAs This increase m
polyadenylatlon efficiencies is reliant on the IE63 protein, but its precise mode
of action 1sunclear. HSV infection also causesan mhlbltion of RNA splicing,
both m vitro and m vlvo (5,6), which required IE63. This mhlbltory effect 1s
thought to be caused by an effect whereby the splicing snRNPs are redistrib-
uted from a general diffuse speckled pattern to that of a distinctly punctate
pattern wlthm the infected cell nucleus: IE63 1snecessary and sufficient for
this effect (7,8) In this way, the snRNPs are removed from the active sites of
transcription and splicing, and a consequent mhlbltlon of splicing would have
a severe effect on expression of the extensively spliced host genome while
having relatively little effect on processmg of viral mRNAs.
The establishment of in vitro systemsthat faithfully reproduce the events of
cleavage, polyadenylation, and splicing m crude cell extracts using RNAs that
resemble authentic in vlvo pre-mRNAs was a huge step forward on the road to
deciphering the mechanism and components required for pre-mRNA process-
mg. These in vitro systemsfacilitate the analysis of effects of virus gene prod-
ucts on these processes.
2. Materials
2.1. Cells and Media
Mammalian cell nuclear extracts permissive for HSV- 1 infection and active
for posttranscriptional processes,such aspolyadenylatlon and sphcmg, are nor-
mally generated from HeLa cells.
HeLa cells are maintained m Dulbecco™s modified Eagle™s medium (DMEM)
supplemented with 5% newborn calf serum, 2 mM L-glutamme, 1000 p.g/mL
pemcillm, and 100 pg/mL streptomycin. HeLa cells are grown at 37™C m an
atmosphere containing 5% (v/v) carbon dioxide. Cell monolayers m plastic dishes
(approx 2.5 x lo7 cells/l40mrn plate) are seeded the day before required. Fol-
lowing infection with HSVl, the cells are harvested as described later.
2.2. Other Reagents
1 Nuclear extraction buffer A* 10 mA4HEPES,pH 8.0, 1.5 mM M&l,, 10 mM
KCI, mM dlthlothreitol (DTT).
2. Nuclear extraction buffer C: 20 mM HEPES,pH 8.0, 1.5 & MgCl,, 25% (v/v)
glycerol, 420 mM NaCl, 0.2 mM EDTA, pH 8 0, 1 mM DTT, 0.5 mM phenyl
methyl sulfonyl fluoride (PMSF)
3 Phosphate-buffered salme (PBS)
195
In Vitro Systems
4 Linearized m vitro transcriptton DNA template wtth Sp6, T7, or T3 RNA poly-
merase promoter
5. In vitro RNA transcription stock reagents. bovine serum albumin (type V) 4 mg/mL,
O.lMDTT, 10 rnA4CAP (G[S™]ppp[S]GOH), RNasin (30,000 p/mL), 1 mA4each
of rATP, rGTP, rCTP, and rUTP, 5X Sp6/T7/T3 transcription reaction buffer
(Promega), Sp6/T7 RNA polymerase (15,000 p/mL), DNase (1000 p/mL)
6. Radio-isotope [u˜˜P] rUTP (800 Ci/mmol)
7. Water saturated phenol chloroform.
8. In vitro polyadenylatton stock solutions 25 mM cordycepm, 50 mM creatme phos-
phate, 10 mMTns-HCl, pH 7 6,25% PEG, proteinase K 20 mg/mL, tRNA 20 mg/mL
9 2Xprotemase K buffer. 100 mMTns-Hcl, pH 8.0,20 mMEDTA, 20 mMNaC1, 0 4% SDS.
10 Sequencing gel loading buffer 98% deionized formamide, 10 mM EDTA, pH
8 0,0.25 mg/mL XCFF, 0 25 mg/mL bromophenol blue.
11. In vitro RNA splicing stock solutions: 20 mA4MgC12, 15 mM ATP/SO mMcreat-
ine phosphate, RNasm (30,000 u/mL), 100 mM DTT
12. Splicing reaction stop solution: 6% SDS, 250 mM EDTA, pH 8 0, 250 mM Tris-
HCl, pH 8 0
13. 6% Sequencing gel mix: 6% bzs.acrylamrde (1:30), 7Murea, 1X TBE gel buffer
14. Bmdmg buffer. 60 mMKCI,20 mMHEPES, pH 8.0, 1 mMMgCl,, 10% glycerol
15 RNase A 10 mg/mL stock.

3. Methods
3.1. Preparation of HeLa Cell Nuclear Extracts
Efficient in Pre-m RNA Processing
In order to generate an active nuclear extract which will function in posttranscrip-
bona1 processing assays, It is essential that extracts contam all the necessary process-
mg components required for both polyadenylation and splicing. These factors include
the cellular splicing snRNPs and snRNP-associatedproteins, polyadenylation fac-
tors CstF and CPSF, poly (A) polymerase, cleavage factors, and so forth. Nuclear
extracts active for these processes are routinely prepared from HeLa cells. Extracts
from other cell types, such as baby hamster kidney (BHK) cells, have only been
shown to have limited activity m the in vitro assaysdescribedhere.
This procedure is a modified version of the Dignam method as described by
Lee and Green (9).
A minimum of three 140˜mm dishes of 70-80% confluent HeLa cells are
required to make a useful amount of active extract. Between 5 and 10 plates are
routinely used. If HSV-l-infected cell extracts are to be made, the infections
(MO1 10 PFU/cell) are allowed to proceed for 8-16 h before the cells are har-
vested. If any longer, the acttvtty of the extracts will be severely reduced.
1. Harvest the cells by removmg the medmm and rinsing the monolayer with ice-
cold complete PBS.Scrapethe cells off the plastrcinto a final volume of approx
50 mL PBS, and place on ice Immediately.
Phelan and Clements
196
2 Pellet the cells by spinning at -2000 rpm for 5 mm at 4°C
3. Wash the cells gently, but thoroughly with -30 packed cell volumes (PCV) of
ice-cold PBS, using a IO-mL glasspipet.
4 Pellet cells by spinning at 2000 rpm for 5 mm at 4°C
5. Resuspendthe pellet in 1 PCV of buffer A, transfer to an Eppendorf tube, and
allow to swell on ice for 15 mm
6. Lyse cells by rapidly pushing through a narrow-gage needle.
a. Using a 1-mL syringe and a 23- to 26-gage needle, draw up and push out
buffer A to remove asmuch air aspossiblefrom the syringe.
b Draw the extract slowly mto the syringe, and then eject with a smgle rapid
stroke. Six rapid strokes normally provide 80% of cell Iysls, but this can be
checked under a light microscope, and further strokes through the needle can
be made if required.
7. Centrifuge the homogenate for 20 s (12,000g) m a mlcrofuge to produce a crude
nuclear pellet (room temperature).
8 Resuspend this nuclear pellet in two-thirds original PCV (established m step 5)
of buffer C.
9 Incubate at 4™C for 30 mm with contmuous stlrrmg.
10 Pellet nuclear debris by spuming for 5 min (12,000g) m a mlcrofuge at 4°C
11. The supernatant is the nuclear extract
12 The extract is then aliquoted and stored at -70°C
13. The extract can be dialyzedmto bmdmg buffer if required.
3.2. In Vitro RNA Transcription
to Generate Precursor RNA Templates for Processing
In vitro transcrlption allows the generation of 32P-labeled precursor mRNA
from a plasmld borne DNA template using either Sp6, T7, or T3 RNA poly-
merase The average precursor RNA should be between 400 and 800 bases m
length, since longer transcripts (> 1.5-2 kbp) are poorly synthesized. For the
purposes of posttranscrlptlonal3™ RNA processing assays,precursor RNAs are
synthesized that contam a polyadenylation signal (AAUAAA) with surround-
mg GU- and U-rich sequences.This precursor can then be processed by cleav-
age immediately downstream of the polyadenylation signal and addition of a
poly(A) tail. The poly(A) tail adds stability to the RNA and is important for its
subsequent transport to the cytoplasm for translation.
To generate precursor mRNAs for m vitro sphcmg assays,a template is used
that contains a coding gene interrupted by a noncoding intervening sequence or
mtron. The termmi of introns are recognized by highly conserved 5™ (AG . .) and
3™ (GU . ,) splice site recognition sequences.A complex array of splicing proteins
interact with these sequencesto form the spllceosome, the protelrrRNA com-
plex responsible for accurate excision of the mtrons and rejoining of the exon
ends. These spliced mature mRNAs are then ready for translation.
197
In Vitro Systems
To generate a precursor mRNA, linearized plasmid DNA template is required,
since the precursor RNA molecules are synthesizedas runoff transcripts:
1 The components required for pre-mRNA synthesis are defrosted and placed on
ice. The reaction mix is prepared in an Eppendorf tube at room temperature by
adding the reagents as follows*
0.5 pL 4mg/mLBSA
5.0 pL 5X SP6 RNA polymerase buffer
2.5 p.L O.lMDTT
2.5 pL 10X rNTPs (10X mix IS 1 mM each of rATP, rGTP, rCTP, and
rUTP)
1.25 @ lo&CAP
1.O p.L RNasm (30,000 p/mL)
4.25 /AL dHzO
2. The reaction mix can be divided into two ahquots of 8.5 mL and used for the
synthesis of two precursors.
3. To the 8.5 mL add
40 mCi (2 pL) a3*P-UTP (800 Cl/mmol)
1 mg (1 pL) lmearlzed DNA
15 U (1 pL) SP6 RNA polymerase (or T7/T3 polymerase as appropriate)
4. Incubate the mix at 37°C for 1 h
5 Add 2.5 U (2 5 pL) RNase-free DNase Incubate at 37™C for 10 mm.
6 Add 185 FL dH20 to bring the volume to 200 pL.
7 Perform one phenol/chloroform extraction followed by a chloroform extractlon
8 Remove the supernatant to an Eppendorf tube with:
2.0 pL tRNA
20 pL 6M ammomum acetate
600 yL ethanol
9. Precipitate RNA at -20°C for 4 h or overnight If more convenient.
10. Pellet RNA by spmnmg for 10 min m a mlcrofuge (12,000g).
11 Wash pellet thoroughly with 70% ethanol.
12. Resuspend RNA to gave 200-30 cp&L
3.3. In Vitro 3™ R/VA Processing Reaction
Normal 3™ RNA processing occurs as a two-step reaction. Initially, a precur-
sor RNA is cleaved 10-30 bases downstream of its canomcal polyadenylatlon
signal (AAUAAA), and then a poly(A) tall of 200-400 adenosme residues 1s
added by the enzyme poly (A) polymerase (10) The process of polyadenylation
1s achieved primarily by two protein complexes, cleavage stimulation factor
(CstF) and cleavage and polyadenylation specificity factor (CPSF), together
with other poorly defined cleavage factors (12,22) These proteins bind spe-
c&ally to the highly conserved polyadenylatlon site sequences, and 3™ RNA
processing occurs rapidly after transcription. The poly (A) tall adds stability to
Phelan and Clements
198

the RNA and ts thought to have a role m the efficient transport of mature RNA
to the cytoplasm for translation (I I)
During this assay, rather than allowmg normal polyadenylation to occur,
immediately after the RNA cleavage step, an analog of ATP called
cordycepin (3™ dATP) is incorporated mto the growing adenosme tail. This
ATP analog inhrbrts any further extension of the poly(A) tall. The result IS
that any processed RNA products can be resolved as a dtstmct band of a
known size on a gel, rather than a smear of RNAs wtth varying poly (A) tall
lengths The precursor constructs commonly used tn our group are Sau 5
(13,14), a tandem construct conststrng of poly(A) sites from an immediate
early gene upstream of a late gene poly(A) sate, together wrth a number of
single constructs encoding representative poly(A) sites from all the tempo-
ral classes of HSVl (unpublished data). At the end of a processing reac-
tion, the products are resolved on a 6% sequencing gel. A precursor band IS
still clearly vtstble, since the reaction is not 100% efficient The processed
RNA product is resolved as a smaller drstmct band on the gel, (An example
of this assay IS shown m Fig 1.)
1 The nuclear extractis defrosted and kept on Ice unttl reqmred.
2 Incubate the nuclear extract at 30°C for 30 mm. This depletes any ATP present m
the extract
3. Prepare the reactron mtx by addmg in the followmg order:
1 0 pL 25 mA4 3™ dATP (cordycepin)
2 5 pL 50 mA4 creatme phosphate
6.5 FL 10 MTrts-Hcl, pH 7 6
3 0 pL 25% PEG
4 To the 13 pL reaction mix, add 11 pL nuclear extract and 200-300 cps (1 pL) 32P-
labeled RNA precursor (see Section 3 2 ).
5 Incubate at 30°C for 2 h
6. To the reaction mix add:
100 ltL 2X protemase K buffer
75 pL dH,O
2 PL Protemase K (20 mg/mL)
2 pL tRNA (20 mg/mL)
7 Incubate at 30°C for 15 mm
8 Phenol chloroform extract once.
9. Chloroform extract once.
10 To precipitate RNA, add 600 pL ethanol and 20 pL ammonium acetate Incubate
on dry ice for 15 mm.
11. Pellet the processed RNA by spinning m a mtcrofuge (12,000g) for 10 min
12. Wash pellet wrth 70% ethanol.
13. Add 5 pL loading buffer, boll for 5 mm, and place on ice.
14 Resolve the bands on a 6% urea acrylamtde sequencing gel.
In Vitro Systems 799

P MI INF
Pre I




IE




Fig. 1. The in vitro polyadenylation assay. Precursor RNA from the tandem HSV- 1
poly (A) site construct SauS, was processed with nuclear extracts from mock-infected
(MI) and wild-type HSV-1 (INF)-infected HeLa cells. The cleaved products were
resolved on a 6% acrylamide sequencing gel. “Pre” indicates the position of the initial
precursor RNA. “IE” represents the cleavage product from the 5™-most immediate early
poly (A) site, and “L” represents the cleavage product from the 3™-most late poly (A)
site. HSV-1 infection results in increased usage of the late poly (A) site at later times
postinfection.


3.4. In Vitro RNA Splicing Assay
The removal of introns to produce mature mRNA molecules ready for transla-
tion requires a very complex and accurate sequence of events. This procedure is
reliant on a large number of small nuclear ribonuclear protein particles (snRNPs)
and associated proteins, which interact to form the spliceosome. These riboproteins
recognize and bind to the 5™ and 3™ RNA splice site sequences, and by proteirr-
protein and protei*RNA interactions, bend the RNA to bring the two ends of the
exon together. The RNA is then cleaved and the two exons joined via two distinct
transestetification steps, which result in the formation of branched lariat RNAs as
intermediates and the mature RNA product. The excised intron remains complexed
with the snRNPs until it is degraded (for a review, see ref. 15).
Phelan and Clements
200

MI I

Lariat
2

IE----cI Precursor
lntron

Product
m

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( 61 .)



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