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Journal of Virology, August 2001, p. 7193-7197, Vol. 75, No. 15
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.15.7193-7197.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Context-Dependent Phenotype of a Human
Immunodeficiency Virus Type 1 Nucleocapsid Mutation
Andrea
Cimarelli
and
Jeremy
Luban*
Departments of Microbiology and Medicine,
College of Physicians and Surgeons, Columbia Univeirsity, New York,
New York, 10032
Received 31 October 2000/Accepted 3 May 2001
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ABSTRACT |
The human immunodeficiency virus type 1 (HIV-1) nucleocapsid
mutation R10A/K11A abolishes viral replication when present in proviral
clone HIV-1HXB-2, but it was found to have minimal effect on replication of the closely related HIV-1NL4-3.
Functional mapping demonstrated that a nonconservative amino acid
change at nucleocapsid residue 24 (threonine in HIV-1HXB-2,
isoleucine in HIV-1NL4-3) is the major determinant of the
different R10A/K11A phenotypes in these two proviruses.
Threonine-isoleucine exchanges appear to modify the R10A/K11A phenotype
via effects on virion RNA-packaging efficiency. The improved packaging
seen with hydrophobic isoleucine is consistent with solution structures
localizing this residue to a hydrophobic pocket that contacts guanosine
bases in viral genomic RNA stem-loops critical for packaging.
| |
TEXT |
Retroviral nucleocapsid (NC) carries
out important functions throughout the entire retroviral life cycle
(2, 19). Upon translation as part of the Gag
polyprotein, NC mediates Gag multimerization and virion
assembly and directs the packaging of two copies of viral genomic RNA
into virions. After Gag polyprotein processing by the
virus-encoded protease, NC coats viral genomic RNA and subsequently
influences early events in the viral life cycle such as reverse
transcription and possibly even integration (3, 4, 8, 12).
Each of these functions seems to require RNA binding on the part of NC.
Genetic and structural studies indicate that conserved Cys-His boxes of
NC mediate specific viral genomic RNA packaging by pairing with
cis-acting stem-loops on the RNA (1, 9, 14,
18). The specificity of RNA binding seems less important for
other NC functions, such as virion assembly and reverse transcription;
here, NC basic residues mediate nonspecific binding of NC to RNA via
electrostatic interactions with the phosphodiester groups of the RNA
(1, 7, 9).
Context-dependent replication of the R10A/K11A mutation.
To
elucidate the function of human immunodeficiency virus type 1 (HIV-1)
NC basic residues, we and others previously characterized a panel of
alanine-scanning mutations (7, 17). Among these mutations,
R10A/K11A was introduced into the HIV-1HXB-2 provirus and
found to disrupt viral replication. This mutation substitutes alanine
at positions that are invariably basic among different HIV-1 isolates
(15). We therefore expected to observe similar negative
effects on viral replication when we introduced this mutation into
other proviral clones.
Using standard techniques (20), R10A/K11A was introduced
into HIV-1NL4-3, a proviral clone closely related to
HIV-1HXB-2. Replication studies were performed as
previously described (7): virions produced by transfection
of proviral DNAs into 293T cells were normalized by exogenous reverse
transcriptase (RT) activity and used to initiate infection of Jurkat T
cells (22). Every 2 days cells were passaged and
supernatant was collected. Evidence for virus spread through the
culture was obtained by measuring exogenous RT activity in the culture
supernatant. Levels of wild-type HIV-1NL4-3 peaked about 8 days postinfection (Fig. 1). Levels of
wild-type HIV-1HXB-2 peaked slightly later, an observation consistent with the fact that this clone has defects in three accessory
genes: vpu, nef and vpr (10, 11). As
previously reported, replication of the HIV-1HXB-2
R10A/K11A mutant was abolished (17) and no exogenous RT
activity above the background could be detected (Fig. 1). Much to our
surprise, when the R10A/K11A mutation was introduced into proviral
clone HIV-1NL4-3, the virus was able to replicate robustly
(Fig. 1), albeit with slower kinetics than wild-type
HIV-1NL4-3.
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FIG. 1.
Replication of wild-type (WT) and R10A/K11A mutant
viruses in the HIV-1NL4-3 and HIV-1HXB-2
proviral backgrounds. Jurkat T cells were infected with virus stocks
(as indicated) produced by transient transfection and normalized by
exogenous RT activity. After infection, cells were passaged every 2 days and supernatant was collected. The accumulation of exogenous RT
activity in the supernatant of infected cells (ordinate) is shown for
the indicated day postinfection (abscissa).
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Mapping of the difference between HIV-1NL4-3 and
HIV-1HXB-2 that determines the phenotype of the R10A/K11A
mutation.
The striking difference in replication between
HIV-1NL4-3 and HIV-1HXB-2 clones harboring the
R10A/K11A mutation could not be explained by differences in accessory
genes or other sequences 3' of pol. First, exchanging these
sequences between HIV-1NL4-3 and HIV-1HXB-2 did
not modify the R10A/K11A phenotype (data not shown). Second, we have
previously shown that transfer into HIV-1NL4-3 of a
SpeI-EcoRV fragment from HIV-1HXB-2
(nucleotides 1507 to 2977 from the middle of CA to the middle of RT) is
sufficient to render R10A/K11A unable to replicate (8).
This indicates that the determinant of the R10A/K11A phenotype lies
within the SpeI-EcoRV fragment (Fig.
2a).
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FIG. 2.
Effect of the R10A/K11A mutation on replication of
HIV-1NL4-3/HXB-2 chimeric viruses, indicated here as
NL4-3/HX. (a) Schematic representation of gag and
pol in the virus chimeras used here. Sequences from
HIV-1NL4-3 or HIV-1HXB-1 are represented by
black and white bars, respectively. The proviral nucleotide numbers
flanking the regions exchanged in the chimeras are indicated on the
left. The major domains of the proteins encoded by gag and
pol are indicated as follows: MA, matrix; CA, capsid; NC,
nucleocapsid; PR, protease; RT, reverse transcriptase; IN, integrase.
(b) Replication of wild-type (WT) HIV-1NL4-3 and various
chimeric viruses, as indicated, in Jurkat T cells. The accumulation of
exogenous RT activity in the supernatant of infected cells (ordinate)
is shown for the indicated day postinfection (abscissa).
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To restrict the determinant of the R10A/K11A phenotype further, a
second chimeric virus was engineered in which a
SpeI-
ApaI
fragment from
HIV-1
HXB-2 (nucleotides 1507 to 2006) was substituted
for corresponding sequences in HIV-1
NL4-3 (Fig.
2a).
Virions were
used to infect Jurkat T cells, and infections were
analyzed as
above. Replication of either "wild-type" chimeric
virus, NL4-3/HX
(1507-2977) or
NL4-3/HX
(1507-2006), was similar to that of
wild-type
HIV-1
NL4-3 (Fig.
2b), as previously described
(
8). In contrast,
when the R10A/K11A mutation was present
in either of these two
chimeric viruses, replication was severely
impaired, as it is
when the mutation is present in
HIV-1
HXB-2 (Fig.
2b). These results
indicate that the
determinant for the R10A/K11A replication phenotype
is within sequences
encoding the C terminus of CA through the
first zinc finger of NC. When
HIV-1
NL4-3 and HIV-1
HXB-2 sequences
from
this region were compared, eight amino acid differences were
found: two
in CA, one in SPI, and five in NC. Three of the latter
are conservative
changes, while two are nonconservative changes
(Fig.
3a).
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FIG. 3.
Effect of NC residues that are not conserved between
HIV-1NL4-3 and HIV-1HXB-2 on the phenotype of
the R10A/K11A mutation. (a) Amino acid sequence alignment showing NC
residues that are not conserved between HIV-1NL4-3 and
HIV-1HXB-2. Dashes indicate residues identical to
HIV-1NL4-3. Amino acid differences are indicated by
lowercase letters (b to d). Replication kinetics following infection of
Jurkat T cells with an HIV-1NL4-3/HXB-2 chimera (b),
complete HIV-1HXB-2 provirus (c), or complete
HIV-1NL4-3 provirus (d) bearing the indicated mutations.
The accumulation of exogenous RT activity in the supernatant of
infected cells (ordinate) is shown for the indicated day postinfection
(abscissa). WT, wild type.
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To determine if any of these nonconserved amino acids is sufficient to
determine the phenotype of the R10A/K11A mutation,
we changed
HIV-1
HXB-2 residues to their HIV-1
NL4-3
counterparts
and asked if these would rescue replication of the
HIV-1
NL4-3/HXB-2 R10A/K11A chimera. We focused our
attention on the two nonconservative
amino acid changes present at
positions 12 and 24 of NC (Fig.
3a). Individual substitutions in NC
were engineered by PCR as
previously described (
6), and
the amplified products, digested
with the restriction enzymes
SpeI and
ApaI, were used to replace
the
corresponding fragment of the specific proviral clone. Jurkat
T cells
were then infected with mutant viruses, and viral replication
was
examined (Fig.
3b). Changing the isoleucine residue encoded
by
HIV-1
HXB-2 at NC position 12 to the threonine encoded by
HIV-1
NL4-3 failed to rescue replication of
NL4-3/HX
(1507-2977) R10A/K11A
(Fig.
3b). In contrast,
NL4-3/HX
(1507-2977) R10A/K11A/T24I
replicated quite well
(Fig.
3b), suggesting that NC residue 24
could be the major determinant
of the R10A/K11A
phenotype.
The experiments described above tested the importance of NC residue 24 in the context of HIV-1
NL4-3/HXB-2 chimeric viruses.
To
formally demonstrate that residue 24 is responsible for the
context-dependent phenotype of the R10A/K11A mutation, residue
24 substitutions were introduced into nonchimeric HIV-1 proviruses.
Change
of the T24 residue to an isoleucine was sufficient to rescue
replication of the HIV-1
HXB-2 R10A/K11A mutant to a level
similar
to that of wild-type HIV-1
HXB-2 (Fig.
3c). In
addition, the reciprocal
change of I24 to threonine impaired the
replication of HIV-1
NL4-3 R10A/K11A (Fig.
3d). None of the
solo changes at position 24 in
the absence of other mutations affected
viral replication (Fig.
3c and d). These results formally demonstrate
that the difference
between NC residue 24 in HIV-1
HXB-2 and
HIV-1
NL4-3 is the primary
determinant of the R10A/K11A
phenotype. However, since exogenous
RT activity clearly distinguishable
from the background accumulated
in cultures infected with
HIV-1
NL4-3 R10A/K11A/I24T, these data
show that other
primary sequence differences between the two viral
clones must
contribute to the replication of the HIV-1
NL4-3 R10A/K11A
mutant.
Characterization of mutant virions.
The effect on the
phenotype of retroviruses harboring the R10A/K11A mutation of changing
the identity of NC residue 24 between isoleucine and threonine was
examined next in a single-cycle replication assay. Virions produced by
transfection of 293T cells were purified by ultracentrifugation through
25% sucrose, normalized by exogenous RT activity, and then used to
infect CD4+ HeLa cells containing a 
-galactosidase
reporter (13). The infectivity of the virion preparations
was then quantitated as previously described (8).
Consistent with the delayed kinetics shown in Fig. 1, wild-type
HIV-1HXB-2 virions were found to be two- to three-fold less
infectious than wild-type HIV-1NL4-3 virions (data not
shown); again, the decreased infectivity of HIV-1HXB-2 might be explained by the nonfunctional Vpu, Vpr, and Nef in this virus
(10, 11). For clarity, then, the infectivity of each mutant shown in Fig. 4a is presented as a
percentage of the infectivity of the respective wild-type provirus.
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FIG. 4.
Characterization of HIV-1HXB-2 and
HIV-1NL4-3 virions bearing NC mutations. 293T cells were
transfected with the indicated proviral DNAs. Virions were purified
from culture supernatant by ultracentrifugation through 25% sucrose
and then normalized by exogenous RT. (a) Infectivity of the virion
preparations was determined by counting -galactosidace-positive
(-gal+) cells 2 days after infection of CD4+
HeLa reporter cells (MAGI assay). Data for each mutant are presented as
the percentage with respect to the respective wild-type (WT) virus. The
bar graph shows results obtained from six independent experiments with
standard errors of the mean. NL4-3 env- indicates a virus lacking a
functional env. (b) Western blot probed with anti-CA
antibody. 293T cell lysates are shown in the upper panels.
Virion-associated proteins are shown in the lower panels. The positions
of migration of the various gag products recognized by this
antibody are indicated. (c) Virion-associated RNA prepared from
normalized amounts of virions was loaded onto a nylon membrane and
probed with a 32P-end-labeled DNA oligonucleotide specific
for viral genomic RNA. The signal obtained after hybridization was
quantified with a phosphorimager. A representative standard curve
obtained after dilution of genomic RNA is shown here and illustrates
the linearity of the method used. Results are presented as percentage
with respect to the wild-type viruses. The bar graph presents results
obtained from two to four independent experiments with standard errors
of the mean. myr indicates a control preparation from
cells transfected with HIV-1NL4-3 bearing the Gag G1A
mutation that disrupts Gag myristylation. This preparation does not
contain virions and is used here to monitor for DNA contaminants
derived from transfection.
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Virions containing the R10A/K11A mutation in the context of
HIV-1
HXB-2 were about 30-fold less infectious than
wild-type HIV-1
HXB-2 (Fig.
4a) or 90- to 100-fold less
infectious than wild-type HIV-1
NL4-3.
Changing T24 to the
isoleucine found at this position in HIV-1
NL4-3 increased
the infectivity of HIV-1
HXB-2 R10A/K11A virions more
than 10-fold. In the context of HIV-1
NL4-3, the R10A/K11A
mutation
caused only a two- to threefold reduction in the number of
-galactosidase-positive
cells. Changing I24 to the threonine found
at this position in
HIV-1
HXB-2 decreased the infectivity of
HIV-1
NL4-3 R10A/K11A further
(five- to six-fold lower
infectivity than for wild-type HIV-1
NL4-3).
The effect of NC residue 24 on the biochemistry of virions harboring
the R10A/K11A mutation was examined next. HIV-1
HXB-2 and
HIV-1
NL4-3 proviral DNAs were transfected into 293T cells.
Cell lysates were analyzed by Western blotting, as previously
described
(
5), using an anti-CA antibody (Intracel, Cambridge,
Mass.) that also recognizes p55 (the Gag polyprotein), p41 (a
processing intermediate containing MA, CA, and the SP1 spacer
peptide),
and p25 (an intermediate containing CA and SP1). Although
differences
were observed between HIV-1
HXB-2 and HIV-1
NL4-3
in
terms of the accumulation of Gag-processing intermediates (Fig.
4b),
the accumulation of these products did not correlate with
the presence
of the R10A/K11A mutation or with the identity of
NC residue 24. Western blot analysis of virions purified by ultracentrifugation
through 25% sucrose did not reveal appreciable differences in
the
virion yield or in the degree of processing of Gag products
at steady
state (Fig.
4b). Similar results were obtained when
Western blot
analysis was carried out using anti-RT or anti-NC
antibodies or after
pulse-chase analysis followed by immunoprecipitation
with serum from an
HIV-1-infected individual (data not shown).
These results suggest that
the R10A/K11A mutation has no obvious
effects on virion yield or on the
stability and processing of
viral
proteins.
Finally, the amount of viral genomic RNA packaged into mutant virions
was quantified using previously described methods (
7).
Virions produced as above were purified by ultracentrifugation
through
25% sucrose, normalized by exogenous RT activity, and
then transferred
to a nylon membrane by using a dot-blot apparatus
(Bio-Rad). RNA was
detected by hybridization with a
32P-end-labeled DNA
oligonucleotide (5'-CGCGCCTTGGTTCTCTCATCTGGCCTGG-3',
antisense orientation, nucleotides 1459 to 1482) that hybridizes
with a conserved portion of HIV-1 genomic RNA. Wild-type
HIV-1
HXB-2 virions were two- to threefold less efficient
than wild-type HIV-1
NL4-3 at incorporating viral
genomic RNA. The difference in packaging
efficiency between these two
clones is unlikely to be due to differential
annealing of the probe
since the target RNA sequence is identical
in the two
clones.
Compared to wild-type HIV-1
HXB-2, the R10A/K11A mutation
caused a fivefold decrease in viral genomic RNA packaging (Fig.
4c).
In
contrast, compared with wild-type HIV-1
NL4-3, no major
effect
on viral genomic RNA packaging was observed with the
HIV-1
NL4-3 R10A/K11A mutant. The decrease in RNA
packaging of HIV-1
HXB-2 R10A/K11A was corrected twofold by
changing the threonine at residue
24 to isoleucine. RNA incorporation
into HIV-1
NL4-3 R10A/K11A
virions was decreased
approximately twofold when the isoleucine
at residue 24 was changed to
threonine. Introduction of changes
at position 24 in the absence of
other mutations did not affect
viral genomic RNA incorporation. Thus,
variations in RNA packaging
correlated with, and possibly explain, the
replication behavior
of the different proviruses bearing the R10A/K11A
mutation.
In conclusion, our results indicate that the context-dependent
replication phenotype of the R10A/K11A mutation depends mainly
on the
identity of the amino acid present at position 24 of NC.
Our data
suggest that effects of this residue on viral replication
are exerted
at the level of viral genomic RNA packaging. NC position
24 is a
hydrophobic residue in almost all HIV-1 isolates (
15);
the
hydrophilic threonine in HIV-1
HXB-2 is a rare
exception.
Solution structures have been determined for two
cis-acting,
HIV-1 packaging-signal stem-loops (SL3 and SL2) bound to
HIV-1
NL4-3 NC (
1,
9). The isoleucine at
HIV-1
NL4-3 NC position 24 is
part of a hydrophobic cleft
that contacts the guanosine bases
at position 9 of SL3 or
position 11 of SL2. Based on these structural
data and the observed
effects on RNA packaging and viral replication
of the mutations
described here, it is reasonable to propose that,
compared to
HIV-1
HXB-2 NC threonine 24, HIV-1
NL4-3 NC
isoleucine
24 confers tighter binding of the NC zinc finger domain to
guanosine
bases. Substitution of isoleucine by threonine would lead to
weaker
binding, reductions in packaging efficiency, and decreased
infectivity.
The NC basic residues R10 and K11 have electrostatic
interactions
with SL3 (
9). If RNA binding by
HIV-1
HXB-2 NC were inherently
weaker due to threonine 24, disruption of residues R10 and K11
by mutation would result in a
noticeable replication phenotype
only in the context of this
provirus.
Amazingly similar to the findings presented here, a
threonine-to-isoleucine change at position 24 of NC was previously
reported
as a second-site suppressor mutation that contributed to the
rescue
of replication and RNA packaging in an HIV-1
HXB-2
derivative bearing
a deletion in the RNA dimerization initiation site
(
16,
21).
Residue 24 is the major determinant of the
different R10A/K11A
phenotypes reported here, but RT activity above the
background
accumulated in cultures infected by
HIV-1
NL4-3 R10A/K11A/I24T,
indicating that residue 24 differences are not sufficient to explain
the different phenotypes with
the two proviruses. Less significant
contributions might be made by
other differences in coding sequences
or perhaps in 5' leader
sequences.
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ACKNOWLEDGMENTS |
We thank Cagan Gurer and Michael Summers for critical reading of
the manuscript.
This work was supported by grant AI 41857 (J.L.) and by shared core
facilities of the Columbia-Rockefeller Center for AIDS Research (P30
AI42848), both from the National Institutes of Health.
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FOOTNOTES |
*
Corresponding author. Mailing address: Departments of
Microbiology and Medicine, Columbia University, College of Physicians and Surgeons, 701 W. 168th St., New York, NY 10032. Phone: (212) 305-8706. Fax: (212) 305-0333. E-mail: JL45{at}columbia.edu.
Present address: Ecole Normale Supérieure de Lyon, 69364 Lyon, France.
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Journal of Virology, August 2001, p. 7193-7197, Vol. 75, No. 15
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.15.7193-7197.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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