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Journal of Virology, October 1999, p. 8527-8540, Vol. 73, No. 10
Departments of
Microbiology1 and
Medicine,2 Columbia University, College
of Physicians and Surgeons, New York, New York 10032
Received 26 January 1999/Accepted 2 July 1999
The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein
directs the formation of virions from productively infected cells. Many
gag mutations disrupt virion assembly, but little is known
about the biochemical effects of many of these mutations. Protein-protein interactions among Gag monomers are believed to be
necessary for virion assembly, and data suggest that RNA may modify
protein-protein interactions or even serve as a bridge linking Gag
polyprotein monomers. To evaluate the primary sequence requirements for
HIV-1 Gag homomeric interactions, a panel of HIV-1 Gag deletion mutants
was expressed in bacteria and evaluated for the ability to associate
with full-length Gag in vitro. The nucleocapsid protein, the major
RNA-binding domain of Gag, exhibited activity comparable to that of the
complete polyprotein. In the absence of the nucleocapsid protein,
relatively weak activity was observed that was dependent upon both the
capsid-dimer interface and basic residues within the matrix domain. The
relevance of the in vitro findings was confirmed with an assay in which
nonmyristylated mutant Gags were assessed for the ability to be
incorporated into virions produced by wild-type Gag expressed in
trans. Evidence of the importance of RNA for Gag-Gag
interaction was provided by the demonstration that RNase impairs the
Gag-Gag interaction and that HIV-1 Gag interacts efficiently with Gags
encoded by distantly related retroviruses and with structurally
unrelated RNA-binding proteins. These results are consistent with
models in which Gag multimerization involves indirect contacts via an RNA bridge as well as direct protein-protein interactions.
The major gag product of
human immunodeficiency virus type 1 (HIV-1) and related retroviruses is
a cytoplasmic polyprotein necessary and sufficient for the assembly,
budding, and release of virions It is generally believed that HIV-1 virion assembly requires
noncovalent interactions among Gag polyprotein monomers. Retroviral Gag
polyprotein monomers form homomultimers (7, 12, 32, 35, 37, 51,
55), but little is known about the stoichiometry of the resulting
complex or the forces that drive its formation. Purified HIV-1 CA forms
dimers (23), higher-order oligomers (18), and,
under certain conditions, lattices of hexamers and trimers
(2). Similar information is not available for the Gag polyprotein.
The CA dimer interface has been pinpointed (23), but the
identity of amino acid residues required for Gag polyprotein
multimerization is unknown. Many gag mutations disrupt
virion assembly (9, 16, 22, 28, 29, 48, 57-59), but it has
not been directly demonstrated that these mutations disrupt Gag
multimerization. The primary effect of these mutations could equally
well be the disruption of other processes, such as the interaction of
Gag with an unknown cellular factor required for targeting the plasma membrane or the release of virions with proper density (3). Therefore, it has not been conclusively demonstrated that virion assembly requires protein-protein interactions between Gag monomers. Some data even suggest that heterologous bridging molecules such as
nucleic acids or ubiquitous RNA-binding proteins may be required for
Gag-Gag interaction (7, 38, 64).
We previously reported attempts to map the primary sequence
requirements for HIV-1 Gag polyprotein multimerization using the two-hybrid system (21). A gag fragment retaining
coding sequences for the major homology region and extending through
the complete NC domain was sufficient for full activity in this assay
(21). Unfortunately, assessment of the activity of larger
deletion mutations was not possible due to technical limitations.
Therefore, definition of the minimal domain required for activity was
not possible with the two-hybrid system.
In the experiments presented here, a large panel of HIV-1 Gag deletion
mutants was evaluated in a more direct assay of HIV-1 Gag-Gag
interaction using recombinant protein in solution. NC, the major
RNA-binding domain of Gag, exhibited activity almost equivalent to that
of the complete polyprotein. In the absence of the NC domain,
relatively weak activity was observed that was dependent on residues
forming the CA-dimer interface and upon basic residues within the MA
domain. Additional experiments demonstrated that RNase disrupts the
Gag-Gag interaction and that Gag interacts with heterologous
RNA-binding proteins. Finally, an in vivo assay in which
nonmyristylated mutant Gags are tested for the ability to be
incorporated into wild-type virions expressed in trans was used to confirm the relevance of the in vitro findings. The results presented here are consistent with models in which Gag multimerization requires RNA (perhaps as a bridge between Gag monomers) as well as
direct protein-protein interactions.
Bacteria, yeast, and transformations.
All plasmid DNAs were
propagated in Escherichia coli DH5 Cloned DNAs and plasmids.
Nucleotide positions in
gag are relative to the 5' edge of the 5' long terminal
repeat (LTR) in the HIV-1 proviral clone HXB2C; gag-encoded
proteins were either expressed as GST fusion proteins or with the HA1
epitope (amino acid residues YPYDVPDYA) from the hemagglutinin (HA)
protein of influenza virus (47) appended to the amino terminus.
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Human Immunodeficiency Virus Type 1 Gag Polyprotein
Multimerization Requires the Nucleocapsid Domain and RNA and Is
Promoted by the Capsid-Dimer Interface and the Basic Region of
Matrix Protein
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
albeit noninfectious particlesfrom
expressing cells (for reviews, see references 12,
28, and 30). Concurrent with
virion assembly, the Gag polyprotein incorporates several viral
elements, including viral genomic RNA, the Env glycoprotein, and the
pol-encoded enzymes, into nascent virions. As virions are
released from the cell surface, the Gag polyprotein is cleaved by the
viral protease to form the matrix protein (MA), which lines the virion
envelope, the capsid protein (CA), which forms the outer shell of the
virion core, and the centrally located nucleocapsid protein (NC), which
coats the genomic RNA.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
. Glutathione
S-transferase (GST) fusion proteins and HIV-1 Gag protein derivatives were expressed in E. coli BL21(DE3)pLysS
(Novagen Inc., Madison, Wis.); this strain contains the 
DE3 lysogen
which expresses the T7 polymerase from the lacUV5 promoter,
as well as pLysS, a plasmid expressing low levels of T7 lysozyme
(45).
831, 5'1632, and 5'1712 were created by deleting
nucleotides at the 5' end of the gag coding sequence by
exploiting unique ClaI, BsmI, or
HindIII sites, respectively. Mutations 5'906 and
5'1509 were created similarly by using previously described XhoI sites that had been engineered at the indicated
positions (40). The 5'1184 mutation was subcloned from a
CA expression plasmid that was previously described (38).
Details of the isolation of deletion mutation 5'1320 in a genetic
screen were described previously (12). 5'1920, 5'1962,
and 5'2004 were generated by PCR using oligonucleotide
5'-GCAACGACCCCTCGTCACAATAAGAATTCGCGC-3' in combination,
respectively, with the following oligonucleotides: 5'-CGCGGATCCATGCAGAGAGGCAATTTTAGGAAC-3',
5'-CGCGGATCCTGTTTCAATTGTGGCAAAGAAGGG-3', or
5'-CGCGGATCCAGGGCCCCTAGGAAAAAGGGC-3'.
Mutations 3'2093, 3'2007, 3'1906, and 3'1787 were
previously described (21), and all were constructed by the
insertion of an Xba linker into restriction endonuclease
recognition sites at the indicated nucleotide positions. This linker
contains nonsense codons in all three reading frames (linker number
1062; New England Biolabs, Inc., Beverly, Mass.). Engineering of the
3'1681 mutation was described previously (12). The
termination codon of the CA coding sequence in 3'1878 was
constructed by PCR amplification of gag sequences using the
oligonucleotides 5'-CGCGCCATGGGTGCGAGAGCGTCAG-3' and
5'-GCGCGGATTCTTACAAAACTCTTGCCTTATGGCCGGG-3'. The
termination codon of the MA coding sequence in 3'1184 was
constructed by PCR of gag sequences using the
oligonucleotides 5'-CGCGCCATGGGTGCGAGAGCGTCAG-3' and
5'-CGCGAATTCTTAGTAATTTTGGCTGACCTG-3'. The 3'1715 and
3'1757 mutations were engineered by PCR using
5'-CCAGTGCATGCAGGGCCTATTGC-3' and either
5'-GCGCCTCGAGCTAAGCTTGCTCAGCTCTTAGAGTTTTATAG-3' or 5'-GCGCCTCGAGCTAGACCAACAAGGTTTCTGTCATCC-3', respectively.
The 3'1862 mutation was engineered by PCR using oligonucleotides 5'-GCGCGGATCCATAAGACAAGGACCAAAGGAGCCC-3' and
5'-GCCGCTCGAGTTAATGGCCGGGTCCTCCTACTCC-3'.
Double mutants 5'831-3'2093, 5'831-3'2007,
5'906-3'2093, 5'906-3'2007, 5'1320-3'2093, and
5'1320-3'2007 were constructed by combining single mutations by
standard cloning methods. To construct the HA-NC fusion protein, NC
coding sequences were amplified with the oligonucleotides
5'-CGCGGATCCATGCAGAGAGGCAATTTTAGGAAC-3' and
5'-CGCGTCGACTTAATTAGCCTGTCTCTCAGTACAATC-3'.
Mutant 5'850-884, previously called dB5 (63), was a
generous gift of Max Essex. This mutant deletes 11 amino acids
encompassing the basic region of MA. Mutants W184A and M185A
(23) were generous gifts from Uta VonSchwedler and Wesley
Sundquist; the numbering of these two mutants is with respect to the
amino terminus of CA as previously described (23).
Gag polyprotein coding sequences from the simian immunodeficiency virus
strain MAC239 (SIVMAC239) and the feline immunodeficiency virus strain Petaluma (FIVPETALUMA) were subcloned into a
modified pGEX vector (54) from previously described plasmids
(21) to generate GST-fusion protein expression vectors. A
plasmid for bacterial expression of GST-Rous sarcoma virus (RSV) Gag
(34) obtained from the pATV8 clone was a gift from Stephen
Goff. To engineer a GST-visna virus Gag fusion protein expression
plasmid, Gag coding sequences (GenBank accession no. L06906) were PCR amplified from a plasmid kindly provided by Janice Clements by using
oligonucleotides 5'-CGCCCATGGCGAAGCAAGGCTCAAAGG-3' and
5'-GCGAGATCTTTACAACATAGGGGGCGCGGACGG-3'. The product was
subcloned into a pGEX vector. The human foamy virus (HFV) Gag
polyprotein coding sequences (GenBank accession no. U21247) were PCR
amplified with oligonucleotides
5'-CGCCCCGGGGGATCCATGGCTTCAGGAAGTAATGTTGAAG-3' and
5'-GCGGAATTCTTACAATTTGTATACTGGCTTTGCC-3' using
pHSRV13 as template (obtained from Stephen Goff). The product
was subcloned into a pGEX vector. The HFV NC domain (62) was
subcloned by using an ApoI restriction fragment
encompassing nucleotides 1394 to 2436 (numbering with respect to the 5'
end of gag).
The HIV-1 Rev cDNA was subcloned into a modified pGEX vector
(54) from pT7Rev, a generous gift of Martin Andreansky and Eric Hunter. The cDNA encoding the human ribosomal L8 protein (GenBank
accession no. Z28407) was subcloned from a two-hybrid library vector
(38) to generate a GST-fusion protein expression plasmid.
The cDNA encoding the human autoantigen small nuclear ribonucleoprotein
Sm-D (GenBank accession no. J03798) was PCR amplified from a
two-hybrid library plasmid (38) by using the oligonucleotides 5'-GCGGGATCCATGAAGCTCGTGAGATTTTTGATG-3' and
5'-GCGGAATTCTTATCGCCTAGGACCCCCTCTTCC-3' to generate a
GST-fusion protein expression construct.
Constructs for HIV-1 gag expression in mammalian
cells.
To express the HIV-1 gag cDNA in mammalian cells
in the absence of viral regulatory proteins, we used gag
sequences provided by George Pavlakis (National Cancer Institute,
Frederick, Md.). These sequences generate wild-type protein but contain
multiple, conservative mutations that act to render the mRNA Rev
independent (53). To express the Rev-independent
gag such that the C terminus of Gag is fused with the
Myc-epitope tag (EQKLISEEDL), the cDNA was amplified by PCR by using
the oligonucleotides 5'-CGCGCCATGGGTGCGAGAGCGTCAG-3' and
5'-GCGCGAATTCGAACCGGTCTACATAGTCTC-3'. The product was
subcloned into pBluescript (Stratagene), and its identity was confirmed by dideoxy-sequencing. The product was then cloned into the unique NcoI and XhoI sites of pEF/myc/cyto (Invitrogen)
to generate pGag-myc. This permitted expression of the Rev-independent
gag as a C-terminal fusion with the Myc-epitope tag from the
EF-1 promoter. Similar Rev-independent expression plasmids were
generated with cDNAs for wild-type HA-Gag, HA-Gag-3'1878,
HA-Gag-W184A, HA-Gag-3'1878/W184A, and HA-NC, except that
these contained the normal gag stop codon and so were not
fused to the Myc-epitope.
In vitro binding experiments. Bacterial lysates containing recombinant proteins were prepared as described previously (38). All binding steps were performed in a 200-µl reaction volume with TK buffer (20 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 5 mM dithiothreitol, 0.5% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, and 5% glycerol). Twenty microliters of a 50% (vol/vol) slurry of glutathione-agarose beads (Sigma), prepared as described (38), was added to each reaction mixture, as described below.
Bacterial lysates containing equivalent amounts of GST-fusion protein (as normalized by staining polyacrylamide gels with Coomassie blue) were adsorbed to glutathione-agarose beads at 4°C for 30 min. Beads were pelleted by a 5-s spin in a microcentrifuge, and unbound protein was removed by washing with TK buffer. The washed beads containing the preloaded GST proteins were then resuspended in TK buffer containing the indicated HA-Gag fusion proteins and were incubated for 1 h at 4°C on a nutator (Becton-Dickinson, Parsippany, N.J.). Beads were again pelleted and then washed with TK buffer three times. Beads were resuspended in 35 µl of 2× sodium dodecyl sulfate (SDS) sample buffer (52), boiled for 5 min, and pelleted. Aliquots (8 µl) of supernatant were then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were either stained with Coomassie blue or processed for Western blot analysis.Western blot analysis. A murine monoclonal antibody (12CA5) (19) raised against the 9-amino-acid HA1 epitope from the influenza virus HA protein was purchased from Berkeley Antibody Company, Berkeley, Calif. A murine monoclonal antibody directed against the 10-amino-acid Myc-epitope tag (EQKLISEEDL) was purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, Calif. A rabbit anti-RNase A antibody was purchased from Cortex Biochem, San Leandro, Calif. Western blot analysis was performed as described (38).
RNase A treatment. To examine the effect of RNase A treatment on the in vitro binding reaction, a slightly modified protocol was developed. GST-fusion proteins were bound to glutathione-agarose beads as described above and then washed once with TEK buffer (20 mM Tris-HCl [pH 7.5], 100 mM KCl, 5 mM EDTA, 5 mM dithiothreitol, 0.5% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, and 5% glycerol). The beads were resuspended in TEK buffer containing 100 ng of RNase A per ml and 1 mg of bovine serum albumin per ml and then incubated for 1 h at 37°C. Simultaneously, bacterial lysate containing HA-Gag protein was diluted at a ratio of 1:100 TEK buffer containing 100 ng of RNase A and 1 mg of bovine serum albumin (1:100) per ml, and the mixture was incubated for 1 h at 37°C.
At the conclusion of the 1-h incubation at 37°C, the glutathione-agarose beads with bound GST-fusion protein were pelleted, and the buffer was removed. The beads were resuspended in the buffer containing RNase-treated HA-Gag protein and were incubated at 4°C for 1 h. At the conclusion of the 1-h binding reaction, the supernatant was removed and processed in two ways. Fifty microliters of the supernatant was mixed with 1 ml of 10% trichloroacetic acid, and the mixture was incubated at 4°C for 30 min and accelerated in a microcentrifuge for 30 min at 4°C to precipitate any proteins that had failed to bind to the beads. The pellet was boiled in SDS and processed for Western blotting. As a rough guide to the effectiveness of the RNase treatment, 100 µl of the supernatant was extracted with phenol-chloroform and extracted with chloroform, and then nucleic acids were precipitated with ethanol. The pellet was electrophoresed on a 2% agarose gel, and the nucleic acid was visualized with ethidium bromide. After removal of the supernatant, the glutathione-agarose beads were washed three times with TEK buffer. Beads were resuspended in 35 µl of 2× SDS sample buffer (52), boiled for 5 min, and pelleted. Aliquots (8 µl) of supernatant were then subjected to SDS-PAGE. Gels were either stained with Coomassie blue or processed for Western blot analysis as described above.Production of HIV-1 Gag virions. Human fibroblast 293T cells were maintained in Dulbecco modified Eagle medium-F12 (1:1) supplemented with 10% fetal calf serum. HIV-1 Gag proteins were expressed transiently by calcium phosphate transfection of 10 µg of supercoiled pGag-myc with 10 µg of carrier DNA into 293T cells using the Mammalian Cell Transfection Kit (Specialty Media, Lavallette, N.J.). Cotransfections of pGag-myc with the HA-Gag expression plasmids contained 10 µg of each plasmid.
Forty-eight hours posttransfection, 8 ml of supernatant was collected from the transfected 293T cells. The cells were placed on ice, washed with phosphate-buffered saline, and lysed in radioimmunoprecipitation assay buffer. The supernatant was centrifuged at 1,000 rpm for 5 min and passed through a 0.45-µm-pore-size filter to remove cellular debris. The filtrate was layered onto 2 ml of 25% sucrose in TNE (10 mM Tris-HCl [pH 7.5], 100 mM NaCl, and 1 mM EDTA) and centrifuged at 80,000 × g for 2 h in a Beckman SW41 rotor. The pellet was resuspended in 30 µl of 2× SDS sample buffer and was processed for Western blot analysis.| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Establishment of an in vitro assay for Gag-Gag interaction. To map primary sequence requirements for Gag-Gag interaction, we developed an in vitro assay by using recombinant proteins expressed in bacteria. The HIV-1 Gag polyprotein was first expressed as a GST-fusion protein in E. coli (Fig. 1A) and was purified from total bacterial lysate in a single step with glutathione-agarose beads as previously described (12, 38). As a negative control for the binding experiment, GST was expressed without Gag residues fused to it (Fig. 1A).
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Construction of HA-tagged Gag deletion mutants.
To determine
the primary sequence requirements for Gag-Gag interaction in vitro, a
panel of mutations, each encoding a different, truncated Gag protein,
was constructed. Mutants were named for the gag nucleotide
where the deletion or disruption of coding sequence begins.
Nucleotides were numbered with respect to the 5' end of the 5' LTR of
HIV-1HXB2 (20). For example, 5'1184 deletes
all coding sequences 5' of nucleotide 1184, and 3'1184 deletes
all coding sequences 3' of nucleotide 1184.
Effect of amino-terminal truncations on HA-Gag interaction with GST-Gag. The first set of deletion mutations to be tested encoded Gag polyproteins with truncated amino termini (Fig. 2). The coding sequences retained or deleted by the amino-terminal truncation mutants are shown schematically in Fig. 2A. Binding strength of individual mutant Gags was estimated in Western blots by first normalizing the quantity of the mutant protein added to the binding reaction with the input for the wild-type protein. Then, the signal intensity of the mutant Gag bound to GST-Gag was compared with serial dilutions of the wild-type Gag bound to GST-Gag as has been reported previously to quantitate signals on Western blots (6, 61). Figure 2B to F shows Western blots obtained from binding experiments with critical mutant proteins; primary data for the remaining mutants is not shown due to lack of space.
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indicates activity less than 5% of
wild type. (B to F) Immunoblots showing the HIV-1 Gag
polyprotein-binding activity of select amino-terminal deletion mutants.
Primary data for the remaining mutants is not shown due to lack of
space. GST or GST-Gag was purified from bacterial lysates by incubation
with glutathione-agarose beads. Beads with associated proteins were
washed and then incubated with lysate from bacteria expressing the
indicated HA-Gag mutant proteins: 5'831, 5'906, 5'1184, 5'1320, 5'1509, 5'1632,
5'1712, and 5'1920). In other words, in our semiquantitative
assay, these proteins bound to GST-Gag with strength comparable to that
of the wild type. These results indicate that the MA, CA, and p2 domains of HIV-1 Gag are not required for association with GST-Gag in vitro.
When 5' deletion mutations extended into sequences encoding the NC
domain, interaction with GST-Gag was attenuated. Mutant 5'1962
deletes nucleotides encoding NC residues up to the amino-terminal zinc
finger; this mutant protein possessed binding activity barely detectable above the background (Fig. 2F). Mutant 5'2004 extends further into NC, deleting all of the amino-terminal zinc finger and
possessing no detectable binding activity (Fig. 2A). These results
suggest that the NC domain is critical for interaction with the Gag
polyprotein in vitro.
Effect of carboxyl-terminal truncations on HA-Gag interaction with
GST-Gag.
The identity of the carboxyl-terminal truncation mutants
tested here is shown schematically in Fig.
3A. Mutant 3'2093 prematurely terminates translation before the beginning of the p6 domain and just
after the end of the NC domain. This protein bound to GST-Gag with
strength comparable to that of the wild type (Fig. 3B). This result
demonstrates that the p6 domain is dispensable for interaction with the
Gag polyprotein in vitro.
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2007 in Fig. 3C), a
significant reduction in binding activity was observed. Compared with
the wild-type protein, or the protein encoded by 3'2093, the binding
strength of this protein was 10- to 20-fold less, as determined by
comparing the signal of 3'2007 bound to GST-Gag with serial
dilutions of the sample containing 3'2093 bound to GST-Gag (data not
shown). With further truncation of carboxyl-terminal residues by
mutations 3'1906, 3'1878, and 3'1862, weak binding activity
comparable to that of 3'2007 was detected (Fig. 3). 3'1878
expresses a protein with the authentic CA carboxyl terminus that
results from viral protease cleavage of the Gag polyprotein.
Further truncation revealed a free carboxyl terminus that was not
stably expressed in bacteria (3'1787 and 3'1757). With truncations that extended beyond this point (3'1715, 3'1681, and
3'1184), stable proteins were expressed, but no binding activity was
detected (Fig. 3F). 3'1184 was constructed to encode a protein with
the same carboxyl terminus as the MA viral protease cleavage product.
The results with the carboxyl-terminal deletion mutants support the
contention that NC is the major Gag-interacting domain. The fact that
mutants 3'2007, 3'1906, 3'1878, and 3'1862 exhibit weak
binding activity suggests that a contribution to the interaction is
also made by carboxyl-terminal CA residues encoded between nucleotides
1715 and 1862.
The CA-dimer interface contributes to Gag polyprotein
multimerization in vitro.
To better pinpoint the residues at the
carboxyl terminus of CA that contribute to Gag multimerization, we have
attempted to generate several mutations with stop codons between
nucleotides 1715 and 1862. Unfortunately, these expression constructs
fail to produce stable protein products in bacteria that can be
reasonably normalized to the full-length polyprotein (for example,
mutants 3'1757 and 3'1787 in Fig. 3).
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Contribution of MA basic residues to Gag multimerization.
Mutant 3'2093 bound to GST-Gag with wild-type activity (Fig.
3B). Though significantly decreased with respect to 3'2093, mutant 3'2007 retained modest, though clearly detectable,
Gag-binding activity (Fig. 3C). When these mutants were expressed in
cis with mutants that deleted residues from the amino
terminus, a different result was observed: mutant 5'1320/3'2093
exhibited binding activity comparable to that of the wild type,
but mutant 5'1320/3'2007 exhibited no detectable activity (Fig.
5B and C). These results suggested
that, in the absence of NC, sequences at the amino terminus of the Gag
polyprotein are required to detect GST-Gag binding activity.
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2093 and 3'2007 was
examined. Mutants 5'831/3'2093 and 5'831/3'2007 retained binding activity, demonstrating that coding sequences 5' to nucleotide 831 do not encode residues that contribute significantly to the stabilization of binding (Fig. 5A). In contrast, when the 5' deletion was extended to nucleotide 906 (5'906/3'2007), no binding
activity was detected (Fig. 5A), indicating that the stabilizing
activity was encoded by nucleotides located between nucleotides 831 and 906.
The amino acids encoded by nucleotides 831 to 906 include a cluster of
basic amino acids (Fig. 5A). A previously characterized mutation
(63) that deletes 11 amino acids encompassing the basic cluster (mutant 5'850-884 in Fig. 5A) was tested next, and
mutants 5'850-884 and 5'850-884/3'2093 retained full
Gag-binding activity (Fig. 5D and E). Mutant
5'850-884/3'2007 had no detectable binding activity (Fig. 5F),
indicating that the basic residues in MA contribute to the
stabilization of Gag binding when NC residues are deleted.
The role of NC in Gag-Gag interaction. The deletion analysis presented in Fig. 2 and 3 indicates that NC is the major domain contributing to Gag multimerization. To determine if the isolated NC domain is able to associate with Gag, the 55-amino-acid NC protein was expressed in bacteria as an HA-fusion protein and as a GST-fusion protein. The ability of HA-NC (Fig. 6A and B) or HA-Gag (Fig. 6C and D) to bind to GST, GST-Gag, or GST-NC was then compared directly. The quantity of the GST-fusion proteins loaded on the beads was normalized by measuring signal intensity on Coomassie blue-stained SDS-PAGE gels (Fig. 6A and C). Comparable amounts of HA-NC were recovered by GST-Gag or GST-NC (Fig. 6B). Similarly, comparable amounts of HA-Gag were associated with either GST-Gag or GST-NC (Fig. 6D). These results confirm the conclusion from the deletion analysis that the NC domain is sufficient to account for the majority of Gag multimerization activity in vitro.
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Establishment of an in vivo assay for Gag-Gag interaction. To test the significance of the in vitro mapping results, an assay for HIV-1 Gag-Gag interaction was established in human fibroblasts. To express HIV-1 gag in human cells in the absence of other viral components, we used a construct provided to us by George Pavlakis that contains multiple, conservative mutations that render HIV-1 gag mRNA Rev independent without changing the primary amino sequence (53). This construct was used to generate plasmids for the expression of two Gag-fusion proteins, Gag-Myc and HA-Gag, shown schematically in Fig. 7A. Gag-Myc is the complete HIV-1 Gag polyprotein with a Myc-epitope tag fused at the carboxyl terminus so that it can be distinguished from other Gag proteins (e.g., HA-Gag) expressed in the same cells. Gag-Myc was well expressed in transfected 293 cells and produced extracellular virions that pellet through 25% sucrose (Fig. 7B, lane 5). HA-Gag has an HA-epitope tag fused at the amino terminus, precluding recognition by the host N-myristyl transferase; though produced in significant quantity in the cytoplasm of transfected cells, HA-Gag was found to be incapable of directing the formation of extracellular virions (Fig. 7C, lane 1).
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Effect of Gag mutants on Gag-Gag interaction in vivo.
With the
establishment of an in vivo assay for Gag-Gag interaction, mutations
exhibiting significant phenotypes in the vitro assay were subcloned
into the HA-Gag expression vector. The mutant proteins were then tested
for the ability to be incorporated into virions when Gag-Myc was
expressed in trans. HA-Gag-W184A was tested first. As with
HA-Gag, when expressed by itself, this protein was unable to direct the
assembly of virions (Fig. 7C, lane 2). When Gag-Myc was expressed in
trans, HA-Gag-W184A was incorporated into virions (lane 7).
Compared with HA-Gag, there was a slight reduction in incorporation
efficiency. HA-Gag-3'1878 was also well expressed but unable
to produce virions when expressed by itself (lane 3). This
mutant exhibited a significant reduction in the ability to be
incorporated in trans into Gag-Myc virions (lane 8),
consistent with its effect in the in vitro assay. No detectable
incorporation in trans into Gag-Myc virions was observed with the double mutant HA-Gag-3'1878/W184A (lane 9), consistent with
the complete disruption of Gag-Gag interaction by this mutant. In the
next experiment, an HA-NC expression construct was shown to be
expressed in the cytoplasm, but the protein was not released from the
cell (lane 10). When Gag-Myc was expressed in the same cells, HA-NC was
efficiently incorporated into virions (lane 11). The results with the
in vivo assay correspond to those obtained in vitro: NC is the major
Gag polyprotein domain required for Gag-Gag interaction, and, to a
lesser extent, the CA-dimer interface contributes to the interaction.
RNA is required for Gag polyprotein multimerization. The NC domain of Gag possesses a high percentage of basic residues and two zinc fingers and has been shown to bind to RNA (for review see reference 4). The basic residues in MA that contribute to Gag multimerization in the context of an NC deletion (Fig. 5A) also have the potential to interact with RNA (36). In response to the results of the deletion analysis presented above, experiments were initiated to test the hypothesis that Gag multimerization requires RNA-protein interactions as well as protein-protein interactions.
A modified binding assay was established in which the two recombinant proteins were treated with RNase A prior to mixing them together. GST-Gag was bound to glutathione-agarose beads. The beads were washed three times before they were resuspended and incubated in a buffer containing RNase A for 1 h at 37°C. After the RNase treatment, the beads were again washed three times. Simultaneously, bacterial lysate containing HA-Gag protein was added to a buffer containing RNase A, and this mixture was also incubated for 1 h at 37°C. The RNase-treated HA-Gag lysate was then added to the RNase-treated GST-Gag that was bound to glutathione-agarose beads and was incubated at 4°C for 1 h. The postbinding supernatant was saved, and the proteins that remained associated with the beads after another three washes were processed by SDS-PAGE. Recovery on the beads of GST-Gag was monitored by staining the gel with Coomassie blue; recovery of HA-Gag was monitored by Western blot with an anti-HA antibody. As a gross measure of RNase activity, an aliquot of the RNase-treated, HA-Gag-containing bacterial lysate was run on an agarose gel which was then stained with ethidium bromide. It was discovered that the majority of the RNA had been degraded, while the bacterial chromosomal DNA had remained intact (Fig. 8A). The Coomassie-stained gel of the products of the final binding reaction indicated that the GST-Gag remained intact through all the incubation steps (Fig. 8B). A Western blot of the postbinding supernatant indicated that the HA-Gag protein had also remained intact through all of the incubation steps (Fig. 8C). Upon examination of the proteins that remained associated with the glutathione-agarose beads at the end of the binding experiment, it was evident that RNase A had significantly impaired the interaction of HA-Gag with GST-Gag (Fig. 8D). The products of the binding reaction were probed in a Western blot with an RNase A antibody. With the concentration of RNase A used here, no RNase A was found associated with GST-Gag (data not shown), indicating that RNase A was not disrupting the interaction by competing for binding to GST-Gag. These results demonstrate that Gag multimerization in vitro is dependent upon RNA.
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The HIV-1 Gag polyprotein interacts in vitro with the Gag polyproteins encoded by other retroviruses. It had been previously shown with the yeast two-hybrid system that HIV-1 Gag interacts with the Gag polyproteins encoded by two SIV isolates and FIV (21). These results were confirmed here with the in vitro binding assay: HA-HIV-1 Gag associates with GST fusions to the Gag polyproteins encoded by SIVMAC239 and FIV (Fig. 9A and B). HIV-1 Gag was also shown to interact with the Gag polyproteins of the visna lentivirus, RSV, and even HFV (Fig. 9A and B).
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The HIV-1 Gag polyprotein interacts in vitro with heterologous RNA-binding proteins. Since HIV-1 Gag interacts with HFV Gag, a collection of heterologous RNA-binding proteins was expressed as GST fusions to determine if they also interact with HIV-1 Gag. The first of the heterologous RNA-binding proteins to be tested was HIV-1 Rev, and it was found to associate with HIV-1 Gag in our assay (Fig. 10).
|
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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Numerous reports in the literature have shown that the NC domain plays an essential role in retroviral assembly. Studies with RSV and HIV-1 have identified three discrete domains essential for virion assembly (13), and one of these assembly domains overlaps NC. A number of groups have shown that the deletion of nucleotides encoding NC disrupts normal particle production (8, 14, 25, 31), as does the mutation of basic residues in NC (10, 14) or the simultaneous mutation of homologous residues in both zinc fingers (17).
In the experiments presented here, any deletion that encroached upon sequences encoding NC residues resulted in a significant decrease in the ability to interact with the Gag polyprotein. In addition, the Gag-binding activity of isolated NC was comparable to that of the full Gag polyprotein. These studies greatly refine previous deletion mapping studies reported with the two-hybrid system (21). NC-NC interaction has been observed in the two-hybrid system, though the activity of the isolated NC domain was not compared with that of the full Gag polyprotein or of other Gag fragments (56). Also, the major activity in a ligand affinity blot examining HIV-1 Gag-Gag interaction was found to disappear when sequences encoding NC were deleted (8).
Our observation that NC is incorporated into HIV-1 virions in trans is consistent with a number of published observations suggesting that one of the major roles of NC in virion assembly is to promote intermolecular interactions among Gag polyprotein monomers. Budding deficient HIV-1 gag mutants can be efficiently rescued in trans by wild-type gag as long as they carry NC sequences (3, 60). Cysteines within the NC domain of the Gag polyprotein can be crosslinked to form dimers (43, 49, 50).
Though NC appears to be the major domain driving Gag-Gag association, other domains appear to contribute to the interaction. Analysis of the three-dimensional structure of a protein fragment encompassing the carboxyl-terminal third of HIV-1 CA identified a hydrophobic dimer interface (23). In the context of an otherwise wild-type provirus, the dimer interface mutants are associated with a fourfold reduction in virion assembly (23); this result correlates with the magnitude of the reduction of Gag-Gag interactions that we observed, suggesting that these mutants disrupt Gag-Gag interaction as well as CA dimer formation. On the other hand, the affinity of the dimer interface is not very high (23), and the activity of the dimer interface that we observed in our assay for Gag-Gag interaction was relatively weak when compared with that of NC. In fact, in the absence of the basic region of MA, we are unable to detect an interaction due to the CA-dimer interface in our assay.
Deletion of MA caused no detectable reduction in Gag-binding activity, and the isolated MA domain was unable to associate detectably with GST-Gag. Though MA trimers have been observed in solution (46), our results are in agreement with reports from other groups that MA is primarily monomeric in solution, even at millimolar concentrations (41, 42). Binding activity associated with the patch of basic residues in MA was revealed in our assay when NC was deleted. Deletion of these exact MA residues prevents viral assembly (63), presumably by disrupting plasma membrane association (65) or, perhaps, because they make a contribution to the Gag-Gag interaction.
NC binds specifically to HIV-1 RNA in vitro and is required for the encapsidation of viral genomic RNA into virions (for a review, see reference 4). The basic residues in MA that contribute to Gag multimerization in the context of an NC deletion (Fig. 5A) also have the potential to interact with RNA (36). These facts, along with the observations that RNase disrupts Gag-Gag interaction and that Gag interacts with heterologous RNA-binding proteins, suggest that RNA plays an important role in virion assembly.
A Gag fragment consisting of CA and NC assembles structures in vitro, in an RNA-dependent manner (7, 24). Some researchers have reported that NC mutations which disrupt packaging of viral RNA attenuate virion assembly (14, 17), but others disagree (1, 27). These conflicting results might be explained by the fact that none of these groups examined the effect of the mutations on the packaging of the heterologous RNAs that might substitute for viral genomic RNA in the assembly function. Nonspecific RNA can substitute for the assembly function in vitro (7), and substitution of NC and p6 by the Bacillus subtilis MtrB tryptophan leader RNA-binding protein domain released particles efficiently (64). Also, specific NC mutants which are defective in the packaging of viral genomic RNA but which package increased amounts of heterologous cellular RNAs have been described (44).
Perhaps interaction with RNA alters the structure of Gag to a form that is permissive for multimerization. RNA might promote protein-protein interactions among Gag polyproteins by neutralizing charge repulsions between the basic residues. This latter possibility is supported by the observation that very high concentrations of salt will substitute for RNA in the in vitro assembly of an HIV-1 CA-NC fragment into virion-type structures (24). The finding that purified NC in complex with the HIV-1 SL3 RNA stem-loop is a monomer in solution (15) suggests that RNA does not promote protein-protein interactions among NC monomers. Rather, RNA may serve as a thread on which NC monomers are strung, thereby promoting protein-protein interactions involving other domains of the Gag polyprotein.
In a previously reported two-hybrid screen of a cDNA library for encoded proteins that interact with HIV-1 Gag, we isolated a large number of RNA-binding proteins (38). At the time we proposed that RNA might serve as a bridge, in effect linking Gag polyprotein molecules to these heterologous RNA-binding proteins. The fact that Gag interacts with such a structurally diverse group of RNA-binding proteins suggests that the interactions among Gag monomers may similarly occur via an RNA bridge, with a smaller contribution from protein-protein interactions. In support of this hypothesis, and indicating that these results are not simply artifacts of an in vitro system, it has been observed that two cellular RNA-binding proteins, elongation factor 1-alpha (11) and histidyl-tRNA synthetase (33), each interact with Gag in vitro, and both proteins are incorporated into HIV-1 virions.
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ACKNOWLEDGMENTS |
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We thank Martin Andreansky, Janice Clements, Max Essex, Eric Hunter, George Pavlakis, Wesley Sundquist, and Uta VonSchwedler for plasmid DNAs and Thomas Bertsch, Philippe El-Helou, and Julie Harris for technical assistance.
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 |
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* Corresponding author. Mailing address: Departments of Microbiology and Medicine, Columbia University, College of Physicians and Surgeons, 701 West 168th St., New York, NY 10032. Phone: (212) 305-8706. Fax: (212) 305-0333. E-mail: Luban{at}cuccfa.ccc.columbia.edu.
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80: 10478-10486
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Dye, B. T., Miller, D. J., Ahlquist, P.
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79: 8909-8919
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Roldan, A., Warren, O. U., Russell, R. S., Liang, C., Wainberg, M. A.
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Marozsan, A. J., Fraundorf, E., Abraha, A., Baird, H., Moore, D., Troyer, R., Nankja, I., Arts, E. J.
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Melamed, D., Mark-Danieli, M., Kenan-Eichler, M., Kraus, O., Castiel, A., Laham, N., Pupko, T., Glaser, F., Ben-Tal, N., Bacharach, E.
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78: 9675-9688
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Perez-Caballero, D., Hatziioannou, T., Martin-Serrano, J., Bieniasz, P. D.
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Derdowski, A., Ding, L., Spearman, P.
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Ott, D. E., Coren, L. V., Chertova, E. N., Gagliardi, T. D., Nagashima, K., Sowder, R. C. II, Poon, D. T. K., Gorelick, R. J.
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von Schwedler, U. K., Stray, K. M., Garrus, J. E., Sundquist, W. I.
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Holm, K., Weclewicz, K., Hewson, R., Suomalainen, M.
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Liang, C., Hu, J., Whitney, J. B., Kleiman, L., Wainberg, M. A.
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Liang, C., Hu, J., Russell, R. S., Roldan, A., Kleiman, L., Wainberg, M. A.
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Saphire, A. C. S., Bobardt, M. D., Gallay, P. A.
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Hill, M. K., Hooker, C. W., Harrich, D., Crowe, S. M., Mak, J.
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Gonsky, J., Bacharach, E., Goff, S. P.
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Bacharach, E., Gonsky, J., Alin, K., Orlova, M., Goff, S. P.
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Rumlova-Klikova, M., Hunter, E., Nermut, M. V., Pichova, I., Ruml, T.
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Mouland, A. J., Mercier, J., Luo, M., Bernier, L., DesGroseillers, L., Cohen, E. A.
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