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J Virol, February 1998, p. 1691-1698, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Implication of a Central Cysteine Residue and the
HHCC Domain of Moloney Murine Leukemia Virus Integrase Protein in
Functional Multimerization
George A.
Donzella,1,
Oscar
Leon,2 and
Monica J.
Roth1,*
Department of Biochemistry, University of
Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical
School, Piscataway, New Jersey 08854,1 and
Instituto de Bioquimica, Facultad de Ciencias, Universidad
Austral de Chile, Casilla 567, Valdivia, Chile2
Received 11 July 1997/Accepted 21 October 1997
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ABSTRACT |
Moloney murine leukemia virus (M-MuLV) IN-IN protein interactions
important for catalysis of strand transfer and unimolecular and
bimolecular disintegration reactions were investigated by using a panel
of chemically modified M-MuLV IN proteins. Functional complementation
of an HHCC-deleted protein (N
105) by an independent HHCC domain
(C232) was severely compromised by NEM modification of either
subunit. Productive N105 IN-DNA interactions with a disintegration
substrate lacking a long terminal repeat 5'-single-stranded tail also
required complementation by a functional HHCC domain. Virus encoding
the C209A M-MuLV IN mutation exhibited delayed virion production and
replication kinetics.
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TEXT |
Productive retroviral infections
require the stable insertion of the reverse-transcribed viral genome
into the host chromosome, a step that is mediated by the virus-encoded
IN protein. Following reverse transcription of the retrovirus genome,
the IN protein processes the termini of the viral long terminal repeats
(LTRs) by endonucleolytic cleavage of the 3'-terminal dinucleotides. This 3'-processing reaction exposes a 5'-CA-3' dinucleotide that is
subterminally embedded in the LTRs and is conserved among known retroelements (3, 27, 28, 31, 41, 45). The 3' processing of
the viral DNA termini leaves behind a 5' overhang or single-stranded (ss) tail which may impart IN-DNA complex stability in vitro (16, 48). Integration of the viral DNA copy occurs by a staggered transesterification of the recessed viral 3' ends and the
phosphodiester backbone of the target DNA (19). Repair of
the resultant single strand gaps creates the hallmark duplication of
target DNA sequence flanking the retroviral integration site (4,
28, 45).
Biochemical and genetic studies suggest that IN functions as a multimer
which coordinates the integration of both viral DNA termini (18,
23, 24, 26, 28, 35, 37, 44, 46). The retroviral IN protein
contains three functionally important domains: an N-terminal HHCC
domain, a central catalytic core, and a C-terminal domain implicated in
nonspecific DNA binding (2, 34, 47, 49, 50). The results of
chemical cross-linking, equilibrium sedimentation, gel filtration, and
yeast two-hybrid studies suggest that regions throughout IN are
mediators of homomeric IN-IN interactions (1, 7, 23, 26,
46). The crystal and solution structures of the N-terminal HHCC
(containing zinc), central catalytic core, and C-terminal subdomains of
IN have substantiated the dimeric nature of these subdomains
independent of DNA (5, 7, 15, 33). Furthermore, recent
findings indicate that metal-dependent IN-IN (17, 52) and
IN-DNA interactions (38) are involved in functional,
higher-order multimerization of the IN protein.
Various assays for IN function have been developed including
integration (3' processing and strand transfer) (12, 29), disintegration (10), and coordinated disintegration (8,
13, 35). For disintegration, the substrate is unimolecular;
strand transfer and coordinated disintegration are dependent on
bimolecular assembly of the substrate mediated by multimeric IN-DNA
interactions. The domains of IN which are important for these
interactions vary among IN proteins and are implicated in such
interactions through mutational analysis (6, 24, 30, 43, 46,
47), analysis of modified substrates (9, 14, 42), and
the ability of defective IN mutants to complement each other in
trans (8, 18, 24, 35, 44).
In previous studies, the C terminus of M-MuLV IN could only be
minimally truncated (28 amino acids) in vitro and in vivo (24, 39). The M-MuLV HHCC domain was shown to be essential for 3' processing (24). M-MuLV IN lacking the HHCC region could
catalyze unimolecular disintegration (24) and single and
double disintegration on a tailed crossbone substrate (13).
Under less-saturating enzyme conditions, double-end disintegration was
strictly dependent on trans-subunit IN interactions promoted
by the HHCC domain (13). With the crossbone substrate, the
intramolecular "foldback" reaction is mechanistically similar to 3'
processing (13). The foldback reaction was found to be
dependent on both the LTR 5'-ss tail and N-terminal HHCC domain of
M-MuLV IN, whereas either determinant sufficed to promote
intermolecular activity (13). Collectively, these data
suggested differing roles of the HHCC domain in the formation of the
initial IN-DNA complexes and coordinated IN-IN interactions.
Prior studies had indicated that the M-MuLV IN was sensitive to
N-ethylmaleimide (NEM) modification (25).
Complementation studies performed with human immunodeficiency virus
type 1 IN have identified an NEM-sensitive site (Cys 56) which is
required in trans to the HHCC domain (17). In the
present work, multimeric M-MuLV IN-IN interactions important for strand
transfer and unimolecular and coordinated (bimolecular) disintegration
reactions were studied by complementation analysis of NEM-modified
M-MuLV IN subunits. A central cysteine within the catalytic core
domain, C209, and the HHCC domain were identified as NEM-sensitive
sites, both of which were necessary to mediate functional
complementation. Furthermore, a critical requirement of the C209
NEM-sensitive site for efficient viral replication was demonstrated,
suggesting a novel model for multimeric IN function.
NEM modification of wild-type (WT) and mutant M-MuLV IN
proteins.
Several M-MuLV IN mutants, which have been previously
characterized (13, 24), were used to probe the NEM
sensitivity of the IN-IN and IN-DNA interactions (Fig.
1). C232 is an inactive C-terminal
truncation that contains the HHCC domain and terminates just beyond the
first aspartate residue of the active site (Fig. 2 and 3, lanes 3 and
4) (24). A point mutant that eliminates the sole non-HHCC
cysteine residue located within the central catalytic core domain of
M-MuLV IN, C209A, is active for integration and disintegration
reactions (Fig. 2 and 3, lanes 5 and 6) (24). N105 lacks
the N-terminal HHCC domain and is inactive for 3' processing but is
active for unimolecular disintegration (Fig. 2B, lanes 7 and 8) and
coordinated disintegration of a tailed crossbone substrate (Fig. 3B,
lane 3). In strand transfer assays, however, N105 retains a limited
capacity to mediate integration into a single target site (Fig. 2A,
lanes 7 and 8) (24). Full integration activity of N105
can be restored by complementation with C232 (Fig. 2A, MOCK, lane
11) or by increasing the reducing conditions and protein levels in the
assay (24).
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FIG. 1.
M-MuLV IN mutants used for NEM alkylation studies. The
conserved domains and residues of IN are depicted, with the cysteines
highlighted in italics (top); the black box represents a C-terminal
insertion of 36 amino acids unique to M-MuLV IN. Cysteine targets of
NEM modification in the WT, C209A, N105, and C232 proteins are
highlighted in italics (bottom). Residues at which N105 and C232
initiate or terminate are also indicated.
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To investigate the nature of the IN-IN interactions important for
functional complementation, each of the M-MuLV IN proteins
was
subjected to NEM alkylation followed by quenching with excess
dithiothreitol (DTT). Strand transfer and unimolecular and bimolecular
disintegration assays were performed in parallel. A total of 120
pmol
of each protein to be treated was separately diluted to a
final
concentration of 2 to 2.5 pmol/µl with ice-cold diluent
(20 mM HEPES
[pH 7.4]-20% glycerol) and equilibrated on ice for
5 min. As a
control, mock reactions were performed in which each
of the M-MuLV IN
proteins were preincubated on ice with 40 mM
DTT for 20 min, followed
by incubation with 10 mM NEM (freshly
prepared; Sigma Chemical Corp)
for a further 20 min. For the NEM
reactions, proteins were first
incubated on ice in 10 mM NEM for
20 min, followed by a 20-min quench
with 40 mM DTT on ice. The
NEM and mock-treated M-MuLV IN proteins were
then assayed individually
and for functional complementation. A final
DTT concentration
of 40 mM was present in both mock and NEM reactions.
In complementation
assays where only one of the IN protein pairs was
alkylated, DTT
was present at a 20 mM concentration. Reactions were
incubated
at 37°C for 1.5 h and terminated. Similar results were
obtained
when the nonalkylated protein in the mixture was either
untreated,
mock treated (indicated in the figure legends), or DTT
treated
(data not shown).
Effects of NEM modification on strand transfer and unimolecular
disintegration reactions.
The results of the NEM treatment and
complementation of the M-MuLV IN proteins for strand transfer and
disintegration are shown in Fig. 2A and
B, respectively. Compared to the control mock reactions (Fig. 2A, MOCK, lanes 1 and 2), NEM-treated WT protein
was inactive for strand transfer (Fig. 2A, NEM, lane 2). Similar to WT
IN, the strand transfer activity of N105 protein was severely
compromised by alkylation (Fig. 2A, NEM, lane 10 versus Mock, lane 7).
N105 (at 20 pmol) remained sensitive to NEM (data not shown). Since
N105 contains only a single cysteine, the loss of N105 strand
transfer activity indicates that residue C209 in the central catalytic
core is NEM sensitive. These results indicate that modification of C209
blocks the catalytic functions of IN. C209 per se is not essential for
catalysis in vitro, since the mutant C209A is capable of mediating
single-end strand transfer (Fig. 2A, lanes 5 and 6) and unimolecular
(Fig. 2B, lanes 5 and 6) and bimolecular (Fig. 3A, lanes 5 and 6)
disintegration reactions. This is supported by NEM treatment of C209A
(Fig. 2A, NEM, lane 6), which cannot be modified in the catalytic core
and maintained activity, albeit yielding reduced levels of strand
transfer, double disintegration, and foldback product. The partial
diminution of activity implies that the HHCC domain may serve as an
additional target for NEM modification.
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FIG. 2.
Activity of NEM-alkylated M-MuLV IN proteins in strand
transfer and unimolecular disintegration reactions. (MOCK) Lanes 1 to
8, odd and even numbered lanes are reactions that respectively
contained 10 and 20 pmol of the mock-treated proteins; lanes 9 to 11, complementation reactions which contained 10 pmol each of the indicated
mock-treated proteins. (NEM) Assays were performed with 10 pmol of each
of the indicated proteins. M-MuLV IN proteins that were subjected to
NEM alkylation are indicated by bold underlined characters.
Non-underlined proteins are untreated IN. Lane 1, NEM-alkylated C232
negative-control reaction; lanes 2 to 13, reactions that contained
individual or mixed pairs of treated and untreated proteins. (A) Strand
transfer reactions with substrate 2784/2785 (13, 24). (B)
Unimolecular disintegration reactions with the standard Y substrate
(Y3154ds [13, 14]). The position of the 5'
32P-labeled C strand of oligonucleotide 3152 (14) is indicated ( ).
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Complementation analysis allowed us to directly assess the sensitivity
of the HHCC domain versus that of the central catalytic
core's C209
site to NEM modification since these domains are present
as separate
proteins. Previous analysis of N
105-C209A, a double
mutant that both
lacks the HHCC domain and contains an alanine
substitution for residue
C209, indicated that although it retains
limited integration and
disintegration activity it cannot be functionally
complemented by
C
232 (
24). This implied that residue C209 and
the HHCC
domain may be in close proximity. It was therefore tested
whether the
C
232 protein could protect residue C209 from alkylation
with NEM.
For premixed complementation reactions, 60 pmol of each
protein pair
was mixed together and then diluted immediately to
a total protein
concentration of 2 pmol/µl. Premixing C
232 with
WT IN prior to NEM
treatment did not protect WT or N
105 IN from
alkylation (Fig.
2A,
NEM, lanes 3 and 11) and caused a slight
reduction in the strand
transfer activity of C209A (Fig.
2A, NEM,
lane 7).
The effect of NEM treatment on multimeric IN-IN interactions could
therefore be addressed in complementation reactions in
which only one
of the pairs of M-MuLV IN proteins was alkylated.
Untreated C
232 was
unable to complement NEM-treated WT, C209A,
and N
105 M-MuLV IN for
the strand transfer reactions (Fig.
2A,
NEM, lanes 4, 8, and 12, respectively). When the HHCC domain of
C
232 was alkylated and tested
for complementation, little effect
on the strand transfer activity of
untreated WT and C209A IN proteins
was noted (Fig.
2A, NEM, lanes 5 and
9, respectively). A low level
of strand transfer activity was seen for
complementation of N
105
by NEM-treated C
232 (Fig.
2A, NEM, lane
13); however, this level
of complementation was below that of the
MOCK-treated sample (Fig.
2A, MOCK, lane 11). This is a result of
functional complementation
by a limited number of C
232 molecules
that were resistant to
alkylation (
51).
In the unimolecular disintegration reactions, NEM modification of the
individual M-MuLV IN proteins (WT, C209A, and N
105)
only resulted in
a partial loss of activity (Fig.
2B, NEM, lanes
2, 6, and 10, respectively). For the unimolecular disintegration
reactions, mixing of
C
232 with WT, C209A, or N
105 prior to NEM
treatment did not
protect the NEM-sensitive subunits against alkylation
(Fig.
2B, NEM,
lane 3, 7, or 11, respectively). Premixing C
232
with WT or N
105,
in fact, decreased the yield of joining product.
Under these
conditions, the HHCC domain may assist in exposing
the cysteines for
NEM modification. For complementation, the untreated
C
232 protein
stimulated the disintegration activity of NEM-treated
WT and C209A by
1.5- to 2-fold and that of N
105 by 4-fold (Fig.
2B, NEM, lanes 4, 8, and 12, respectively). The alkylated C
232
HHCC domain did not
interfere with productive unimolecular IN-DNA
interactions mediated by
the WT, C209A, and N
105 proteins (Fig.
2B, NEM, lanes 5, 9, and 13, respectively).
NEM alkylation and multimeric IN function: effects on bimolecular
coordinated disintegration reactions.
The NEM-treated M-MuLV IN
proteins were assayed for coordinated disintegration to more directly
address the effect of alkylation on the productive assembly of
bimolecular IN-DNA complexes. In the bimolecular disintegration
reactions, four major products have been identified by using tailed
crossbone substrates: intermolecular single disintegration and circular
double disintegration products, a foldback product formed by an
intramolecular attack by the target DNA 3'-OH, and an LTR hydrolysis
product (Fig. 3) (13). The catalytic properties of WT IN,
N105, C209A, and C232 on this substrate have previously been
characterized (13).
NEM treatment of WT (Fig.
3A, NEM, lane 2 and Fig.
3B, NEM, lane 1) and N
105 (Fig.
3B, NEM, lane 3) clearly
compromised activity.
This point is illustrated by the severe reduction
of single disintegration
and by the total ablation of double
disintegration and foldback
reactions compared to the control WT (Fig.
3A, NEM, lanes 1 and
2) and N
105 (Fig.
3B, NEM, lane 3). In
contrast, C209A was only
moderately affected by NEM alkylation, with a
slight decrease
in the level of double disintegration and foldback
products (Fig.
3A, NEM, lane 6). C209A has been previously
characterized as catalyzing
intramolecular foldback reactions less
efficiently than WT (
13).
To identify targets of NEM
important for coordinated disintegration
and to test if the loss of IN
function could be rescued, different
combinations of treated and
untreated proteins were assayed for
complementation. Mixing of C
232
with WT, C209A (Fig.
3A and B,
NEM, lanes 3 and 7), or N
105 (Fig.
3B, NEM, lane 4) prior to
NEM treatment did not protect the
NEM-sensitive subunits against
alkylation. The activity of NEM-treated
C209A was not changed
by complementation with untreated C
232 (Fig.
3A, NEM, lane 8).
Significantly, the coordinated disintegration
activity mediated
by NEM-alkylated WT (Fig.
3A, NEM, lanes 4) and
N
105 proteins
(Fig.
3B, NEM, lane 5) could not be restored by
complementation
with an untreated C
232 HHCC domain. The
NEM-alkylated C
232 HHCC
domain did not alter the activity of
untreated WT (Fig.
3A, NEM,
lane 5) or C209A (Fig.
3A, NEM, lane 9),
indicating that the loss
of complementation functions was not due to
incomplete quenching
of unreacted NEM. Importantly, NEM-treated C
232
was unable to
complement untreated N
105 (Fig.
3B, NEM, lane 6). The
untreated
C
232 protein did function as a multimer with N
105, as
indicated
by the restoration of double disintegration and foldback
products
to WT levels in the control reactions (Fig.
3B, MOCK, lane 4,
compared to N
105, lane 3 and WT IN, lane 1).
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FIG. 3.
Activity of NEM-alkylated M-MuLV IN proteins in
coordinated bimolecular disintegration reactions. Reaction products of
coordinated disintegration are indicated at the left of each panel. The
input crossbone oligonucletide substrate consists of tailed
half-crossbones 4166-4167 (13). M-MuLV IN proteins that were
subjected to NEM alkylation are indicated by bold underlined
characters. Non-underlined proteins are untreated IN. (A) Tailed
coordinated disintegration reactions mediated by WT and C209A M-MuLV
IN. (MOCK) Lanes 1 to 6, odd and even numbered lanes are reactions that
respectively contained 10 and 20 pmol of the mock-treated proteins;
lanes 7 and 8, complementation reactions which contained 10 pmol each
of the indicated mock-treated proteins. (NEM) Assays were performed
with 10 pmol of each of the indicated proteins. Lane 1, NEM-alkylated
C232 negative-control reaction; lanes 2 to 9, reactions that
contained individual or mixed pairs of treated and untreated proteins.
(B) Tailed coordinated disintegration reactions contrasting WT and
N105 M-MuLV IN. The activity of complementation mixtures containing
10 pmol of each indicated protein pair (MOCK, lane 4, and NEM, lanes 4 to 6) is compared to the activity of reaction mixtures containing 10 pmol of the individual proteins (lanes 1 to 3).
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Thus, when either residue C209 or the HHCC domain was alkylated by NEM
treatment, functional complementation could not be
achieved. These data
collectively suggest that residue C209 of
M-MuLV IN, in conjunction
with the HHCC domain, participates in
the multimeric assembly of
bimolecular IN-DNA complexes and coordinated
IN-IN interactions.
The HHCC domain of M-MuLV IN is required for productive
interactions with an untailed unimolecular disintegration
substrate.
The function of the HHCC in retroviral integration
remains obscure. Its proposed role in IN-IN interactions, LTR
positioning, or in the maintenance of stable IN-LTR DNA complexes
(13, 16, 17, 24, 25) has been inferred from biochemical
analysis of mutant proteins through multiple assay systems. The
importance of the HHCC domain in the absence of the LTR 5'-ss tail has
been documented for coordinated disintegration reactions
(13). The requirements for bimolecular substrate assembly in
coordinated disintegration, however, is distinct from that of a
unimolecular disintegration reaction. In the context of unimolecular
disintegration, the HHCC domain is not critical when an LTR 5'-ss tail
is present on the Y substrate (24). To further define the
function of the HHCC domain, conditions were identified which strictly
require the HHCC for unimolecular disintegration (Fig.
4A).
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FIG. 4.
Influence of the HHCC domain, residue C209, and the LTR
5'-ss tail on N105. The structures of the 5'-32P-labeled
untailed dumbbell substrate (7440, 5'-TGAAAGCGTAAGCTTTCAACCTGCGTAAGCAGGTAGACCGCAAGGTCT-3') and
the reaction product of unimolecular disintegration are indicated on
the left of each panel. (A) Requirement of the HHCC domain in
trans to N105 for unimolecular disintegration of an
untailed dumbbell disintegration substrate. Lane 1 contains a control
buffer reaction. The molar quantities of the proteins and their ratios
in each reaction are respectively indicated above and below each lane.
(B) Effect of NEM alkylation of residue C209 or the HHCC domain on
productive interactions with an untailed dumbbell substrate. Individual
M-MuLV IN proteins and complementation protein pairs are indicated as
in Fig. 2 and 3. NEM-treated samples are indicated in bold and are
underlined.
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In the absence of both the HHCC domain and the LTR 5'-ss tail, no
disintegration activity of the N
105 protein was detected
by using an
untailed dumbbell substrate (Fig.
4A, lane 2). The
critical requirement
of the HHCC domain for productive interactions
with the untailed
disintegration substrate was demonstrated by
functional complementation
with the C
232 protein (Fig.
4A, lanes
3 to 7), with maximal
disintegration occurring at a 1:1 molar
ratio of C
232 to N
105
protein. This suggests that in the absence
of the 5'-ss tail,
productive unimolecular IN-DNA interactions
are dependent on a
functional IN multimer containing an HHCC domain
in stoichiometric
levels with the catalytic core and C terminus
of M-MuLV IN. In this
simplified assay, the HHCC domain is required
directly for LTR
stabilization and not bimolecular substrate assembly.
This necessity of the HHCC domain for productive IN-DNA interactions in
the absence of the LTR 5'-ss tail was further investigated
by NEM
alkylation of the N
105 and C
232 complementation pairs
(Fig.
4B).
In comparison to the complementation of N
105 by C
232
(Fig.
4B,
lane 2), NEM treatment of the C
232 protein resulted
in complete loss
of functional complementation (Fig.
4B, lane
3). When the C209 site of
N
105 was alkylated, complementation
by untreated C
232 was also
inhibited (Fig.
4B, lane 4). These
data indicate that both the HHCC
domain of C
232 and residue C209
of N
105 represent NEM-sensitive
sites when present as independent
domains. In the context of WT M-MuLV
IN (Fig.
4B, lane 5), NEM
alkylation also caused a complete loss of
function with the untailed
dumbbell substrate (Fig.
4B, lane 6).
Residue C209 of M-MuLV IN influences the retrovirus life cycle in
vivo.
The results of NEM alkylation on multimeric M-MuLV IN
function in vitro prompted an investigation of its importance to viral replication in vivo. M-MuLV proviral DNA clones (pNCA-C [11, 20]) that encoded either WT or C209A IN proteins were
transiently transfected into Rat-1 cells by the DEAE-dextran method
(36). Rat-1 cells were maintained in 5% CO2 at
37°C in Dulbecco's modified Eagle's medium supplemented with 10%
bovine calf serum (HyClone), 2 mM glutamine, 50 U of penicillin/ml, 50 µg of streptomycin/ml, and 100 µg of gentamycin sulfate/ml. Viral
propagation was measured over time by assaying supernatant medium for
reverse transcriptase (RT) activity (21). In comparison to
WT M-MuLV, C209A IN virion production was delayed 2 to 3 weeks (Fig.
5A, WT and C209A). RT activity levels of
the C209A IN producer cells remained low throughout the time course.
This implied that the C209A mutant virus was infecting cells extremely
poorly and was hindered by dilution of the low level of infected cells
during passage. To test this possibility, plates from passage on day 21 were maintained through day 40 with changes of the medium every 1.5 weeks and assayed for viral spread. Under these conditions, the C209A
IN-infected cells showed increasing RT activity levels on days 29 and
40 (Fig. 5B). No virus was detected in the control cells (Fig. 5A and
B, MOCK), and the WT maintained the high level of viral release under these conditions.
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FIG. 5.
In vivo analysis of the effects of the C209A IN mutation
on viral replication. (A) Time course of viral RT release into medium
by Rat-1 producer cells. Days posttransfection are indicated on the
left. Passages of the transfected producer cell line are indicated by
arrows. (B) Accumulation of C209A viral RT in medium of plates
maintained from passage on day 21 through day 40. (C) Time course of
viral spread in Rat-1 cells. WT and C209A (pNCA-c/C209A IN) viruses
collected at 40 days posttransfection were normalized for RT activity
and used to infect fresh Rat-1 cells. RT activity was quantitated on a
Molecular Dynamics PhosphorImager (Sunnyvale, Calif.). The pixel
densities for each time point during the infection represent relative
RT activity and are indicated on the y axis. The drop in RT
activity at day 4 was due to passage of the infected cell lines.
d.p.i., days postinfection.
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The delayed spread of the viral C209A IN mutant suggested a defect in
replication kinetics affected by the C209A IN mutation.
Supernatants
from the WT and C209A IN producer cells at 40 days
postinfection were
normalized with respect to RT activity levels
and used to infect fresh
Rat-1 cells to address this possibility.
The kinetics of C209A IN viral
spread showed a 1.5- to 2-week
delay compared to WT M-MuLV (Fig.
5C).
To confirm that the phenotype
of the C209A IN virus was not due to
reversion or second-site
mutations, low-molecular-weight viral DNA was
isolated from infected
cells (
22), and the full IN coding
region was amplified by PCR
(data not shown). Sequencing of the 1.2-kb
IN coding region confirmed
that the C209A mutation was retained in the
producer cell virions
at 40 days postinfection (data not shown). Though
a single nucleotide
change was found, this was in a codon wobble
position which did
not affect the amino acid sequence of the IN protein
(ACT
ACG,
Thr
112).
Immunoprecipitation of C209A M-MuLV IN viral particles produced in the
RT time course (Fig.
5B) indicated that normal levels
of proteins
(i.e., CA, MA, TM, SU, RT, and IN) are incorporated
into the C209A
M-MuLV IN virions (data not shown). Similarly,
Southern blot analysis
of unintegrated DNA indicated the levels
of linear viral DNA from C209A
were similar to those of the WT
virus at days 2 and 4 (data not shown).
PCR amplification of C209A
and WT M-MuLV regions of
pol
occurred with equivalent levels of
input Hirt DNA. These results
indicate that the replication defect
probably does not reflect aberrant
levels of viral protein synthesis,
protein incorporation into mature
viral particles, or subsequent
viral DNA synthesis (
32,
40).
Taken together with the in vitro
data, the replication-defective
phenotype of the C209A IN virus
suggests that residue C209 may affect
in vivo multimeric IN functions
that are important for coordinated
integration (
37).
Model for assembly of IN multimers.
The in vitro integration
and unimolecular and bimolecular disintegration reactions mediated by
M-MuLV IN have been shown to be subtly influenced by substrate
determinants and domains of the IN protein (13, 14, 24, 25).
Thus, the assembly of different productive IN-DNA complexes may be
achieved through complicated and distinct multimeric IN-IN and IN-DNA
interactions. The combined approach of complementation and chemical
modification of M-MuLV IN mutants in each of the in vitro reactions was
used to investigate these different states of IN function.
A model for the assembly of functional multimers for each of the
integration and disintegration substrates is presented in
Fig.
6. Two symmetric dimers of M-MuLV IN bind
independent LTRs
in a fashion that is analogous to how one holds a pen:
the thumb,
index finger, and middle finger each contribute to the
mechanism
and stability of the grasp. In the working model, each of
these
stabilizing factors are represented by the HHCC domain of IN,
the
CA dinucleotide of the LTR, and the LTR 5'-ss tail. Elimination
of one
or more of these factors would cripple the stability of
LTR binding to
various degrees. This is exemplified in Fig.
4,
where unimolecular
disintegration of a 5'-untailed substrate is
dependent on the HHCC
domain.
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FIG. 6.
Model of multimeric M-MuLV IN functions in vitro and in
vivo. An illustration of M-MuLV IN domains and multimeric subunit
interactions is shown. A dimeric interface is formed by parallel
association of M-MuLV IN monomers, reflecting the structure studies of
the catalytic core and C terminus of human immunodeficiency virus type
1 and avian sarcoma virus IN (5, 15, 33). DNA is depicted as
striped bars. The N terminus of M-MuLV IN through the HHCC region is
shaded. (Top) LTR coordination and multimeric assembly of subunits for
integration. (Bottom) Substrate coordination and subunit interactions
for unimolecular disintegration (left) and bimolecular coordinated
disintegration (right).
|
|
We have previously demonstrated that under nonsaturating conditions,
coordinated disintegration mediated by the HHCC-deleted
N
105 protein
was strongly dependent on
trans-subunit interactions
promoted by an independent HHCC domain (
13). The model
presented
here proposes that coordinated IN activity is arbitrated by
an
interaction of the HHCC domains at the multimerization interface,
in
the vicinity of the C209. Two LTR strand transfer and coordinated
disintegration would be dependent on this assembly, whereas the
unimolecular disintegration substrate is essentially
"preassembled,"
with the attacking nucleophile position maintained
by the substrate
structure, and may be supported by a minimal
protomeric subunit.
The sensitivity of the C209 and the HHCC domain to NEM modification can
be visualized in this model. Both the C209 and the
HHCC region
participate in the productive assembly of multimeric
IN-IN complexes.
For unimolecular disintegration, the structural
arrangement of the
attacking target 3'-OH and scissile bond diminishes
any steric
constraints that could be imposed by NEM alkylation.
These would only
be visible under conditions of suboptimal LTR
recognition, such as the
absence of the 5'-ss tail. C209 is not
essential for catalysis; the
C209A full-length protein as well
as the C209A-N
105 truncated
protein can catalyze single LTR strand
transfer (
24). The
addition of the large maleimide group onto
the C209 blocks IN-DNA
and/or IN-IN interactions required for
the strand transfer and
coordinated disintegration reactions.
While NEM treatment of the core C209 site greatly reduced catalytic
activities of the M-MuLV IN proteins, variation was detected
in the
sensitivity of the HHCC domain to NEM. The N-terminal HHCC
domain may
represent multiple functional domains. C
232 consists
of the
N-terminal 176 amino acids of M-MuLV IN, of which the HHCC
structure
per se only accounts for 41 amino acids. For M-MuLV,
the N terminus of
IN contains an additional 56 amino acids which
are not conserved among
related retroviral IN proteins. For HIV-1,
the nuclear magnetic
resonance solution structure (
7) identified
the dimer
interface within the HHCC domain. For M-MuLV, an HHCC
construct smaller
than C
232 is a dimer as well (
51). The IN
constructs
which are catalytically active were not renatured in
the presence of
exogenously added zinc. Preliminary data (
32a)
indicate an
inverse relationship between NEM sensitivity and zinc
coordination. The
absence of zinc may also explain the inability
of the HHCC domain to
complement LTR hydrolysis in the coordinated
disintegration reactions.
It is possible that NEM may inhibit
one function of the HHCC, such as
LTR positioning, while maintaining
a second function, such as
dimerization. Additional studies to
separate these functions are under
way.
| |
ACKNOWLEDGMENTS |
This work was supported by American Cancer Society grant
RPG-95-056-03-VM and NSF-International Program
NSF-INT-9408501/Fundacion ANDES (travel grant). G.A.D. was supported by
NIH training grant 5T32AI07043.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Ln., Piscataway, NJ 08854. Phone:
(732) 235-5048. Fax: (732) 235-4783. E-mail:
Roth{at}mbcl.rutgers.edu.
Present address: Aaron Diamond AIDS Research Center, New York, NY
10019.
| |
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J Virol, February 1998, p. 1691-1698, Vol. 72, No. 2
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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