Vol.

170,

August

No.

3, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

16, 1990

CHARACTERIZATION

Peter

OF AN INTERNALLY INITIATED OF HIV-l PRODIJCED IN E. COLI Zervos,

Tom

Hassell, Richard Mei T. Lai*

INTEGRASE

Van

Frank,

1061-1066

PROTEIN

and

Division of Molecular and Cell Biology Research, Lilly Research Laboratories, Indianapolis, Indiana 46285 Received

.&me 20,

1990

Summarv: In E. coli cells transformed by an expression vector for the production of the protease (PR) integrase (IN) of HIV-l, three vitally encoded proteins were produced: an II-kDa protein and a 32-kDa protein identified by immunoassays as the mature PR and IN protein, respectively, and an additional protein 15-kDa in size that reacted strongly with an antiserum recognizing a region in the carboxyl half of the IN protein. The kinetics of its synthesis indicated that it was not a degradation product of p32-IN, rather it probably arose from internal initiation at an AUG codon in the middle of the IN gene. Amino terminal sequence analysis of the first 70 residues demonstrated a perfect match with those predicted from the nucleotide with the methionine codon at position 154 of the integrase sequence, beginning gene. @1990 AcademicPress, mc One of the steps critical retroviruses,

including

integration

(1).

Integrase

for the process

in the life

cycle

syndrome

(AIDS),

selective

drugs

Integrase

of HIV-1

HIV-l

protease

obtaining under

large

In cells

from

(residues

251-269)

Because

of the unique agent

target

gag-pal

control by

protein

(-32-kDa)

polyprotein

into

of the pal

the host gene is

by the IN protein

immune

deficiency

an expression

plasmid

an additional

an antiserum

made

that

protein against

of highly

sequence

Nucleotide should

of

We were the IN

the

encoded

processed

interested

by

in

gene in E. coli

bacteriophage a functional

lambda. protease

and

molecule

15-kDa

in size that was

a synthetic

peptide

to a region

end of the 32-kDa

and

(9).

that is specifically (6, 7).

of the PL promoter

in the carboxyl

To whom correspondence

the

for the development

by expressing

expressing

*

is

genome

played

of acquired

may be a good

of the IN protein

we found

and Hughes

RNA

role

of

1 (HIV-l),

by the 3’-end

same size has also been noted in E. coli Hizi

infection

type

HIV- 1.

a 160-kDa

with

encoded

the etiological

quantities

integrase,

virus

copy of the viral

(IN)

is a 288-residue

transformed

immunoreactive

protein

the IN protein against

the transcriptional

the 32-kd

DNA

(2-5).

of HIV-l,

of a productive

immunodeficiency

of the double-stranded

chromosome essential

to the establishment human

IN protein. HIV-l

A protein

integrase

examination

reveals

by Chang

of the et al. (8)

a Shine-Dalgamo

be addressed.

1061

0006-291x/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

like

170,

No.

3, 1990

sequence

gene. position E. coli

upstream

They

In this

and the amino

initiated

of an internal

suggest that in E. coli,

154.

the conclusion

BIOCHEMICAL

terminal

at an internal

protein methionine

BIOPHYSICAL

methionine plS-IN

communication,

that this

AND

codon

may result

we describe

sequence indeed codon

analysis resulted located

RESEARCH

at position from

of the 15kDa from

a de nova

at position

154 of the IN

internal

the time-course

COMMUNICATIONS

initiation production

protein, protein

which

at of IN in led to

synthesis

154 of the integrase

protein.

MATERIALS

AND METHODS

Co struction of Plasmid oHPIa: The expression plasmid pHPIa (Fig. 1) was derived nr,orn a prokaryotic expression vector, pHP10, that produced a functional HIVThe insert in pHPIa contained the pol gene nucleotides 1 protease in E. cofi (10). 2084-2620 linked to nucleotides 4154-5121 of clone HXB-2 of the HTLVIIIB isolate of HIV-l (11). The expression of the inserted sequence was driven by the 4, promoter of the bacteriophage lambda under the regulation of the thermal labile repressor ~1857. All recombinant DNA manipulations were carried out according to standard procedures (12).

Induction and Analvsis of the HIV-l Protease and Interrrs The plasmid pHPIa was transfected into a lon protease deficient E. coli strain, L507 (htpR165am, ion R9, cps3, a derivative of LC137, originally from Professor F. Goldberg, Harvard University) to obtain the LS-HPIa strain. LS-HPIa cells were maintained at 32Oc until To induce gene expression, the growth temperature was quickly mid-log phase. shifted to 4OoC. At different times after induction, aliquots of cells were taken, chilled and pelleted. To analyze the proteins, pelleted cells were resuspended in approximately 5 OD600 ml of SDS gel sample buffer (62.5 mM Tris-HCl, pH6.8, 2% SDS, 2% 2-mercaptoethanol, 10% glycerol and 0.01% bromophenol blue). Cell lysates equivalent to 0.15-0.2 OD6oo were loaded into each well of a 12.5% SDS polyacrylamide gel. After electrophoresis (SDS-PAGE), the proteins were blotted onto nitrocellulose filters. The filters were then reacted with a polyclonal antiserum against an HIV-l protease peptide (residues 17-40) or with a polyclonal antiserum against an HIV-l integrase peptide (residues 251-269) followed by reaction with 125 I-labeled protein A (Amersham). The filters were exposed to X-ray films at -7OoC.

Amino Terminal Seauence Determination; The proteins used in sequence analysis were obtained from LS-HPIa cells harvested two hours after induction. The bacteria were lysed in a SDS gel sample buffer and the proteins were separated by Cell lysates prepared from approximately lSDS polyacrylamide gel electrophoresis. 2 x 108 bacteria (E 0.2 OD6oo) were loaded into another well and five times as much After transferring the proteins onto an Immobilin was loaded into another well. PVDF membrane (Millipore), the strip containing the larger amount of cell lysates was stained with Coomassie brilliant blue, and the other strip containing lesser amounts of bacterial lysates was reacted with the same polyclonal antiserum made against IN protein residues 251-269, as described above. The reaction was then followed by standard Western blot procedure, using a Vectastain ABC kit (Vector Laboratories). Both p32-IN and plS-IN were clearly discerned. Using this strip as a guide, the protein band corresponding to pl5-IN was excised from a CBB-stained strip. The membrane was arranged in a single layer in the upper cartridge block of an Applied Biosystems 470A gas-phase sequenator. A pretreated trifluoroacetic acidetched glass fiber filter containing polybrene was then placed on top of the excised band and the cartridge was reassembled. Sequencing and analysis of the PTHderivatives was performed by standard procedure (13). 1062

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BIOCHEMICAL

AND

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RESULTS AND DISCUSSION The

DNA

493 amino

insert

acids long

for translation mature

(493aa

initiation,

protease

transcriptase primary

in plasmid

(ART),

followed

allow

whether

mid-log

phase

were

cleavage

this were

(IN,

288aa I

amino

and

carboxyl

PR and IN protein

PR must precede

thermally

by SDS-PAGE

filter

antiserum, Methods

induced

to express

followed

protease.

(The

non-specific culture induction,

Figure antiserum

bacterial

and its quantity the mature

up to 120 minutes

2A

with

harboring

was

an 11-kDa

since

with

11 kDa

noted

induction

noted.

5kDa

proteins.

with

Samples

of cells filters,

in the

immunoreactive

At

HIV-l

markers [zero

was a time]

80 minutes increased

after with

time

band seen only in the

32 kDa

w Plasmid map of pHPIa for expression of HIV-l protease and integrase protein. The DNA insert (PI) contained nt 2084-2620 linked to nt 4154-5121 of HIV-l clone HXB2. The expression was driven by PL promoter thermally regulated by repressor ~1857. L: the 56aa of pol gene that precede the mature protease (PR); ART: partial IN: integrase protein. reverse transcriptase; 1063

were

anti-IN

the mature

in uninduced

The other specific

at

the time-course band

and 30-kDa

Its production

To

The proteins

depicting

time).

scheme, cells

A, as described

protein

sites

LS-HPIa

nitrocellulose

representing

46-kDa

it was also

was clearly

blot

protein

(Fig. 2A, lanes 5, 6 and 7).

6kDa

protein major

between

did not increase protease

The

IN protein.

and the other

a Western

cells.

In this

pHPIa,

onto

sites

processing

after induction.

blotting

125 I-labeled

band migrating protein,

After

The

autoprocessing

of HIV-l.

the viral

antiserum

represents

PR in LS-HPIa

intense

gels.

the anti-PR

by reaction

of HIV-l

the anti-PR

with

32-kDa). These

that of the 32-kDa

the case in E. coli

in duplicate

reacted

section.

production with

was

of reverse

protein

the

one added

of the pol gene, the

site of the IN protein.

of authentic

was indeed

(L)

for a polyprotein

at position

and 526-560

taken at 0, 20, 40, 60, 80, 100 and 120 minutes

separated one

contained

terminal

of the mature

examine

l-24

integrase

capacity

a methionine

56 residues

residues

product

the production

the production

It included

by the first

and the entire

translational

(Fig. 1) had a coding

I 54-kDa).

(PR, 99aa E 11-kDa),

of PR and the amino would

pHPIa

Vol.

170,

No.

3, 1990

(FE)

1

BIOCHEMICAL

2

3

4

5

6

7

AND

BIOPHYSICAL

,!&

1 2

66L

46-

RESEARCH

3

4

5 6

COMMUNICATIONS

7

4630-

-

P32-IN

-

P15lN

21.5-11

14.3-

PW

E&I-& Time-course induction of HIV-l protease and integrase in L507 E. coli cells transformed by pHPIa (Fig. 1). After temperature shift to 400 (induction), samples were taken at 0, 20, 40, 60, 80, 100 and 120 minutes (lanes 1, 2, 3, 4, 5, 6 and 7, respectively), and analyzed by Western blot analysis using (A) an antiserum made against residues 17-38 of HIV-l PR; and (B) and antiserum made against residues 251269 of HIV-l IN protein.

induced

and migrating

samples

processing

intermediate.

(-54-kDa)

was observed

autoprocessed Figure

2B

IN protein

representing

approximately

a Western

cells.

2%kDa

excess was not

depicting

antibody

against

primary

represented

a

translational

suggesting

product

that PR probably

protein

the full-length

a degradation

its nature

suggested

pl5-IN

may

that codon

have

as early

protein IN

was produced

protein

indicated

of the

detected

three

protein

most

A minor

band

Most

striking

as 20 minutes

(Fig. 2B, lane 5), its yield

32-kDa

product

251-269

as a 32-kDa

was not known.

that was detected

after

was more than lomuch that

earlier

and

pl5-IN

of p32-IN.

out previously

of sequence

IN residues

production

2B, lanes 5, 6 and 7).

The fact that the 15-kDa than

the time-course

One migrating

(Fig.

By 40 minutes

of a methionine

4641

blot

IN protein

bases upstream that

little

in size was also noted;

It has been pointed is a stretch

probably

of induction,

cultures.

of a 15-kDa

fold that of p32-IN. probably

The

the mature

(Fig. 2B, lane 2).

in greater

very

the course

in the induced

was the appearance

there

during

protein

rapidly.

represents

proteins

induction

Interestingly,

in LS-HPIa

IN-related likely

very

as a 2%kDa

(8, 9) that in the middle

resembles at position

resulted

from

the Shine-Dalgamo

of the integrase sequence

154 of the IN gene (Fig. 3). internal

initiation

gene

located It

11

was

at met-154.

AATTTGGAATTCCCTACAATCCCCAAAGTCAAGGAGTAGTAGAATCTATGAATAAAGAAT

4700

FGIPYNPQSQGVVESMNKEL 4701

TAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAA KKIIGOVRDQAEHLKTAVQ

w The nucleotide sequence and the deduced amino acid sequence around the Shine-Dalgamo-like sequence in the middle region of the integrase gene of HIV-l clone HXB-2. The Shine-Dalgamo-like sequence and the methionine codon at position 1.54 are underlined.

1064

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BIOCHEMICAL

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RESEARCH

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Table 1. Amino Terminal Sequence Analysis of p15-IN of HIV-l Produced in E. coli

Gycle

PTH-aa

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

MET ASN LYS GLU LEU LYS LYS ILE ILE GLY GLN VAL ARG ASP GLN ALA GLU HIS LEU LYS

*Deduced from the nucleotide Initiation

at met-154

encompassing To

residues

determine

produced

excised

procedure.

sequence of HIV-l

MET ASN LYS GLU LEU LYS LYS ILE ILE GLY GLN VAL ARG ASP GLN ALA GLU HIS LEU LYS

clone HXB2.

lead to the production

whether

plS-IN

cells

and blotted

pl5-IN

18 6 18 14 17 13 14 22 20 14 16 11 5 6 5 7 6 2 3 3

of a 135aa protein

(zlS-kDa)

135 to 288 of the IN protein.

by LS-HPIa

SDS-PAGE The

would

Predicted* aa

pMOLES

indeed

harvested

onto an Immobilin

from

The results

represented

at two

the filter

hours

PVDF

such

after

as described

was microsequenced

of the first 20 cycles

a protein,

induction

plS-IN

was separated

in the Methods

according

of sequencing

by

section.

to the standard

is shown

PTH

in Table 1.

For

comparison, the first 20 amino acid residues (starting from met-154) predicted from the DNA sequence are listed in parallel. It is clear that the first 20 amino acids of pl5-IN

matched

demonstrated met-154

perfectly

with

unequivocally

that

of the IN protein.

against

resiclues

the carboxyl

In summary, protein

that

It contained diagnosis

suggested

represented

the

identified

protein

pl5-IN

IN

DNA

sequence.

represented

This

a protein

strongly

contained

with

result

that

began

the antiserum

the sequence

at made

extended

to

protein.

by immunoassay

carboxyl

immunoreactive

of the integrase

indeed

that

of the mature

we had

a highly

pl5-IN

from

The fact that it reacted

251-269

terminus

those predicted

terminal region

half

and of the

of the IN

in individuals

microsequencing

infected

HIV-l

protein with

a 15-kDa

integrase

and may

protein.

be useful

for

HIV-l.

REFERENCES (1)

Varmus, H. and Swanstrom, R. (1984) In “RNA Tumor Viruses” (Weiss, R., Teich, N., Varmus, H. and Coffin, J., eds.) 2nd Ed. p. 75-135. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1065

Vol.

(2) (3) (4) (5)

(6) (7)

(8)

(9) (10) (11)

(12) (13)

170,

No.

3, 1990

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Schwartzberg, P., Colicelli, J. and Goff, S.P. (1984) Cell 37, 1043-1052. Donehower, L.A. and Varmus, H.E. (1984) Proc. Natl. Acad. Sci. USA 81, 64616465. Panganiban, A.T. and Temin, H.M. (1984) Proc. Natl. Acad. Sci. USA 81, 7885 7889. Quinn, T.P. and Grandgenett, D.P. (1988) J. Virol. 62, 2307-2312. Gendelman, H.E., Theodore, T.S., Willey, R., McCoy, J., Adachi, A., Mervis, R.J., Venkatesan, S. and Martin, M.A. (1987) Virology 160, 323-329. Jacks, T., Power, M.D., Masiarz, F.R., Luciw, P.A., Barr, P.J. and Varmus, H.E. (1988) Nature (London) 331, 280-283. Chang, N.T., Huang, J., Ghrayeb, J., McKinney, S., Chanda, P.K., Chang, T.W., Putney, S., Samgadharan, M.G., Wong-Staal, F., Gallo, R.C. (1985) Nature 315, 151-154. Hizi, A. and Hughes, S.H. (1988) Virology 167, 634-638. Lai, M.T., Dee, A.G., Zervos, P.H., Heath, W.F. and Scheetz, M.E. (1990) In “Retroviral Protease: Control of Maturation and Morphogenesis” (Pearl, L. ed.). Stockton Press, London. (in press). Ratner, L., Haseltine, W., Patarca, R., Livak, K.J., Starcich, B., Josephs, SF., Doran, E.R., Rafalski, J.A., Whitehorn, E.A., Baumeister, K., Ivanoff, L., Petteway, S.R., Pearson, M.L., Lautenberger, J.A., Papas, T.S., Ghrayeb, J., Chang, N.T., Gallo, R.C. and Wong-Staal, F. (1985) Nature (London) 313, 277-284. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Hewick, R.M., Hunkepillar, M.W., Hood, L.E. and Dreyer, W.J. (1981) J. Biol. Chem. 256, 7990-7997.

1066

Characterization of an internally initiated integrase protein of HIV-1 produced in E. coli.

In E. coli cells transformed by an expression vector for the production of the protease (PR) integrase (IN) of HIV-1, three vitally encoded proteins w...
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