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
Vol.
170,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
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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Vol.
170,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
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
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
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.
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