The preparation and biochemical characterization of intact capsids of equine infectious anemia virus

Vol. 160, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages

April 28, 1989

486-494

THE PREPARATION AND BIOCHEMICAL CHARACTERIZATION OF INTACT CAPSIDS OF EQUINE INFECTIOUS ANEMIA VIRUS MICHAEL M. ROBERTS AND STEPHEN OROSZLAN LABORATORY OF MOLECULAR VIROLOGY AND CARCINOGENESIS, BRI-BASIC RESEARCH PROGRAM, NCI-FREDERICK CANCER RESEARCH FACILITY, P. 0. BOX B, FREDERICK, MARYLAND 21701 Received

March

8,

1989

Capsids of equine infectious anemia virus have been isolated as coneshaped particles 60x120 nm in size. Detergent treatment of whole virus followed by two cycles of rate-zonal centrifugation in Ficoll produces these capsids in a yield of approximately 10%. The major protein components are the gag-encoded pll nucleocapsid protein and p26 capsid protein, which are present in eauimolar amounts. Substantial cleavaae of $1 to p and p4 can be observed under conditions where the viral p&ease packaged in the capsid is enzymatically active. 0 1989 Academic Press. Inc.

anemia Ew ne infectious sub-classified as a lentivirus (4-6)

to human

elongated

Cores partially

no lentivirus

core

cores

have

myeloblastosis

(14). electron

virus

microscopy

positively

identified

micrograph

(15).

isolated,

by the

visualized

with

nonionic

(AMV)

been

studied

and their

protein

spherical

of the

core

leukemia

and shown

to be similar

shell

virus

(MuLV)

most

of To date,

retrovirus

detergent. the

Those

of

extensively

AMV cores and the seen

an

bilayer

(3,12,13).

composition,

of murine

lipid

Other

by treatment

contain

by EM studies

detergent

the

related

Lentiviruses

characterized.

have

has been

seen

by

p27 was

in the

electron

and AMV were

in morphology

and

(16).

investigated

Abbreviations: immunodeficiency leukemia virus; microscopy. 0006-291x/89

chemically

that

to be genetically

surrounded

by nonionic

as a component Cores

a retrovirus (7-9).

was made between

(EM)

characterized

In this been

has been

correlation

composition

(HIV)

structure

disrupted

been prepared

Direct

virus

core

is

and found

of EIAV and HIV have been

virus

avian

(EIAV)

(l-3)

immunodeficiency

electron-dense

(l,lO,ll). whole

virus

study

the

conditions

for

the

as a means to locate

the

preparation viral

of EIAV capsids

RNA, structural

have

protein

EIAV, equine infectious anemia virus; HIV, human virus; AMV, avian myeloblastosis virus; MuLV, murine NC, nucleocapsid protein; CA, capsid protein; EM, electron

$1.50

Copyright 8 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

486

BIOCHEMICAL

Vol. 160, No. 2, 1989

components

and replication

can be well-defined core

by physical

preparation

developed

enzymes

and using

for

MuLV (17),

a homogenous

preparation.

and ~'26 (CA)

(for

the

EIAV cores.

purified

capsids.

Since

the

gag-encoded

HIV,

and both

capsids

is

the

AND BIOPHYBICAL RESEARCH COMMUNICATIONS

within

a macromolecular

and chemical

methods.

Ficoll

we have

gradient

succeeded

nomenclature

see ref.

18)

Therefore

these

extensive

sequence

homology

structural

proteins

viruses

a model

have study

for

similar

purification

that cores

the

in a 1:l

molar

pll ratio

can be appropriately

has been

demonstrated enzymes

this

AMV

EIAV capsids

proteins

and the pal-encoded morphology,

the

method

structural

exist

that

By modifying

in purifying

We determined

assembly

investigation

to (NC) in

named between of

EIAV and of EIAV

HIV. MATERIALS

AND METHODS

EIAV-producing Cf2Th cells (Wyoming strain) were grown as Virus: monolayers (19) at 37'C in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum containing L-glutamine (4 pmoles/ml), penicillin (100 units/ml), and streptomycin (100 pg/ml) in a humidified 5% CO, atmosphere. Confluency was attained after four days. Cell debris was removed from the medium by centrifugation on a Sorvall centrifuge with an SS-34 rotor for 10 min at 12,000 x g. Whole virus was harvested from the clarified medium by pelleting through a layer of 5% Ficoll 400 (Sigma Chemical Co., St. Louis, MO) for three hours at 100,000 x g using a Beckman L3-50 ultracentrifuge with a SW27.1 rotor. Densitv uradient centrifuoation: Gradients of 40-60% w/w sucrose or 5-30X w/v Ficoll 400 in 10 I# TriseHCl, 1 mM EDTA (TE buffer pH 7.6 or pH 9) were prepared in 12 ml centrifuge tubes at room temperature then equilibrated at 4°C for 1 hour. Centrifugation of detergent treated or untreated virus was carried out on a Beckman L3-50 ultracentrifuge at 100,000 x g for five hours on Ficoll gradients (rate-zonal) and 200,000 x g for 64 hours on sucrose gradients (equilibrium density determination) using a SW41 rotor. Equal fractions of 0.5-l ml were collected by side puncture just clear of any pelleted material at the bottom of the tube. These were diluted to 12 ml with TE buffer and centrifuged at 100,000 x g for four hours on a L3-50 Beckman ultracentrifuge using a type 65 rotor. The pellets containing cores were drained, then resuspended in 0.5 ml of TE buffer to be rebanded through a second Ficoll gradient, followed by another pelleting through TE buffer. After resuspension in TE buffer, fractions from the first and second banding were ready for analysis. Sodium dodecvl sulfate oolvacrvlamide qel electroohoresis (SDS-PAGE): This was performed on 10 to 20% gradient gels by the method of Laemmli (20). Samples were boiled 15 mins in disruption buffer (2% SDS/2% P-mercaptoethanol/2D% glycerol/100 mM Tris HCl pH 6.8/20 bg of bromophenol blue per ml). Gels were stained with Coomassie brilliant blue R-250 (Bio-Rad Laboratories, Richmond, CA). Protein determination: The total protein content of viral preparations were determined by calorimetry using the Bio-Rad dye reagent (21). acid compositions of purified proteins were determined with a Durrum amino acid analyzer (ninhydrin detection) after hydrolysis in vacua HCl containing 0.1% liquid phenol for 24 hrs. Relative proportions proteins were also estimated by densitometry of stained bands after PAGE. 487

Amino D500 with 6N of SDS-

Vol. 160, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Determination of NC:CA ratio: Both pll (NC) and p26 (CA) were purified by reverse-phase high pressure liquid chromatography (RP-HPLC) (6) from a capsid preparation. These were loaded onto a gel in a 1:1 molar ratio. After electrophoresis, stained gel bands were scanned using a Zeineh Soft Laser scanning densitometer to determine their relative integrated intensities. These were compared to the densitometric values obtained for the same protein bands after electrophoresis of capsid preparations. Electron buffer, electron

microscopv (EM): Samples were submitted for and evaluated by conventional negative staining microscopy with 2% phosphotungstic acid (22).

EM studies in TE transmission

RESULTS The nonionic used

to prepare

that

of Sterox

(14-16). pelleted

SL,

used

from yield.

through

Ficoll

revealed

1 liter

the

(see

probe

Materials

(see

below).

This

contained

Viral

for

corresponding

to the that

through

after

the

pictures

ratio

upper

part

lc).

a 32P-labeled

(see

Fig.

in the

when

the

bar-shaped

DNA

2)

bar-shaped

gradient. cores

were

particles

of the

was

cone-

gradient

The bar-shaped

g/cm3.

collected

and the

density

of the

cone-shaped

(Fig.

The density

by equilibrium

middle

observed

of cone-to by EM.

to be 1.27

capsids

which

resulted

banding at the

there from

were shown

reveal fat

appear

90-180’,

rebanded

in further

are

of EIAV capsids

varies

the

gradient

in the

cores

experiments,

counting

shaped

second

capsid

In initial

of the

particles

were

further.

60 nm in width of the

bands

banded

of fractions

regions

capsids

then

the

RNA using

in two peak

in

of the

appeared

of viral

scattering

RNA.

sucrose

characterized

centrifugation

that

viral

in

The cone

bar-shaped

cone-shaped

was determined

centrifugation not

light the

by particle

capsids

band

mainly presence

30% sucrose,

to be -3:l

shaped

two

both

package

pelleted found

the

below

location

A broader

RNA was located

This

particles

band just

50 1.c1 of

detergent

10 min on ice

to the

to

of AMV

capsids

Illumination

blue

to correspond

similar

(w/v)

producing

for

was

5 mg of protein

A 0.5%

for

TX)

by adding

containing

medium.

optimum

scattering

Houston,

substructures virion

lb and d) and SDS-PAGE analysis

tested

suggests

viral the

and Methods).

light

gradient

(23).

culture

Co.,

has a composition

from

was incubated

band was shown

were

Oil

to prepare

as the

The mixture

gradient.

Fractions

45-12T

was removed

of tissue

by EM (Fig

of the

(Shell

to 1 ml of an EIAV suspension

a distinct This

capsids

previously

was determined

maximal

45-12T

Neodol

bilayer

45-12T

concentration

tube.

Neodol

EIAV capsids.

The lipid

10% Neodol

from

detergent

end,

in a second purification.

in Fig.

particles

le.

approximately

and 25 nm at the

to be three depending

vertices

on the 488

Negative narrow sharply

orientation

cycle

of rate-zonal

Particles stained

recovered EM

120 nm in length, end. defined of the

At the

fat

end

by an angle particle

on

Vol.

160,

No.

BIOCHEMICALAND

2, 1989

BIOPHYSICALRESEARCH

COMMUNICATIONS

Figure 1. Negative-stained electron micrographs (~150,000) of (a) intact EIAV (b) cone-shaped capsid pelleted through 30% sucrose (c) bar-shaped core from which capsids were separated, and (d) capsids obtained after first and (e) second banding through Ficoll. Some changes in surface geometry are evident after the second banding, including a cleaner background.

the with

little

the

capsids

that

grid.

microscope

variation have

show more

The capsid in

shape

a more angular

surface

detail.

preparation or dimensions. appearance There

are 489

appears After against

to the

be homogeneous, second

a cleaner

some suggestions

banding,

background of faces,

edges

Vol. 160, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Capsids

1

2

3

CAPSID Preparation

02

4

5

6

7

6

9

10

11

Virus

M

12

*9,adlB*

EQWO

..

Control

Figure 2. Dot-blot test for viral RNA on nitrocellulose paper with 32Plabeled complementary DNA probe (23). The fractions were taken from the first 5-3D% Ficoll gradient banding at pH 7.6, run at 1O’C for 5 hours at 100,000 x g. Capsid fraction 5 and control fraction 10 were not tested (NT). SDS-PAGE analysis of the purified capsids prepared at pH 9, ws own a ongside starting whole virus and molecular weight markers (M).

and vertices the acid

caPsid

( see Fig to be pll

analysis

*

le). and p26

of the

M

la

SDS-PAGE (see

2a

the

3).

This

Fig.

RP-HPLC purified

lb

showed proteins

B

2b

92 -

major

was confirmed (see

M

Materials

la

and Methods).

2a

2b

-

p66 p51

45 -

45 -

- p32 - p26

30 -

-

21.5 -

6-

lb

by amino

66 -

- p66 - p61

-

components

92 -

66-

14.4

protein

-

e!sm

30 0

P22

21.5

--

-

-

14.4

--ppi

ab* r,

6-

+

w Ib

-‘_ -

-

-

P32 ~26

-

P22

-

P15

z

g’

Figure 4. SDS-PAGE to illustrate the effect of proteolytic cleavage in EIAV capsids. Samples were prepared and incubated at pH 7.6 at 4'C in the absence (lanes a) and presence (lanes b) of pepstatin (0.5 mg/ml). Samples were electrophoresed before (frame A) and after (frame 8) incubation overnight at 37-C in TE buffer at pH 7.6. Lanes M, molecular weight standards; lanes 1, whole EIAV before banding (-35 Ag loaded); lanes 2, capsid band of second gradient (-25 c(g (a) and 16 Ag (b) loaded). The cleavage of pll to p6 and p4 is inhibited by the presence of pepstatin (lane 2b). 490

of

BIOCHEMICAL

Vol. 160, No. 2, 1989

Other

proteins

after

the

minor

protein

including

second

66K bands

the

banding

(see

Fig.

We have

RT assay

intact

which

have been

(manuscript

to p26 in the The

percentage approximately

capsids

of capsids were

as the

a short

time,

incubation

capsids partial

period were

known (25)

Fig.

to pll

exposed the

used

active

in the in the

densitometry the

of pll

virus

indicated This

of

RP-HPLC

to p26 in the

(Table

the

1) which

Fig.

protease

with

TABLE 1.

almost

is

was

the

cleaved arrested

Fig.

has been

It

capsids

have

(manuscript

Yield of viral coomassie-stained

Protein content ofa ~26 band (rra)

Western

definitely

also

the

protease confirmed

blotting

in preparation).

inhibitor.

proteases

The pll

of cleavage

of

Total p26 content in oreoaration (Iral

0.005

158.4

Capsids

0.86

0.060

14.3

491

a

Cleavage

0.77

p26 standards.

pll

presence

association

Whole virus

on precalibrated

with

by pepstatin,

capsids determined from densitometry p26 band from gel in Figure 3 Fraction of total loaded on se1

from

retroviral the

pH 7.6)

when the

derived

was inhibited

including

by PMSF, a serine

substrates

3).

(at

shown to be due to the

capsid.

even for

In an extended

completely

p6 is

preparation

unchanged.

was completely

at pH 9 (see that

the

remained

at room temperature,

pll

proteinases,

4) but not synthetic

to pH 7.6

to p6 occurred.

in the

of aspartic

Provided

components

of pll

cleavage

packaged

with

'Based

as

intensities

used to determine

and pH 9.

protein

The cleavage

and kept

(6)

inhibitor (see

were

4).

prepared

protease

studies

the

cleavage

not-shown).

of viral

Using

ratio

from whole

at pH 7.6

times,

at 37'C,

to p6 and p4 (see

(data

molar

normally

200 pg of viral

to be 1:3 as for

a 1:l

in TE buffer

incubation

integrated

p26 band was also

obtained

prepared

at 4°C at all

antiserum

implies of the

(24).

10%.

As soon

capsids

relative

and

known.

itself

salts

about

preparation.

the

51K,

not

enzymatically

Usually

capsid

32K,

EIAV capsids

by EM after

was determined

This

The capsids was kept

each

and Methods

intensity

yield

visualized

other

gag-p07

(RT)

or other

RT is

in preparation). from

standards.

capsids.

the

are

are

gag and/or

intact

the detergent be that

22K,

of these

of the in

removed

There

transcriptase

activity

could

3.

capsids:

of each

products

without

It

in Materials

purified

viral

but

was recovered

described pll

Mg+',

buffer.

buffer

protein

cleavage

RT endogenous

capsids,

assay

The nature

completely

in Fig.

in the

The 66K may be reverse

detected

almost

as shown

remained

3 and 4).

(6).

at pH 7.6 with in

Ficoll,

which

They may be intermediate polyproteins

p9 and p15 were

in

components

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 160, No. 2, 1989

which

occurs

in the

components remains

capsid

has not packaged

incubated

pll,

BIOCHEMICAL

been

observed

pll

in the

Capsids does

to those

prepared

sequence

is

take in

that

subviral

the

protease

at 4°C and subsequently permitting

place,

Fig.

now being

and other

implying

and pH 7.6,

not

shown

RESEARCH COMMUNICATIONS

envelope

virus,

or at 37'C

cleavage

similar

from

in whole

form.

temperature

or at pH 9 where

morphologically cleaved

when separated

in latent

at room

AND BIOPHYSICAL

cleavage

of

appeared

le.

The peptide

bond

investigated.

DISCUSSION We have established component whole is

of the

EIAV

likely

(6)

suggested

virion. is

that

that

(26).

viral

The p26 of

nucleocapsid important

enzymes.

The role

of the

noted

that

is

now under

in the

capsid cycle

RT, are

The isolated

capsids

both

CA coat

functional

to affect

the

the

is

and as

within

an

passage

of

of these

protease

step

integration

in the

that in the

of viral

the

and pepstatin

conceivable

in a critical

on

and other

function

by viral

It

investigation.

around

protease,

allows

lost

by nucleases

(RT substrates)

structure

involved

p27 is

protease,

that

it

as

layer

(RT),

in

(10,ll).

CA protein,

demonstrated the

including

(CA)

results

exterior,

to digestion

transcriptase

pll + p6 cleavage

is

replication

From our

a protective

as deoxynucleotides the

(NC) and p26

on the

as the

form

the

the

stable

on EM and immunolocalization

Apparently

of

features

we have

suggest,

a structurally

capsids.

susceptible

RNA, reverse

into

is

of pll

ratio

therefore

However,

such

inhibitor)

packaged

based

EIAV would

molecules

cycle

models

structure.

(protease life

geometrical

more

experiments

smaller

the

has been

protein,

capsid

of EIAV

recovered

RNA becomes

enzymes.

preliminary intact

in the

p26 forms

In the case of AMV it the

capsid

The equimolar

retained

by structural

banding,

the

the

protease

early

DNA into

viral phases

the

host

cell.

important from

viral

other

capsids

enzymes

viral

contaminants.

crystals

procedures

described

the

in their

can be obtained

investigate would

should

make it capsid,

example. formidable Focusing

here

which

has already

obstacle on capsid

to the

potential

of HIV envelope development

components

proteins

as targets

objective. 492

HIV capsids.

could

drug

aid

binding

HIV p24 (27), is

of a therapy

be possible

to

The preparative

to prepare the

of

of these

should

diffraction.

for

assays interference

yields it

antiviral

proposed

for

without

high

by X-ray

be adapted been

system

environment preparation,

material

could

a good

sufficiently

a purified

to locate

The variability

natural If

in of the

possible

now provide

for

currently AIDS

in achieving

This sites

on

for posing

a

(8,28,29). such

an

BIOCHEMICAL

Vol. 160, No. 2, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGMENTS We thank Kalmar

Sally for

Lockhart

help

in tissue

HPLC, and Robert grateful

for

Our appreciation

for

Nagashima finally

Research-sponsored

with

dot-blot

Shawver

Cancer

for

and Akos with

viral the

RNA.

the

RP-

We are

electron

typing

Institute,

Research,

Cassell

assistance for

Gonda for

to Carol

National

for

test

and Matthew

Bionetics

Beth

Ray Sowder

the

goes

by the

NO. NOl-CO-74101

assistance,

culture,

Stephens

to Kunio

technical

also

microscopy.

the

manuscript.

DHHS, under

contract

Inc.

REFERENCES

1. Gonda,

M.A., Charman, H.P., Walker, J.L., and L. Coggins. (1978) Am. J. Vet. Res. 39, 731-740. 2. Haase, A,T. (1986) Nature 322, 130-136. F., Matheka, H.D., Coggins, L., and Htirtner, D. (1977) Arch. 3. Weiland, Virol. 55, 335-340. 4. Chiu, I.-M., Yaniv, A., Dalberg, J.E. et al. (1985) Nature 317,

366-368. 5. 6. 7.

Stephens, 594. Henderson, J. Virol.

R.M.,

Casey,

J.W.,

L.E., Sowder, 61, 1116-1124.

Barre-Sinoussi,

F.,

and Rice,

R.C.,

Smythers,

J.C.,

Chermann,

(1986) Science

N.R. G.W.,

Rey,

F.,

and Oroszlan, et

al.

231,

589-

(1987)

S.

(1983) Science

220,

868-870. 8. 9.

Popovic, Science Ratner,

M., Sarngadharan, 224, 497-500. L., Haseltine, W.,

M.G.,

Read,

Patarca,

E.,

and Gallo,

R. et al.

284.

R.C.

(1985) Nature

(1984) 313,

277-

10. Gelderblom,

H.R., Hausmann, E.H.S., Ozel, M. et al. (1987) Virology 156, 171-176. 11. Gonda, M.A. (1988) J. Electron Microsc. Tech. 8, 17-40. 12. Palmer, E., Sporborg, C., Harrison, A. et al. (1985) Arch. Virol.

85,

189-196. 13. Stannard, Gen. Virol.

L.M., 68,

de St. J. van der Riet, 919-923.

(1972) J. Virol.

F, and Moodie,

14. Stromberg,

K.

15. Stromberg, 16. Bolognesi, 549-564.

K. (1974) J. Virol. 13, 513-528. D.P., Luftig, R, and Shaper, J.H.

17. Oroszlan,

S.,

Johns,

(1987) J.

9, 684-697.

Jr.,

L.W.,

J.W.

and Rich,

(1973) Virology

M.A.

(1965) Virology

56, 26,

638-

645. 18. Leis,

J.,

Baltimore,

D.,

Bishop,

J.M.

et al.

(1988) J. Virol.

62,

1808-

1809. 19. Benton,

C.V., Intervirology

20. Laemmli, 21. Bradford, 22. Anderson, Microscopy Press.

U.K.

Brown, B.L., 16, 225-232.

(1970) Nature

Harshman, 227,

J.S.,

and Gilden,

R.V.

(1981)

680-685.

M.M. (1976) Anal. Biochem. 72, 248-254. N., and Doane, F.W. (1987) In: Doane, F.W. in Diagnostic Virology, pp. 14-31. Cambridge

493

(ed.), Electron University

Vol. 160, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

23. Selden, R.F. (1987) In: Current Protocols in Molecular Biology, unit 4.9; John Wiley & Sons. 24. diMarzo-Veronese, F.D., Copeland, T.D., DeVico, A.L. et al. (1986) Science 231, 1289-1291. 25. Katoh, I., Yasunaga, T., Ikawa, Y., and Yoshinaka, Y. (1987) Nature 329, 654-656. 26. Chen, M.-J., Garon, C.F., and Papas, T.S. (1980) Proc. Natl. Acad. Sci. USA 77, 1296-1300. 27. Rossmann, M.G. (1988) Proc. Natl. Acad. Sci. USA 85, 4625-4627. 28. Fisher, A.G., Ensoli, B., Looney, D., et al. (1988) Nature 334, 444447. 29. Saag, M.S., Hahn, B.H., Gibbons, J. et al. (1988) Nature 334, 440-444.

494