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Fig. 2. The in vitro splicing assay. Precursor RNA from the Adenovirus type 2
major late transcript was processed with mock-infected (MI) and HSV-l-infected (I)
HeLa cell nuclear extracts. The spliced products were resolved on a 10% sequencing
gel. The lariat, precursor, intron, product, and 5™-exon forms of the initial precursor
have been indicated on the figure.

This in vitro splicing assay allows the efficient splicing of 32P-labeled
precursor RNA molecules, resulting in the generation of spliced product,
excised intron, and lariat forms of the spliced intermediate. The template
used commonly in our group is that of the Adenovirus type 2 major late
transcript as has been described by Moore et al. (Z6,27). This construct
contains a single intron with typical splice site recognition sequences. An
example of the splicing assay and the intermediate processed forms can be
seen in Fig. 2.
1. The splicing reactions are set up in Eppendorftubes at room temperature as follows:
11 pL Nuclear extract
30 /AL 20 mMMgClz
15 mM ATPISO m44 CrP04
3 IIL
201
in Vitro Systems
2.0 pL RNasm (60 U)
1.5 pL 100 mMDTT
1.O pL pre-mRNA (200-300 cps)
8.5 pL ddHaO
2. Incubate the mix at 30°C for 2 h
3. Add 3 0 pL of splicing stop solution (see Section 2 2 , Item 12).
4. Add 3.5 mL of proteinase WRNA mix:
16 mg/mL protemase K
2 mg/mL tRNA
5. Incubate at 65°C for 45 mm.
6. Precipitate RNA on Ice with 7 vol of ethanol/ammonmm acetate mix for 30 min.
MIX consists of 86 mL abs. ethanol + 14 mL 4MNH,OAc, pH 5.0.
7. Pellet RNA by spmnmg m a mlcrofuge (12,000g) for 20 mm.
8 Resuspend m 10 pL loading buffer
9. Heat sample to 65°C for 20 min before resolvmg on a 10% acrylamlde
sequencing gel

3.5. UV RNA-Protein X Linking
This assayallows closely associated RNA and protem molecules to be coval-
ently crosslinked by UV ltght (16,18,29). The complexes formed are resistant
to heat, detergent, and alkali treatment, and therefore are very stable. RNA
crosslinked to protein is protected against the action of RNase A RNase A
treatment removes any unprotected RNA leaving the RNA-protein hybrids
radiolabeled. The proteins and their interacting RNA can be resolved on a
1O-l 2% protein gel and visualized by autoradiography.
Precursor RNAs labeled with 32P-UTP (containing RNA processmg motifs
or not) are synthesrzed as described m Sectlon 3.2. and should be resuspended
m binding buffer. The nuclear extracts must be dialyzed agamst binding
buffer to remove the high salt content of buffer C before use m the binding
assay. Protein concentrations were determined by Bradford™s assay (20) and
normalized before performing the binding assay, adding 1O-l 5 pg of protein
to each reaction.
1 Into a 96-well microtlter plate, add:
8 pL binding buffer-see Section 2.2.
1 PL precursor RNA (30&400 cps)
1 pL nuclear extract (10-15 mg protein)
2 Incubate the mix for 15-20 mm at room temperature
3. UV X-link the interacting RNA and protein molecules with 250 mJ/cm2
4. Add RNase A to a final concentration of 1 mg/mL, and Incubate at 37“C for
15 min.
5. Add an equal volume of bollmg mix, boll for 5 mm, and resolve on a lO-12%
SDS polyacrylamide protein gel.
202 Phelan and Clements

4. Notes
1 (a) It is important that the cells are no more than 70-80% confluent if good active
extracts are to be prepared. (b) Solutions A and C should be made fresh every
time a new extract is prepared (c) The final protem concentration of a nuclear
extract should be 15-20 mg/mL.
2 (a) Precursor RNAs should be resuspended in a mmtmum of 50 pL, I pL each of
which should count at least 200 cps Poorly labeled RNA will result m poor RNA
processing results (b) tRNA must be added to aid precipitation of the precursor RNA,
and allow a vistble pellet to be formed (c) A “smeary” precursor, which consists of
mcomplete transcripts can be cleaned up by a gel purlficatton method (I 7)
3 (a) All components of the polyadenylation assay should be stored at -20°C and
allowed to defrost on me. (b) The solutions, such as cordycepm and creatme phos-
phate, should be stored in small ahquots to avoid repeated freeze-thawing (c)
Vtgorous vortexmg of the samples should be avoided
4 (a) It is important only to heat the samples to 65°C prtor to loading on the gel
Higher temperatures can destruct the lariat forms of the incompletely spliced
precursor. (b) The same prmciples apply to storage of the components as for the
polyadenylation assay.
5. (a) The extracts should be thoroughly dialyzed prior to use m the binding assay,
to remove the high salt from buffer C, though it is important that no appreciable
amount of protein is lost at this stage. (b) The bmdmg of many factors to RNA is
ATP-dependent If ATP has been lost from the extract, it may be necessary to add
ATP to the reaction, prior to the mcubation at room temperature

References
1. Roizman, B. and Sears, A E (1991) m Fundamental Vu-ology, Herpes Simplex
Vzruses and Thezr Replzcatzon, 2nd ed., Chapter 34 (Fields, B N. and Kmpe, D
M , eds ), Raven New York
2. Rice, S A, Long, M C , Lam, V , Staffer, P A, and Spencer, C. A. (1995)
Herpes simplex vnus immediate early protein ICP22 1s required for viral modtfi-
cation of host RNA polymerase II and establishment ofthe normal viral transcrrp-
non program J Vu-01 69,555&5559
3. McGeoch, D J , Dahymple, M A., Davison, A J , Dolan, A , Frame, M C.,
McNab, D., Perry, L J , Scott, J. E , and Taylor, P (1988) The complete DNA
sequence of the long unique region m the genome of herpes simplex vnus type-l.
J Gen Vwol 69, 153 I-1574
4 Kwong, A. D and Frenkel, N. (1987) Herpes simplex virus infected cells contain
functton(s) which destabilize both host and viral mRNAs. Proc. Nat1 Acad. Scz
84, 1926-1930
5. Hardy, R W. and Sandra-Goldm, R M. (1994) Herpes simplex vn-us inhibits host
cell splicing and the regulatory protein ICP27 is required for this effect J Vzrol
68,7790-7799
6. Sandri-Goldin, R and Mendoza, G. E ( 1992) A herpes virus regulatory protein appears
to act posttranscriptionally by affecting mRNA processmg. Genes Dev 6,848-863
In Vitro Systems 203
7. Phelan, A., Carmo-Fonseca, M., McLauchlan, J , Lamond, A. I , and Clements, J. B
(1993) A herpes simplex vuus type 1 immediate early gene product, IE63, regulates
small nuclear nbonucleoprotein distribution. Proc. Nat1 Acad Sci. 90,9056-9060.
8. Martin, T. E Barghusen, S C , Leser, G P , and Spear, P. G (1987) Redistribu-
tion of nuclear ribonucleoprotein antigens during herpes simplex vnus infection.
J Cell BEOZ 105,2069-2082.
9 Lee, K A. W and Green, M. R. (1990) Small scale preparation of extracts from
radiolabeled cells efficient m pre-rnRNA splicing. Methods Enzymol. 181,20-3 1.
10 Chrrstofori, G. and Keller, W (1989) Poly (A) polymerase purified from HeLa
cell nuclear extracts is required for both cleavage and polyadenylatron of pre-
mRNA m vitro A401 Cell Blol. 9, 193-203
11 Wahle, E. and Keller, W (1992) The biochemistry of 3™-end cleavage and
polyadenylation of messenger RNA precursors. Annul Rev Btochem 6L419-440.
12. Beinroth, S., Keller, W., and Wahle, E. (1993) Assembly of a processive messen-
ger RNA polyadenylation complex EMBO J. 12,585-594.
13 McLauchlan, J., Simpson, S , and Clements, J B. (1989) Herpes simplex virus
induces a processing factor that stimulates poly (A) site usage. Cell 59,1093-l 105.
14 McLauchlan, J., Phelan, A , Loney, C , Sandra-Goldm, R M , and Clements, J B
(1992) Herpes simplex virus IE63 acts at the posttranscriptional level to stimulate
viral rnRNA 3™ processing J Virol 66, 6939-6945
15 Sharp, P. A. (1994) Split genes and RNA sphcmg Cell 77,805-8 15.
16. Moore, C. L , Chen, and Whorrskey, J. (1988) Two proteins crosslmked to RNA
containing the adenovnus L3 poly (A) site require the AAUAAA sequence for
binding EMBO 7,3 159-3 169
17. DeZazzo, J D., Falck-Pedersen, E., and Imperrale, M. J (199 1) Sequences regu-
latmg temporal poly (A) site switching m the adenovnus major late transcription
unit. Mol Cell Blol 11,5977-5984
18. Vakalopoulou, E , Schaack, J , and Shenk, T (1991) A 32-kilodalton protein binds
to AU-rich domains in the 3™ untranslated regions of rapidly degraded mRNAs.
Mol. Cell Blol 11,3355-3364
19 Pelle, R. and Murphy, N. B. (1993) In vivo UV-crosslinkmg hybridisation, a pow-
erful technique for isolatmg RNA binding proteins. Application to trypanosome
mini-exon derived RNA. Nucleic Aczd Res. 21, 2453-2458.
20. Bradford, M. (1976) A raprd and sensitive method for the quantrtation of micro-
gram quantmes of protem utihsing the principle of protem-dye binding Anal
Blochem. 72,248-254.
14
Analysis of HSV-1 Transcripts by RNA-PCR
Jordan G. Spivack


1. Introduction
Since the herpes simplex virus type 1 (HSV-1) genome has been sequenced
and most HSV- 1RNAs are not splrced (I), detailed mformation about the struc-
ture of many HSV- 1 RNAs can be obtained without the considerable time and
effort that is required to construct and analyze cDNA libraries. Once the 5™ and
3™ ends of an RNA have been mapped precisely, the RNA nucleotide sequence
can be deduced simply from the genomic DNA sequence. However, there are
certain situations, such as the analysis of spliced RNAs or of chimeric RNAs
expressed from foreign genes inserted into HSV- 1 vectors, where cDNA clon-
mg of HSV-1 transcripts may be informative. There are famihes of transcripts
that arise by alternate splicing m several human herpesviruses: HSV-1,
cytomegalovirus (CMV), and Epstein-Barr virus (EBV). For example, HSV-1
encodes several overlappmg latency-associated transcripts or LATs (2-4). The
splice junctions of the intron within the HSV-1 2.0-kb LAT have been deter-
mined by RNA-PCR with primers located on either side of the intron, followed
by direct DNA sequence determination of the PCR product (5). The construc-
tion of partial cDNAs by PCR saves much time-consummg effort and expense
compared with the analysis of cDNA libraries. In addition, by sequencing PCR
products directly, the need to analyze several cDNA clones in order to be
assured of obtaining the consensus sequence is eliminated.
The protocol presented in this chapter uses total RNA as a template for PCR
combined with direct DNA sequence determination of the products. As with
all techniques that involve RNA, the preparation of high-quality nuclease-free
RNA is of crucial importance These techniques are suitable for isolation of
RNA from either tissue culture cells or mammalian tissues. The RNA-PCR
and double-stranded DNA sequencing procedures have been modified specifi-
From Methods m Molecular Medrone, Vol 10 Herpes 8mplex Virus Protocols
Edlted by S M Brown and A R MacLean Humana Press Inc , Totowa, NJ


205
Spivack
206

tally for the high GC content (68%) of HSV-1 DNA. Addtttonal PCR tech-
niques using RNA templates can be found m vol. 15 of this series, PCR Proto-
cols, Current Methods and Applxations (6).
2. Materials
2.1. RNA Isolation (see Notes 1 and 2)
1, 200 mL guamdmmm thiocyanate stock (7,8) Add the following m order.
a 100 g Guamdmmm thlocyanate
b 4 g Sodium-N-lauroyl-sarcmate
c 5 mL lMNaCttrate, pH 7 0
d 1.4 mL P-mercaptoethanol
e 0 67 mL anttfoam A (Sigma, St Louts, MO).
f Add HZ0 to 190 mL
g. Star and warm (35-4O”C)
h Adjust to pH 7.0 with 1NNaOH.
1 Add HZ0 to 200 mL, filter sterilize, store in a brown glass bottle (hght senst-
tive) at room temperature
2 100 mL CsCl cushion.
a. 95 98 g Biochemical grade CsCl
b 10 mL lMEDTA, pH 7 0.
c Add DEPC H,O to 100 mL (see Note 2, check pH [should be 7 01)
d Filter stertlrze, store at room temperature.
3 5M NH4 acetate, store at -2O™C m tightly capped microcentrifuge tubes
4 100% Ethanol, store at -20°C.
5. Phosphate-buffered salme, pH 7.4
2.2. Agarose Gel Electrophoresis of RNA
1 Deionized 6M glyoxal (see Note 3)
2 DEPC-treated H20.
3 DMSO.
4. 0. lMNaH2P04, pH 7.0
5 RNA (5 ug/lane, 10 ug max)
6 Loading buffer. 50% glycerol, 0 OlM, NaH2P04, 0 4% bromophenol blue
7 10 mg/mL acndme orange m H20. Store at 4°C in plastic tube covered with
alummum fotl (light-sensmve).
2.3. RNA and DNA-PCR
1 RNA template (l-2 pg) or DNA template (1 ng)
2 DEPC-treated H20.
3 100 M random hexamer (pdN6, Pharmacia, Uppsala, Sweden)
4. dNTPs (mixture of 2 mM of each).
5 10X Taq buffer (Promega, Madison, WI).
6. Moloney murme leukemia vnus (M-MLV) reverse transcnptase (BRL, Richmond, CA)
207
Analysis of HSV- 1 Transcripts by RNA-PCR
7. RNasin (Promega).
8. PCR primers (6,9; see Note 4)
9. Taq polymerase (Promega)
2.4. Direct Sequence Determination of PCR Products
2.4.1. Double-Stranded DNA Sequencing Reaction
1. Sequenase Version 2 0 kit (United States Blochemlcal, Cleveland, OH)
2. O.lM Dithlothrettol (DTT).
3. 10 mg/mL proteinase K.
4. SeaPlaque (FMC, Rockland, ME) or LMP agarose (BRL).
5. 10X TBE, pH 8.0. 1X TBE = O.O89MTrts-borate, 0.089M EDTA, pH 8.0
6. 50 mMTris-HCl, pH 8.0, 1 rnIr4 EDTA.
7. Phenol.
8. 100% Ethanol.
9. TE* IO nnI4 Trrs pH 8.0 and 0 1 n-&I EDTA
10 [32P]dATP (sequencmg grade, =3000Ci/mmol).
2.4.2. 8% Sequencing Gel
1 0.6 g Bis-acrylamlde.
2. 11 4 g Acrylamtde.
3. 30.68 g Urea
4 Amberhte MB-3 (Mallmckrodt).
5. Dtsttlled H20.
6. 10X TBE, pH 8.0
7. Tetramethylethylenediamme (TEMED)
8 25% Ammonium persulfate (APS). Make fresh.
2.4.3. Pouring Sequencing Gel
1. MIX the bu-acrylamtde, acrylamide, and urea.
2. Add water to approx 130 mL total, stir until acrylamlde has dissolved, add
1 teaspoon Amberhte, stir gently for 5 min, and filter through Whatman (Matdstone,
UK) 1 paper Add 7 5 mL 10X TBE, bring the volume up to 150 mL, and place
on ice for 5 min. Just before pouring the gel add 240 pL APS and 60 pL TEMED.
Pour the gel immediately and polymerize for 2 h at room temperature before
loading sequencing samples The gel can be poured the day before and stored
overnight at 4°C.

3. Methods

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