Electrophoretic separation of the plus and minus strands of rotavirus SA11 double-stranded RNAs

Journalof VirologicalMethods, 13( 1986) 185-190

185

Elsevier JVM 00485

ELECTROPHORETIC

SEPARATION

OF ROTAVIRUS

DOUBLE-STRANDED

JOHN

T. PATTON

SAll

and SANDRINA

OF THE PLUS AND MINUS

STRANDS

RNAs

STACY-PHIPPS

Department of Biology, University of South Florida, Tampa, FL 33620, U.S.A. (Accepted

3 January

1986)

The genome of the rotaviruses To provide

these segments, and the individual

reovirus strand

dsRNAs

genome segments

recovered

at different (Smith

6 M urea, the dsRNAs

patterns

were resolved

under denaturing

by electroelution. were denatured

et al., 1981), rotavirus

plus strand

double-stranded

conditions

on agarose-

on a polyacrylamide

Upon electrophoresis

with complementary

RNA migrates

RNA (dsRNA).

plus and minus strand RNAs within

by electrophoresis

rates. Our results showed that, like cytoplasmic

on agarose-urea

rotavirus

the complementary

their migration

‘H-labelled

gel containing

migrating

segments of completely

and identifying

we have characterized

urea gels. Virion-derived agarose

consists ofeleven

a method for separating

gel

in a low pH 1.75%

plus and minus strands

polyhedrosis

virus but unlike human

faster than its complementary

minus

gels.

agarose-urea

gel electrophoresis

strand

separation

INTRODUCTION

Rotaviruses,

members

pletely double-stranded (Rodger and Holmes, is identical structures

of the family RNA (dsRNA)

contain

eleven segments

each of which encodes

a unique

of com-

polypeptide

1979; Mason et al., 1983). The plus strand found in viral dsRNA

to rotavirus

messenger

and lack 3’4erminal

RNA replication

Reoviridae,

RNA

polyadenylate

of the rotaviruses,

(mRNA); sequences

both

contain

Y-terminal

cap

(Imai et al., 1983). During the

viral mRNA acts as a template

for the synthesis of

minus-strand RNA producing dsRNA (Patton, manuscript in prep.). Studies describing the synthesis of the eleven rotavirus genome segments require a method for separating and identifying the plus and minus strand RNAs contained within them. Previously, Smith et al. (1981) described the separation of the complementary strands of the genome dsRNA segments for cytoplasmic polyhedrosis virus (CPV) and human reovirus by electrophoresis on 1.75% agarose gels containing 7 M urea. The results of the study showed that the plus strands of CPV and most of the minus strands of human reovirus migrated faster than their corresponding, complementary strands on agarose-urea gels. In this communication, we describe the electrophoretic

separation

of the plus and minus strands of each of the eleven simian rotavirus

186

Fig. 1. Separation were prepared analyzed stacking pg/ml genome

of rotavirus

SA

from purified

by electrophoresis

11dsRNAs by polyacrylamide

double-shelled

on a SDS-containing

gels were of 10% and 3%acrylamide, ethidium

bromide

segments

virions

and photographed

gel electrophoresis.

by phenol

extraction

polyacrylamide

respectively.

gel (Laemmli,

After electrophoresis,

in the presence of ultraviolet

3H-labelled

and ethanol

dsRNAs

precipitation

and

1970). The resolving

and

thegel was stained with 10

light. The positions

of the eleven

are indicated.

Fig. 2. Resolution

of rotavirus

urea gels. 3H-labelled

complementary

dsRNAs

recovered

plus and minus strand RNAs by electrophoresis

from polyacrylamide

gel by electroelution

on agarose-

were resuspended

in 10

pl water. To each sample was added 25 pl of 0.25 mM sodium citrate buffer, pH 3.0,6 M urea, 20% sucrose (w/v) and 0.005% bromophenol agarose

slab gel containing

(Lerach

et al., 1977; Wertzand

1979) and exposed

to Kodak

(lane 12) and plus strands parallel

lanes.

micococcal

to electrophoresis

citrate

‘H-labelled

plus strand rabbit

RNAs

reticulocyte

were made lysate

(Patton

in a horizontal

1.75%

buffer, pH 3.0, at 175 V for 22 h at 4°C

Davis, 1979). The gel was then processed XAR-5 film. As markers,

RNAs made in vitro by EDTA-activated

Radio-labelled

nuclease-treated

blue which was then subjected

6 M urea and 0.025 M sodium

for fluorograph

dsRNAs

purified

(Wertzand

Davis,

from rotavirus

virions

virions (lane 1) were electrophoresed in a reaction et al.,

containing

by volume

in 70%

1984) and 30% EDTA-activated

187

1

2

3

4

5

6

7

8

9

10 ,ll 12

,9+

double-shelled creatine

virions (Mason et al., 1980). Reactions

phosphate,

acids, 2 mM dithioerythritol, ICN). RNA product

also contained

66 mM NH,Cl, 2 mM magnesium

was recovered

by phenol

Shown in lane 2are the complementary

extraction

acetate, and 1 uCi/ul

and ethanol

RNAs making up segment

for the eleven dsRNA segments

including termination

those

labelled

during

pH 7.7, 10 mM

A-E

(lane

RNA synthesis.

precipitation

[IH]UTP(40 (Patton

Ci/mmol,

et al., 1984).

1 1 (1 1), lane 3 (lo), lane 4(9), lane 5 (7 and

8), lane 6 (6), lane 7 (5), lane 8 (4), lane 9 (3), lane IO(Z), and lane 1 I strands

50 mM HEPES-HCI,

1 mM ATP, 0.6 mM each CTP and GTP, 0.1 mM UTP, 0.05 mM each of the 20 amino

(1).The positions ofthe plus and minus

is given. The origin of several bands in the plus strand RNA marker 1) is unknown

but may result from

non-specific

initiation

and/or

188

SAll

dsRNAs

in agarose

dsRNAs showed strand RNA. MATERIALS

Simian

gels containing

that the plus strand

6 M urea at pH 3.0. Analysis

migrates

of rotavirus

faster than its complementary

minus

AND METHODS

rotavirus

SAll

was propagated

in monolayers

of fetal rhesus

monkey

kidney cells (MA104) infected with
(MEM) containing 5% fetal bovine serum and 5% newborn bovine serum. cultures were maintained in MEM without serum but containing 5 pg/ml (Difco

1: 250) and 0.5 mCi/ml

[3H]uridine

(30 Ci/mmol,

ICN). When 80-90%

of cells showed cytopathic effects (5 days post infection), infected cell cultures were freeze-thawed 3 times and homogenized in the presence of an equal volume of trichlorotrifluoroethane (Mason et al., 1980). The phases were separated by low-speed centrifugation at 4°C and the organic phase re-extracted with an equal volume of TN buffer (50 mM Tris-HCI, pH 7.5,150 mM NaCl). The aqueous phases were combined, adjusted to 10% polyethylene glycol, and with stirring left overnight at 4°C. Virus was then pelleted by centrifugation at 65,000 X g for 30 min in a Beckman Type 70 Ti rotor at 4°C and resuspended in TN buffer. The virus sample was loaded onto a 4 ml 20-40% CsCl gradient in TN buffer (wt./wt.) and centrifuged for 18 h at 120,000 X g in a Beckman

SW50.1

rotor

(Mason

et al., 1980). Double-shelled

virus

particles

that

banded at a density of 1.36 g/cm3 were collected and dialyzed overnight against TN buffer at 4°C. ‘H-labelled RNA was isolated from virus by phenol extraction and ethanol precipitation and resuspended in 10 ul of water. To separate

rotavirus

SA 11 dsRNA

into individual

genome

segments,

3H-labelled

RNA from double-shelled virus was subjected to electrophoresis on a 10% polyacrylamide gel (PAGE) containing sodium dodecyl sulfate (SDS) (Laemmli, 1970). The position of dsRNAs in the gel was determined by soaking the gel overnight in a solution

of 10 pg/ml

ethidium

bromide

and exposing

to long wavelength

ultraviolet

light. A photograph of the separated dsRNAs is shown in Fig. 1. Bands corresponding to each of the rotavirus SAll genome segments were detected. An additional band found

immediately

above

that

of segment

10 in Fig. 1 is of unknown

origin

and

non-reproducible. To recover the viral dsRNA segments, bands were cut out of the gel and each placed into a separate dialysis tube containing approximately 250 1.11 of buffer (50 mM Tris-HCl, 1 mM Na,EDTA, pH 7.5). Because genome segments 7 and 8 nearly comigrate in this gel system, bands representing these RNAs were recovered in the same gel slice. RNA was eluted by immersing the dialysis bags containing gel slices into a shallow layer of TE buffer in a horizontal gel apparatus and applying a potential of 100 V for 24 h. Afterwards, the voltage was reversed for 15 set and the buffer removed from each bag. The RNA in the buffer was then extracted once with phenol in the presence of 0.5% SDS and precipitated with 20 ug yeast tRNA in 2.5 vol. 95% ethanol.

189

To resolve complementary ed dsRNAs

representing

horizontal

pH 3.0 (Wertz to Kodak

the viral genome segments

1.75% agarose

bromophenol

plus and minus strand RNAs, preparations gel containing

and Davis,

were placed in separate

6 M urea and 0.025 M sodium

1979). The gel was electrophoresed

blue dye front migrated

XAR-5 film (Patton

of 3H-labell-

22 cm, processed

at 150 V until

for fluorography

et al., 1984). A representative

wells of a

citrate buffer,

fluorograph

the

and exposed of the gel is

shown in Fig. 2. All preparations of rotavirus 3H-labelled dsRNA were resolved into at least two bands indicating that under these electrophoretic conditions viral dsRNAs were denatured different rates. RESULTS

with complementary

plus and

minus

strand

RNAs

migrating

at

AND DISCUSSION

Direct comparison

of bands representing

the separated

strands of rotavirus

dsRNAs

with bands of plus strand RNA made by activated virions (Fig. 2, lane 1) allowed polarities to be assigned to the single stranded RNAs generated by electrophoresis of rotavirus genome segments in the presence of urea. The results demonstrate that all the rotavirus SA 11 plus strand RNAs migrate faster than their complementary minus strand RNAs under these electrophoretic conditions (Fig. 2). The basis for differences in migration

rates of complementary

plus and minus strands

RNAs is not known but

cannot be correlated to differences in overall base composition and electrostatic charges (Smith et al., 1981). However, the fact that all the rotavirus and CPV plus strand RNAs migrate faster than their corresponding minus strand RNAs suggest that a common structural feature may exist for all plus or minus strands (Smith et al., 198 1). Together, these data show that electrophoresis on 1.75% agarose gels containing 6 M urea at a pH of 3.0 is a useful method for resolving rotavirus

dsRNAs.

that samples

Previous strand separation

were to be boiled

techniques

in the presence

the plus and minus strands

of

for dsRNA viruses indicated

of urea prior to electrophoresis

and

included 7 M urea in agarose gels as opposed to the 6 M urea that we have employed. However, our results indicate that neither is required to achieve denaturation and resolution

of dsRNAs

into plus and minus strands

ance of these more stringent conditions as a result, provide for better resolution on agarose-urea

on agarose-urea

gels. The avoid-

may reduce the risk of RNA degradation and, and detection of plus and minus strand RNAs

gels.

ACKNOWLEDGEMENTS

This research was supported by grants from the National Institutes 21478, and the USF Research and Creative Scholarship Program.

of Health,

AI

190

REFERENCES

Imai, M., K.A. Kantani, Laemmli,

U.K.,

N. Ikegami

1970, Nature

and Y. Furuichi,

(London)

Lerach,

H., D. Diamond,

Mason,

B.B., D.Y. Graham

and M.K. Estes, 1980, J. Viral. 33, 1111.

Mason,

B.B., D.Y. Graham

and M.K. Estes, 1983, J. Viral. 46, 413.

Patton,

J.T., N.L. Davis and G.W.

Rodger,

J. Wozney

1983, J. Virol. 47, 125.

277, 685.

S.M. and I.H. Holmes,

Smith,

R.E., M.A. Morgan

Wertz,

G.W. and N.L. Davis,

and H. Boedt,

Wertz,

1977, Biochemistry

16, 4743

1984, J. Virol. 49, 303.

1979, J. Viral. 30, 839.

and Y. Furuichi,

1981, Nucl. Acids Res. 9, 5269.

1979. J. Viral. 30, 108.