<<

. 32
( 61 .)



>>

3.1. RNA Isolation
Analysis of RNA begms with the preparation of clean, undegraded RNA.
This extractton procedure, based on the procedure of Chirgwin (3,7,8), should
first be followed precisely, before mtroducmg any modifications.
208 Spivack
1. Rmse mouse ttssues, or tissue culture cells, with phosphate-buffered saline (PBS)
Homogemze tissue or pelleted cells (resuspend by vortexmg) in guamdinium thto-
cyanate solutton for 20 s with a Polytron (Brmkmann) or Teckmar (Cmcmnatt,
OH) homogenizer at setting 5 The solutton to tissue ratto should be 8: 1 or greater
Use a homogenizer probe that is designed to mimmtze foaming of the sample
2 Depending upon sample volume, pellet the RNA through a cushion of 5 7M
CsCl, 0 1M EDTA, pH 7 0, by centrtfugation at 35 K in a Beckman
SW40 1 rotor (1 I-mL sample/2-ml cushion) or 40 K in a Beckman SW55
rotor (3 5 ml-sample/l 5-mL-cushion) for 20-24 h, at 18°C. After centrrfu-
gatton, carefully aspirate the supernatant with a Pasteur ptpet, invert, and
dry the RNA pellet for 2-3 mm (see Note 5) Resuspend the clear glassy
pellet m 100 pL H,O. The amount of RNA 1s estimated by measuring the
absorbance of a 1.100 dilution of each sample at 260 nm (1 OD U = 40 pg
RNA) The RNA yield is -80-100 pg from a cell monolayer in a 75 cm*
flask The purity of the RNA is estimated by the 260:280 nm ratio; for a
clean preparation the ratio IS between 1 8 and 2 0 Ratios below 1 6 indicate
contamination, often with proteins (see Note 6) Ethanol precipitate the
RNA by adding an equal volume of 5MNH4 acetate (100 pL) and 2.5 vol(500
pL) of 100% ethanol and store at -20°C overnight The RNA IS stable at -
20°C for at least several months and for as long as 2-3 yr

3.2. Agarose Gel Elecfrophoresis of RNA
The quality of the RNA can be Judged by agarose gel electrophoresis and
acridme orange staining. Smce approx 80% of mammalian cell RNA is riboso-
mal, promment 28s and 18s rRNAs should be visible on acrldine orange
stained gels, and sometimes 5s RNA can be seen.
1 Vortex the RNA ethanol precrpitate Remove the approprtate volume and centrr-
fuge for 30 mm at room temperature m a mtcrocentrifuge. Carefully remove the
supernatant using a mrcroprpet wtth a dtsposable plastic ttp.
2 Air-dry the pellet in a well-venttlated hood for 15-20 min When the pellet IS dry
resuspend m 3 7 pL DEPC treated Hz0 Denature RNA (5-10 pg/sample) by
mcubatton with 10 mM Na2HP04, pH 7 0 (1 6 pL of 0 1M stock), IM detonized
glyoxal (2.7 pL of 6M stock), and 50% DMSO (8 pL) at 50°C for 1 h. Also
denature RNA markers from BRL (etther 0 16-l 77 or 0.24-9 5 kb) After cool-
mg to room temperature add 4 pL loadmg buffer.
3. Electrophorese the RNA through 1 2% agarose m 10 mA4Na2HP0,, pH 7 0, with
constant buffer recirculation until the dye marker 1s 1 cm from the bottom of the
gel (see Note 7)
4. Stain the RNA by placmg the gel m a glass dish, cover with the running buffer
from the gel, add 1 pL/mL of the acrrdme orange stock, rock gently, and incubate
m the dark for 20 mm (10) Transfer the gel to a white metal enamel baking dish,
add fresh gel buffer and destain for a mmtmum of 1 h at room temperature or
overnight at 4°C (see Note 8). Photograph the gel on a UV ltght box
Analysis of HSV-1 Transcripts by RNA-PCR 209

3.3. RNA- and DNA-PCR
3.3.1. DNA-PCR
1 Before designing RNA-PCR primers, the ends and splice junctions of the HSV-1
transcript(s) of interest need to be at least partially mapped. PCR primers are
chosen upstream and downstream of introns (see Note 4).
2. Optimal amplification conditions can be established empirically with 1 ng plas-
mtd DNA template
3 To ensure uniformity between samples, and to reduce the possibility for contami-
nation, make a master mix containing PCR buffer, the dNTP mixture, H,O, and
Tag polymerase, and aliquot to each tube.
4 Then, with positive displacement pipets, add the primers, and lastly the DNA
template. Always include positive and negative (no DNA added) controls.
5 Perform PCR amplifications with 2 5 U of Tag polymerase, in 1X Promega buffer
with 1 pJ4primers, 100 cuz/r dNTPs, in a 25 pL vol overlaid with mineral oil, by
30 cycles of denaturatton at 96°C for 1 min, annealing at 55-68”C for 2 min, and
extension at 72°C for 3 min (see Note 9).
6. Resolve PCR products by agarose gel electrophoresls, stain with ethidium bro-
mide, and vlsuahze on a UV light box
3.3.2. RNA-PCR
1. For RNA PCR, m 18 uL combme 2 ug of total RNA, 1X PCR buffer, 1 mM
dNTPs and 5 pg pd(N)b, and heat to 80°C for 3 mm, then chill on ice.
2. Add 40 U RNasin and 200 U of M-MLV reverse transcrtptase, incubate the reac-
tion for 1 h at 37™C, heat to 95™C, and chill on ice (see Note 10)
3 To 10 yL of the first-strand reaction add 1 pMPCR primers and 2.5 U Tag and
increase the vol to 100 pL in 1X PCR buffer
4. Amplify as for the DNA-PCR, and resolve PCR products by agarose gel electro-
phoresis, as described.
3.4. Direct Sequence Determination of PCR Products
3.4.1. Isolation of PCR Products for DNA Sequence Determination
The best double-stranded sequencing of PCR products IS obtained with large
amounts of template.
1. Ideally use l-2 ug DNA per sequencing reaction. This usually requires runnmg
several (5-l 0) identical PCRs.
2 Run the PCR products on a low melting point agarose gel.
3. Use a 123-bp ladder (BRL) for DNA size markers.
4. Find the band of interest, excise the smallest gel shce possible, and transfer to a
microcentrifuge tube
5. Add 400 uL 50 mMTris-HCl, pH 8.0,1 mMEDTA and melt gel at 65°C for 10 mm.
6. Extract twice wrth 400 uL phenol, transfer the aqueous phase to a fresh
microcentrifuge tube, and precipitate by addition of 40 uL 3M Na acetate, pH
5.0, and 1 mL of 100% ethanol.
Spivack
210
7 Incubate in a dry ice/ethanol bath for at least 1 h and then microcentrifuge for 30
mm at room temperature
8. Dram the sample well and use a disposable 20-pL pipet tip to remove excess
ethanol.
9. Reprecipttate from 100 pL TE, pH 8 0, and 100 pL SMNH, acetate.
10 Redissolve the sample in 25 PL TE (see Note I 1).
3.4.2. DNA Sequencing Reaction
The DNA sequencing reaction is based on the USB Sequenaseprotocol.
3.4.2 1 ANNEALING REACTION
In a microcentrifuge tube combine: 2 uL 5X Rxn buffer (USB), 1 uL primer
(0.5 pmol/pL), and 7 PL DNA (approx 1 pg)
Heat samples m a boiling water bath for 5 min, chill on ice for 3 mm, quick
spin samples, and incubate at room temperature for 5 mm (see Note 12).
3.4.2.2. LABELING REACTION
To the annealmg reaction, add m order 1 p.L DTT (0. lw, 1 pL Mn buffer
(USB), 2 uL labeling mix (IX), 1 uL dATP32P, 2 uL Sequenase Version 2.0
(see Note 13). Dilute 1:8 with deionized H20.
Incubate at room temperature for 5 min, then proceed with normal termina-
tion reactions, transferring 3.5uL porttons to each of the four termination
reaction tubes (see Note 14).
3.4.3. DNA Sequencing Gel
Good resolution is obtained by using wedge spacers with an 8% acrylamide/
7M urea gel. Heat the gel to 50°C and denature the samples at 95°C for 3 min
before loading. Then load 1.5 uL of the samples onto the gel (see Notes 15 and
16). When gel has run so that the methylene blue is 2-3 cm from the bottom,
fix the gel m 10% acetic acid/l2% methanol for 1 h at room temperature, and
dry the gel at a maximum of 70°C.
Notes
4.
1 For RNA work use materials made of sterile disposable plastic, or glass baked
overnight at 180°C Wear disposable gloves during all of these procedures to
muumize contammation with RNase from hands and fingers.
2. DEPC-treat all solutions (except solutions contammg Tris or guamdmm thiocy-
anate) Add 0.1% dtethylpyrocarbonate (DEPC), shake vtgorously, incubate over-
mght at room temperature, shake again, and autoclave
3 To deionize a 40% glyoxal solution, add 40-45 mL glyoxal to 10 mL Bio-Rad
AG 501-X8 resin m a 50-mL plastic disposable tube. Rock gently for 3-4 mm,
transfer glyoxal to a fresh tube wtth new resm During deiomzation more than
Analysis of HSV-7 Transcripts by RNA-PCR 211

half of the volume IS lost within the resin. After repeating this three times, remove
10-15 PL with a micropipet and check pH wrth a colorpHast indicator strip (EM
Science/E. Merck, Rahway, NJ). Repeat with fresh resin until the pH 1s between
6.0-7 0. Store the deionized glyoxal m filled, tightly capped mlcrocentrifuge
tubes at -20°C Leave a small amount of room in each tube for expansion during
freezing The deionized glyoxal is stable at -20°C for at least several years.
4. Designing PCR primers IS an empirical art (see ref. 6; Chapter 2) Often primers
that are Ideal on paper work poorly m practice. On the other hand, sometlmes
when compromlses are made owing to the need for a primer m a specific loca-
tion, the primers work well. For HSV-1 PCR primers we have been successful
followmg these guidelines.
a. Primers are 22 bases long;
b. Approx 68% GC content (i.e., 15122 bases) with a mix of all four bases;
c. Lack of self-homology or complementanty, determined by visual mspectlon
or computer analysis;
d. Exact alignment m only one position of the HSV- 1 genome,
e Primer pairs are chosen to yield PCR products of different sizes (20&600 bp)
so that the primers can be used in combination;
f Primers span mtrons so that the PCR products from RNA and DNA templates
can be distinguished by size.
This last point is important because it IS difficult to prepare RNA that IS com-
pletely free of DNA.
5. The supernatant is removed by careful aspiration with baked Pasteur plpets. After
removal of the guamdmmm layer, a few seconds are allowed for the walls of the
tube to dram. A fresh plpet IS used to remove the CsCl layer, and after removmg
the viscous DNA layer, and another pause, another fresh plpet IS used These
precautions mmlmlze the contamination of the RNA with DNA or proteins
Before resuspendmg the pellet, the tubes are inverted for 2-3 min and the walls
gently wiped with a KImwipe. If this interval is too long, the glassy RNA pellets
are very difficult to resuspend
6. If the 260:280 ratio IS less than 1.6, sometimes the RNA can be cleaned up by a
series of phenol, phenol/chloroform, and chloroform extractions, followed by
ethanol preclpltatlon However, these mampulatlons can lead to RNA degrada-
tion. Check by agarose gel electrophoresls and ethidium bromide staining
7. These gels can also be used for Northern blot transfer (3,5) For RNA smaller
than 1 kb, better resolution can be achieved with 1 5% agarose
8. The acridine orange binds to the white metal enamel dish, which aids the destanung
process. To clean the enamel dish use ethanol; acridme orange is soluble in ethanol
9 In our hands, Promega™s Tuq polymerase produces a higher yield of PCR prod-
ucts than Tuq from other suppliers. For first strand synthesis with M-MLV reverse
transcriptase, the yield with the Taq buffer from Promega is greater than with the
reverse transcrlptase buffer from BRL.
10. RNA PCR can be performed with polyA+ RNA However, m my hands, satisfac-
tory results have been obtained with total RNA. Although a PCR primer can be
212 Spivack
used in the tirst step reaction to synthesize cDNA, I have obtained better results
with the random hexamer (pdN&
11, Make sure that the sample is completely dry before resuspending m TE
12. PCR primers are also used for DNA sequence determmation. To achieve hrgh
primer and template concentrations, the volume for the annealing reaction always
should be 10 I.˜L
13 At each step, it is important to gently mix all the reagents m the tube without
mtroducmg an bubbles. The Sequenase should be diluted nnmedlately before use
and this diluted mtxture should not be left on ice longer than 20 mm. When start-
mg the reactrons, add Sequenase to one sample, wait 2 mm, and then add
Sequenase to the next sample. This ˜111 allow enough time to terminate each
reaction before starting the terminatton of the next sample
14 Aliquot 3.5 pL of sample into each of the termination reaction tubes containmg
prewarmed 2.5 PL of appropriate ddNTP. Incubate at 37°C for 3 mm, add 4.0 PL
stop solutton, and immediately place on ice. Just before loading samples on the
gel, incubate at 93-95™C for 3 mm Chill on ice, quick spin samples, return
samples to Ice, and load 1 5 uL on sequencing gel.
15 For an 8% urea gel, methylene blue dye (the darker dye) runs at 20 bp and xylene
dye (light blue) runs at 40 bases. Run the gel until the methylene blue is approx
l-2 cm from the bottom of the gel At this time, or up to 1 h later, the samples can
be reloaded to read farther m the DNA sequence.
16 If loading the samples for a second run, reheat them at 95°C for 3 mm

References
1. McGeoch, D J , Cunmngham, C , McIntyre, G , and Dolan, A. (1991) Comparative
sequence analysis of the long repeat regions and adjoining parts of the long umque regions
m the genomes of herpes simplex vnus types 1 and 2 J Gen Vzrol 72,3057-3075
2. Rock, D. L., Nesbum, A B , Ghiasl, H , Ong, J , Lewis, T L., Lokensgard, J. R., and
Wechsler, S. (1987) Detection of latency-related viral RNAs in trigeminal ganglia of
rabbits latently infected with herpes simplex vnus type 1 J Vu-ol 61,3820-3826
3. Spivack, J G. and Fraser, N W. (1987) Detection of herpes simplex vuus type 1
transcripts during latent mfectlon m mice J Viral 61, 3841-3847
4. Stevens, J G., Wagner, E. K., Devt-Rao, G. B., Cook, M. L., and Feldman, L. T
(1987) RNA complementary to a herpesvirus a gene mRNA is prominent in
latently infected neurons. Science 235, 1056-1059.
5. Spivack, J. G., Woods, G. M., and Fraser, N. W. (1991) Identification of a novel
latency-specific sphcing signal wtthm the herpes simplex virus type 1 2.0 kilobase
latency-associated transcript (LAT)* translation mhtbmon of LAT open reading
frames by the mtron wnhm the 2.0 kilobase LAT. J Vu-01 65,6800-X1810.
6 White, B. A. (1993) PCR Protocols, Current Methods and Appbcatzons. Methods
zn Molecular Blo/ogy, vol. 15, Humana, Totowa, NJ
7 Chtrgwm, J M., Przybyla, A E., MacDonald, R J., and Rutter, W. J. (1979)
Orientation of herpes simplex virus type 1 immediate early mRNAs Bzochemzstry
l&5294-5299.
Analysis of HSV- I Transcripts by RNA-PCR 213
8. Maniatis, T., Fritsch, E F., and Sambrook, J. (1982) Molecular Clonzng* A Labo-
ratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
9. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular clonmg. A Labo-
ratory Manual 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
10. Carmichael, G. G and McMaster, G. K (1980) The analysis of nuclerc acids in
gel using glyoxal and acrrdine orange. Methods Enzymol 65,380-39 1
11. Kavaler, J., Caton, A. J , Staudt, L. M., Schwartz, D., and Gerhard, W. (1990) A
set of closely related antrbodies dominates the primary antibody response to
the antrgenlc site CB of the A/PR/8/34 influenza hemagglutinm. J. Immunol
7,2312-2321
15
Transient Assays for HSV Origin
and Replication Protein Function
Nlgel D. Stow


1. Introduction
Investigations of genome replication in DNA viruses involve several facets.
These include characterizing the sites at which synthesis is initiated (origins of
DNA replication), identifying the viral and host protems that participate,
understanding the enzymatic actlvittes of these protems, and elucidating the
mechanisms of DNA synthesis and maturation. For several vuuses cell-free
systems capable of carrying out faithful viral origin-dependent DNA synthesis
have been described that have provided important insights into these areas.
Unfortunately, suchan assayis not yet available for HSV and other approachesthere-
fore have been required. One of the most useful and widely employed has
involved transient assaysfor viral origin-dependent DNA synthesis m trans-
fected tissue culture cells. Such assays played important roles m the initial
identification of the viral replication origins and the virus-coded protems
essential for DNA synthesis and more recently have helped provide detailed
information on the structure and function of these elements. Similar approaches
also have been exploited to study genome replication in other herpesviruses
The HSV-1 genome contains three separate origins of rephcation, a smgle
copy of ori, close to the centre of the UL segment and two identical copies of
oris located in the repeat regions IRs and TRs that flank the Us segment (see
Note 1). A set of seven viral proteins encoded by genes UL5, UL8, UL9, UL29,
UL30, UL42, and UL52 are both necessary and sufficient for viral DNA rep-
lication. The UL30 and UL42 proteins constitutethe catalytic and an accessorysub-
unit of the viral DNA polymerase, the UL29 protein IS a nonsequence-specific
single-stranded DNA bmdmg protein and the UL9 protein binds to specific
sequence elements within the rephcatton origms. The UL5, UL8, and UL52
From Methods m Molecular Med/one, Vol 10 Herpes Bmplex Vws Protocols
Edlted by S M Brown and A R MacLean Humana Press Inc I Totowa, NJ

275
stow
216

<<

. 32
( 61 .)



>>