Methylation of sindbis virus “26S” messenger RNA

BIOCHEMICAL

Vol. 66, No. 4,1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

METHYLATION OF SINDBIS Donald College

Department and Dentistry Piscataway,

of Medicine

Received

August

T. Dubin

26,

VIRUS "26s" and Victor

MESSENGER RNA Stollar

of Microbiology of New Jersey - Rutgers New Jersey 08854

Medical

School

1975

The main virus-specific messenger RNA species of Sindbis virusinfected hamster cells, the "26s" RNA, has been examined with regard to Internal methylated residues and terminal methylated methylation status. The internal methyl residues were present, in approximately equal amounts. groups were almost all in 5-methylcytosine residues and the terminal methyl groups were mainly in 7-methylguanine residues. Evidence is presented that these latter occur in "capped" 5'-termini with the novel structure m7G(5')pppNp. SUMMARY.

INTRODUCTION. slightly

but

--in vitro

under

virus

(7),

Recent significantly

of the cellular

and all

We have,

in addition,

contains

a single

Sindbis “26s”

virus-coded RNA (12,

(l-5),

of cytoplasmic (8),

and vesicular

of the viral 5'-termini found

methyl

(11).

shown that

methylated

virus

at "capped"

m7G(5')pppAp

have

the direction

vaccinia

clustered

studies

group

We now report mRNA species

occurs methylation

in

infected

cellular

mRNA is

the mRNA synthesized

polyhedrosis

virus

stomatitis groups

the general

the virion which

as is

mRNA methyl

with that

mammalian

reo-

virus

(9).

Some

were

found

to be

structure

("42s")

(6),

m7G(5')pppNmp?

RNA of Sindbis

virus

in the 5'-terminus properties hamster

cells,

of the main the so-called

13).

METHODC. Growth of BHR-21 cells as monolayers, infection with Sindbis virus (14), harvesting of infected cells, and phenol extraction of RNA, were essentially as previously described (15). The RNA was also subjected to LiCl precipitation (4", 18h, in 2M LiCl; ref. 16). B-elimination and DEAE cellulose column chromatography were performed according to published procedures (17, 8). Nucleotides were recovered from column fractions by BaC12 precipitation (18). Acid hydrolysis was accomplished by heating in 1N HCl at 100' for 45 min. and digestion with ribonuclease T2 (Sigma Chemical Co.) was according to *Unless otherwise noted, nucleic acid components: nucleoside-3'-phosphate; side.

standard abbreviations (10) have been used for Np, e.g., PN, nucleoside-5'-phosphate; m7G, 7-methylguanosine; Nm, 2'-O-methylnucleo-

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Vol. 66, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Furuichi et al (19), omitting prior denaturation of the RNA. Paper electrophoresis was performed in 0.05 N ammonium formate buffer, pH 3.5, on Whatman 3MM sheets at 3OOOV/90 cm, for 3h. Samples were spotted mid-way between the anode and cathode. Methylated base standards were obtained from Cycle Chemical Corporation, and [methyl-3Hlmethionine (specific (specific activity 55.5mci/mmole) activity 10.5 ci/mmole) and [2- 14Cluridine from New England Nuclear Corp. RESULTS. general

Hamster

cells

RNA label)

and

beginning were hibit purified 13),

added

3h after

RNA.

In view

with

with

RNA.

Fig.

(to virus. purine

of the expectation

was a well-defined that

label

Adenosine rings

viral

(to

RNA methyl

as expected

gradient for

provide

a

groups),

and guanosine

and actinomycin

1 shows a density

peak of 14 C appeared it

14 C-uridine

5h with

Sindbis

of 3H into

entry

A major

for

[methyl-3H]methionine

of cellular

and associated

labeled

infection

to minimize

labeling

were

to inpattern

of

26s mENA (12,

3H peak. mBNA would

contain

methylated

14 C-Uridine-labeled Fig. 1. Density Gradient Sedimentation of 3H-Methyl, RNA from Sindbis Virus-Infected Cells. Five BHK monolayers in lOOmm-petri plates were infected at zero time with Sindbis virus (14), 20 pfu/cell. After lh at 20' for adsorption, the virus suspension was replaced with Eagle's medium (24) containing the non-essential amino acids, adenosine and guanosine (lo-4M), actinomycin D (4 pg/ml) and a lowered level of methionine (0.3pg/ml). At 3h [methyl-3K]methionine (200 uci/3.5pg/ml, final) and [2-14Ciuridine At 8h the cells were harvested and processed as (O.lpc/ml, 10w4M) were added. described in the text. The purified RNA was subjected to density gradient sedimentation (15-30% sucrose in Tris-HCl buffer, pH 7.5, containing 0.1 N NaCl, 0.001 M EDTA and 0.2% sodium dodecyl sulfate) in an SW 27.1 rotor; Samples of 20 1.11were transferred from each 0.6 ml 23,000 RPM, 17h at 20'. fraction to scintillation vials containing T ton X-100 and New England counter. Nuclear Formula 949, and assayed for 3H and rf C in a scintillation The positions of cytoplasmic RNA markers were determined on the basis of 33, (-0-O-J ; 260 mn absorbancy tracings using an ISCO absorbance monitor. 14c, (-x-x-).

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Vol. 66, No. 4,1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Froctian Number

Fig. 2. DEAE Cellulose Chromatography of T2 Eibonuclease Digests of Sindbis Virus 26s RNA. An aliquot (10% of the total) of the 26s cut of Fig. 1 was subjected to B-elimination, followed by T2 ribonuclease digestion in parallel with a second, intact, aliquot. The digests were subjected to DEAE cellulose chromatography as described in Methods and 0.4 ml portions of each 2.0 ml fsc~:~;~~,'~~;-r;;;cJ $2 an tlAquasolfl scintillant. Only the k is pl;;ezd; C appeared in the mononucleotide fractions. arrows indicate the mid-points of the indicated isostiches from pancreatic ribonuclease digests of unlabeled cytoplasmic RNA run with the labeled digests. RNA; B, B-eliminated RNA. A, Intact

5'-termini

such as those

following

experiment.

oxidation

followed

should

release

linkage,

as is

"B-eliminated" with

compounds

with

and Nl is

absent

standard

lose

by aniline

from the the

case for

internal

treatment

m'G in

was then

structure

in

samples (3'-5')

of the

Introduction,

RNA.

("B-elimination"). present

the proposed

digested This

with

linkage

to prior

appear

As shown in Fig.

ZA, intact

chromatography.

One corresponded

This

structure

ENA yielded

( 8, 19).

5'-termini

n can be zero,

one,

g-elimination.

to the mononucleotide

The

in parallel

"capped"

two main peaks

procedure 5'-terminal

T2 ribonuclease,

as mononucleotides

the

to periodate

in "inverted"

enzyme releases

Nl(5')ppp(Nmp)nNp; subjected

we performed

26s ENA was subjected

RNA any ribosides

of intact the

in the

An aliquot

aliquot

an aliquot

described

or two

Residues (19,

as

in

20).

on DEAE-cellumarker

and con-

Vol. 66, No. 4, 1975

tained

BIOCHEMICAL

50% of the

(the

" -4"

second

peak)

3H.

subjecting

hydrolysis

under

toward

(5).

the cathode,

tives

hydrolysis

N2, N$di-,

guanine,

pyrimidine

adenines

also

phoretic

pattern

is

run

obtained

(Fig.

for

-4 peak from DEAE-cellulose. nificant ure,

3H is approx.

adenine

("peak

7-methylguanine;

Detailed

chromatographic

N6-methyladenine

the presumed

indicate

(30%) and adenine amount

of "leakage"

constituent end group

(70%); of peak (Fig.

peak

of 3H into

carbon

1376

of the methylated 3 shows the electro-

("2")

RNA and of containing

ahead

l-4

the Figin the

of

to 7-methylCp (peak

will

skeletons;

"4").

be published

a mixture

presumably

of arising

peak 3 is Peak 2 and

5-methylcytosine.

The 38 recovered contained

the sig-

RNA ran

just

behind

peak 1 is

the latter

3, open circles)

fastest,

corresponding

of peaks

7-methyl-

As shown in

slightly

3 are unidentified.

for

of intact

(70X),

and peak 4 is

tested

strip

length).

that

guanines

of intact of the

("3")

the origin

studies

Fig.

move deriva-

except

and most

hydrolysate

peak

of ribosc

ribose

the next

5).

total

characterizations our

7-methylguanine

the lesser

of the

off

and pyrimidine

and methyl

Adenine

by

at pH 3.5.

as a mixture

the rest;

5% ran as a small

ran just

Briefly,

elsewhere.

appear

the region

35% ran as a major

and 40-45%

mainly

(l/3

1");

guanine;

from a small

Only

illustrated

purines

hydrolysate

10% of the 3H of the acid

region

free

3 and ref.

an acid

was

examined

by electrophoresis

and the methylated

3.

aliquot

at pH 3.5 the purines

than

shown in Fig.

from 42s virion

was further

move as a group

faster

as a group

derived

residues

nucleotides

Guanine

moves slightly

position

2B).

residues

the anode.

N$,-dimethylguanine,

(Fig.

releases

and 7- methylguanine)

which

of this

On electrophoresis

while

move towards

The elution

followed

our conditions

derivatives

marker

of the B-eliminated

methylated

2'-0-ribose-methylated

phosphate

(l-,

to acid

to the trinucleotide

of the end-group

of the -4 peak

of the various

samples

nucleotides;

that

3H.

The DEAE-pattern

(90%) devoid

The nature

Acid

(11).

corresponded

43% of the

resembles

RNA, m7G(5')pppAp essentially

The other

and contained

peak closely

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

only

from

electrophoretic

Vol. 66, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 3. Electrophoretic Analysis of Acid Hydrolysates of Sindbis Virus 26s RNA and of the T2 Eibonuclease-derived End-Group therefrom. Samples of intact 26s RNA and of the labeled oligonucleotide from the -4 isostich of Fig. 2A (bracketed) were subjected to acid hydrolysis followed by electrophoresis at pH 3.5 as described in the text. The ovals indicate markers run with the hydrolysates and the bars indicate parallel markers. Symbols are as follows: A, adenine; 6MA, N6-methyladenine; 7MG, 7-amthylguanine; DMG, Ni-dimethylguanine; G, guanine. The markers for the two hydrolysates ran indistinguishably. One-cm segments were counted in an "NCS" scintillant. The 14C represents the label in Cp in the hydrolysate of intact RNA; no 14C was detectable in the end-group hydrolysate. 3H from hydrolysate of intact RNA, (-O-O-); 14C from hydrolysate of intact RNA, (-x-x-); 38 from -4 peak, (-O-O-).

peaks

2 and 3, in the

5% of the

total

derivatives

again the

3H ran beyond

would

bracket),

same relative

do (5).

when analyzed

no 2'-O-methyl g-eliminated

reaction

the same way, were

completion

(cf

the

mainly

observed.

ref.

20),

failure

small

(Fig.LB,

2 and 3;

residual

of the than

phosphate

sample

peaks

Thus the

rather

No more than

as 2'-O-methyl

%-eliminated

yielded

reflects

RNA.

label

in

B-elimination amounts

of

termini.

The present occur

as internal

other

half

occur

data

indicate

residues, in structures

5'-termini

DISCUSSION.

in

as the intact

the anode,

The -4 peak from

-4 peak presumably

N(5')pppNmpNp

nucleoside

Cp towards

derivatives

to reach

capped

amounts

mN(5')pppNp,

that almost with where

half all

of the methyl of which

properties

are

groups

5-methylcytosine.

resembling

mN is mainly

of 26s RNA

m7G

those

The of the

and the penultimate

is unmodified, Certain

facets

of the 26s RNA methylation

1377

pattern

are

similar

to

Vol. 66, No. 4, 1975

patterns

found

ilar.

BIOCHEMICAL

for

mRNA from other

The presence

of this this

feature

residue

required

One striking viral

systems

in the

Introduction

However,

find

of internal internal

failure

of internal

other

mature

methylation 1) the

2) Sindbis

with these

mkWA differs

from

play

to examine

The pattern distinctive HeLa (4)

other

of internal

and mouse (3)

the role(s)

methylated

cytoplasmic

no internal,

BHK cytoplasmic

internal

methylated

(i.e.,

(23)

than

far

other

ab-

examined

viruses

for

in that

as SV40 mPNA) in that

it

may be correlated (e.g., It would

ref.

4),

be of

regard.

in Sindbis

26s RNA is also

of m5Cp residues. as of SV-40

residues

from

and

in this

Sindbis-specific

1378

indicates

26s RNA differs

as suggested

mRNA, but

obscure.

to

of mRNA polarity);

properties

residues

failure

the absence

mRNA thus

mRNA, as well

of the

mRNA is

respect

residues

base-methylated

The significance of the

1" viruses

proportion

rather

from the

if,

monkey

systems

mRNA precursors.

in the prominence

m5Cp in uninfected

of m6Ap (5).

These

report

mRNA.

mRNA (as well

Nm residues

"class

Perhaps

virus

differs

in processing

, particularly

(211, revealed found

a role

m6Ap.

methylases,

and "plus"

(22).

of penultimate

a high

and viral,

also

methyla-

another

contained

viral

that

the --in vitro

of internal

RNA-synthetic

Sindbis

(20)

on SV40-infected

In this

cellular

in the cytoplasm

residues

interest

virus

from

manuscript

all

certain

cytoplasmic,

single-stranded

the absence

viral

of the

RNA is

synthesized

albeit

evidence

ribosomes.

finding

(21),

Nm residues.

Sindbis

our

of this

from "native"

eukaryotic

status.

virion

residues,

feature

with

the generality

and those

mRNA also

to encapsidate

5'-terminal

results is

in --in vitro

distinctive

of penultimate

our

preparation

methylation

m/G extends

in accord

mRNA appeared

methylated

and some are dissim-

by eukaryotic

SV40-specific

methylation

Another

is

viral

of the virions

sence

all

during

The cytoplasmic

(75%)

translation between

on --in vivo-synthesized cells.

mRNA, and is

for

systems, with

"capped"

difference cited

ted residues.

eukaryotic

of 5'-termini

of eukaryotic is

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

besides at levels pattern,

Analyses

of

cytoplasmic m6Ap. of only

mRNA

We have l/4

and in

in ml?NA in general,

that fact

remain

BIOCHEMICAL

Vol. 66, No. $1975

The present cumber citly

infected cipate

of provocative in the

Sindbis

results,

questions, Others

foregoing.

mRNA play cell? that

together

a role Are the

ongoing

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

studies

will

cited

some of which include:

in subverting pertinent

those

have been

work,

touched

Does the distinctive the protein-synthetic

methylases help

from other

cell-

to answer

a

upon implimethylation

machinery

or virus-coded? these

raise

of of the

We anti-

questions.

by Grant No. GM-14957 of the NatACKNOWLEDGMENTS. This work was supported ional Institute of General Medical Sciences and Grant No. AI-05920 of the National Institute of Allergy and Infectious Diseases, U.S.P.H.S. We thank Mrs. K. Timko for her expert technical assistance. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Perry, R.P. and Kelley, D.E. (1974) Cell, 37-42. Desrosiers, R., Friderici, K., and Rottman, F. (1974) Proc. Natl. Acad. Sci., U.S.A. 71, 3971-3975. Adams, J.M. and Cory, S. (1975) Nature, 255, 28-33. Furuichi, Y., Morgan, M., Shatkin, A.J., Jelinek, W., Salditt-Georgieff, M., and Damell, J.E. (1975) Proc. Natl. Acad. Sci., U.S.A. 72, 1904-1908. Dubin, D.T. and Taylor, R.H. (1975) Nucleic Acids Res., in press. Furuichi, Y. and Miura, K.I. (1975) Nature, 253, 374-375. Furuichi, Y., Morgan, M., Muthukrishnan, S., and Shatkin, A.J. (1975) Proc. Natl. Acad. Sci., U.S.A., 72, 362-366. Wei, C.M. and Moss, B. (1975) Proc. Natl. Acad. Sci., U.S.A., 72, 318-322. Abraham, G., Rhodes, D.P., and Banerjee, A.K. (1975) Cell, 5, 51-58. IUPAC-IUB Commission on Biochemical Nomenclature (1971) J. Mol. Biol., 55, 299-310. Hefti, E., Bishop, D.H.L., Dubin, D.T. and Stollar, V. (1975) J. Virol., submitted for publication. Mowshowitz, D. (1973) J. Virol., 11, 535-545. Simmons, D.T. and Strauss, J.H. (1974) J. Virol., 14, 552-559. Stollar, V., and Shenk, T.E. (1973) J. Virol., 11, 592-595. Stollar, V., Shenk, T.E., and Stollar, B.D. (1972) Virology, 47, 122-132. Baltimore, D. (1966) J. Mol. Biol., 18, 421-428. Fraenkel-Conrat, H. & Steinschneider, A. (1968), in Methods in Enzymology, Grossman and Moldave, Ed., Vol. XIIB, pp. 243-246, Academic Press, New York. D.H.L. (1973) J. Virol., 11, 487-501. ROY, P., and Bishop, Furuichi, Y., Muthukrishnan, S., and Shatkin, A.J. (1975) Proc. Natl. Acad. Sci., U.S.A., 72, 742-745. Muthukrishnan, S., Both, G.W., Furuichi, A.J., and Shatkin, A.J. (1975) Nature, 255, 33-37. Lavi, S. and Shatkin, A. (1975) Proc. Natl. Acad. Sci., U.S.A., 72, 2012-2016. Pfefferkom, E.R. and Shapiro, D. (1974) Comprehensive Virology, Fraenkel-Conrat and Wagner, Ed., Plenum Press, New York. 2, 171-230. Shatkin, A.J. (1974) Ann. Rev. Biochem., 41, 643-665. Eagle, H. (1959) Science, 130, 432-437.

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