Trimer Enhancement Mutation Effects on HIV-1 Matrix Protein
Binding Activities
Ayna Alfadhli,aAndrew Mack,aChristopher Ritchie,aIsabel Cylinder,aLogan Harper,aPhilip R. Tedbury,bEric O. Freed,bEric Barklisa
Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, Oregon, USAa; Virus-Cell Interaction Section, HIV Drug Resistance Program, National Cancer Institute, Center for Cancer Research, Frederick, Maryland, USAb
ABSTRACT
The HIV-1 matrix (MA) protein is the amino-terminal domain of the HIV-1 precursor Gag (Pr55Gag) protein. MA binds to
membranes and RNAs, helps transport Pr55Gag proteins to virus assembly sites at the plasma membranes of infected cells, and
facilitates the incorporation of HIV-1 envelope (Env) proteins into virions by virtue of an interaction with the Env protein
cyto-plasmic tails (CTs). MA has been shown to crystallize as a trimer and to organize on membranes in hexamer lattices. MA
muta-tions that localize to residues near the ends of trimer spokes have been observed to impair Env protein assembly into virus
parti-cles, and several of these are suppressed by the 62QR mutation at the hubs of trimer interfaces. We have examined the binding
activities of wild-type (WT) MA and 62QR MA variants and found that the 62QR mutation stabilized MA trimers but did not
alter the way MA proteins organized on membranes. Relative to WT MA, the 62QR protein showed small effects on membrane
and RNA binding. However, 62QR proteins bound significantly better to Env CTs than their WT counterparts, and CT binding
efficiencies correlated with trimerization efficiencies. Our data suggest a model in which multivalent binding of trimeric HIV-1
Env proteins to MA trimers contributes to the process of Env virion incorporation.
IMPORTANCE
The HIV-1 Env proteins assemble as trimers, and incorporation of the proteins into virus particles requires an interaction of Env
CT domains with the MA domains of the viral precursor Gag proteins. Despite this knowledge, little is known about the
mecha-nisms by which MA facilitates the virion incorporation of Env proteins. To help elucidate this process, we examined the binding
activities of an MA mutant that stabilizes MA trimers. We found that the mutant proteins organized similarly to WT proteins on
membranes, and that mutant and WT proteins revealed only slight differences in their binding to RNAs or lipids. However, the
mutant proteins showed better binding to Env CTs than the WT proteins, and CT binding correlated with MA trimerization.
Our results suggest that multivalent binding of trimeric HIV-1 Env proteins to MA trimers contributes to the process of Env
vi-rion incorporation.
T
he matrix (MA) domain of the human immunodeficiency type
1 (HIV-1) precursor Gag (Pr55Gag) protein serves several
as-sembly-related functions. One role is to direct Pr55Gag proteins
to assembly sites at cell plasma membranes (PMs) that are
en-riched for phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2)
and cholesterol (
1–8
). This function is accomplished in part by
virtue of an amino-terminal myristate moiety and, in part,
through direct binding to PI(4,5)P2 (
1
). Evidence indicates that
the PI(4,5)P2-binding site on MA overlaps a binding site for RNA,
which suggests a chaperone model in which MA-RNA binding
protects the MA domain of Pr55Gag from associating with
inap-propriate intracellular membranes prior to delivery to the
PI(4,5)P2-rich PM assembly sites (
9–16
). In some experimental
systems, it has been shown that Pr55Gag proteins with MA
dele-tions (⌬MA) or swaps of MA with other membrane binding
do-mains are capable of directing the assembly of conditionally
infec-tious viruses, but in these cases, assembly and replication
efficiencies have tended to be compromised (
17–21
).
A second role of HIV-1 MA is to facilitate the incorporation of
envelope (Env) proteins into virions (
22–25
). How MA
accom-plishes this is complicated. One confounding issue is that some
cell surface proteins can be assembled into HIV-1 particles in what
appears to be a passive fashion (
26
). Indeed, this is how
glycopro-teins from other viruses can pseudotype with the cores of HIV-1
(
17
,
27–31
). Presumably, PM proteins that localize to HIV-1
as-sembly sites can be incorporated into viruses as innocent
bystand-ers, and interestingly, several heterologous viral glycoproteins are
assembled efficiently into both wild-type (WT) and
⌬MA HIV-1
viruses (
17
,
31
). The same is not true for the WT HIV-1 Env
protein (
17
,
18
). However, HIV-1 Env proteins that carry
dele-tions of the long Env cytoplasmic tail (CT) can be assembled
effi-ciently into either WT or
⌬
MA HIV-1 particles (
18
), at least in cell
types that permit the efficient cell surface localization of
⌬CT Env
proteins (
32
,
33
).
The above-described results suggest that
⌬
CT Env proteins
assemble passively into HIV-1 virions, whereas WT Env proteins
require a direct or indirect interaction with MA for virion
incor-poration (
17
,
18
,
22–25
). Because of the unusual length of the
HIV-1 Env CT, one can speculate that WT Env trimers are
hin-dered from assembling into lattices organized by PrGag proteins
Received17 March 2016Accepted25 March 2016
Accepted manuscript posted online30 March 2016
CitationAlfadhli A, Mack A, Ritchie C, Cylinder I, Harper L, Tedbury PR, Freed EO, Barklis E. 2016. Trimer enhancement mutation effects on HIV-1 matrix protein binding activities. J Virol 90:5657–5664.doi:10.1128/JVI.00509-16.
Editor:K. L. Beemon, Johns Hopkins University
Address correspondence to Eric Barklis, [email protected].
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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unless they are guided by an interaction with MA. Data supporting
such a hypothesis are available. HIV-1 MA has a propensity to
form trimers (
22–25
,
34–36
), and mutations of residues at the
spokes of those trimers (such as 12LE, 16EK, 30LE, 34VE, and
98EV) (
Fig. 1A
) have been shown to impair WT HIV-1 Env virion
incorporation (
22–25
,
37
). MA also has been shown to assemble
in
vitro
in two forms of hexamer lattices, and in both lattices, residues
12, 16, 30, 34, and 98 point toward hexamer centers (
Fig. 1B
and
C
), suggesting that Env CT tails can be coordinated in these
loca-tions (
38
,
39
).
Unexpectedly, recent investigations have revealed that another
mutation, 62QR, located at the MA trimer interface (
Fig. 1A
),
impacts WT HIV-1 Env incorporation (
22–25
). Cell culture
stud-ies showed that the 62QR mutation suppressed the Env
incorpo-ration defects of the 12LE, 16EK, 30LE, 34VE, and 98EV MA
mu-tations and of an Env CT mutation that has the same phenotype
(
22–25
). How is the 62QR suppression mediated? One possibility
(
22–25
) is that 62QR stabilizes trimers such that MA lattices which
form large hexamer holes (
Fig. 1B
) are favored over those that
feature small hexamer holes (
Fig. 1C
). Such a model is
parsimo-nious in that it does not require specific MA-CT interactions, but
it does not directly address why MA lattices have not been
ob-served in immature HIV-1 virions, why WT HIV-1 Env fails to
incorporate into
⌬MA virions, or why mutations at the spokes of
MA trimers affect Env incorporation (
22–25
,
37
,
40–44
).
Due to the importance of the 62QR mutant, we have examined
its activities
in vitro
in comparison with WT MA. Our results show
that 62QR MA proteins had increased capacities to trimerize
rel-ative to their WT counterparts, but that the WT and 62QR
pro-teins appeared to organize similarly on membranes. The two
vari-ants showed slight differences in binding to PI(4,5)P2-containing
membranes and RNA ligands, but 62QR proteins bound
signifi-cantly better than WT proteins to the HIV-1 Env CT. Our results
suggest a model in which multivalent binding of trimeric HIV-1
Env proteins to MA trimers contributes to the process of Env
virion incorporation.
MATERIALS AND METHODS
Proteins for analysis.WT myristoylated and unmyristoylated [desig-nated Myr(⫺)] MA C-terminally His-tagged proteins were expressed in
Escherichia colistrain BL21(DE3)/pLysS (Novagen) from pET-11a-based vectors and purified and analyzed as described previously (9,11,13,38,
39). The WT MAGFP protein was purified as described above. It was constructed from a pET-11a-based MACA expression vector (38,39) by insertion of the coding region for the green fluorescent protein (GFP) from pEGFP-c1 (Clontech) near the C terminus of MA, as in the previ-ously reported vector HIVgpt-MAGFP (20). The MA-GFP juncture dues are AALALPVATM-GFP-KSGLRSAD, where the first and last resi-dues represent HIV-1 MA resiresi-dues 119 and 120. The 62QR MA and MAGFP variants and the 12LE and 12LE/62QR variants were transferred from the NL4-3 constructs (22–25,37,45) and were purified similarly to their WT counterparts. TheSchistosoma japonicaglutathione S -trans-ferase (GST) and GST-CT proteins also were expressed and purified from
Escherichia coli strain BL21(DE3)/pLysS. GST was expressed from pGEX4T3 (GE Healthcare), and GST-CT was expressed from pGEX4T3-CT, where the cytoplasmic tail coding region of the HXB2 Env protein was amplified with BglII ends by PCR and inserted in frame C-terminally to the GST coding region of pGEX4T3 at the pGEX4T3 BamHI site. The juncture sequence is GGA TCT GTT AAC AGA GTT-ENV CT-ATT TTG CTT TAA AGA TCG, where the underlined codons represent the HXB2 Env codons 705 to 708 and 854 to 856. The GST and GST-CT proteins were purified under nondenaturing conditions from 400-ml cultures (op-tical density at 600 nm [OD600] of 0.6 to 0.9) that were induced for 4 h at
25°C via the addition of 0.5 mM isopropyl-D-1-thiogalactopyranoside (IPTG). Induced cultures were pelleted and resuspended in 5 ml of GST sonication buffer (50 mM sodium phosphate [pH 7.8], 300 mM NaCl) containing protease inhibitors (15g/ml aprotinin, 1.5 mM phenyl meth-ane-sulfonyl fluoride [PMSF], 36g/ml chicken trypsin inhibitor, 36
g/ml soybean trypsin inhibitor, 6g/ml benzamidine, 15g/ml leupep-tin, 7.5g/ml pepstatin A) plus 12.5g/ml DNase 1 (Roche). Suspended bacteria were incubated on ice for 30 min, subjected to two rounds of French press lysis, clarified by a 10-min centrifugation at 4°C and 7,500⫻g, and incubated with rocking for 1 to 3 h at 4°C with 1 to 2 ml of reduced glutathione agarose resin (Sigma, Pierce). Samples and resin then were loaded onto columns, unbound material was collected, columns were washed twice with 10 ml sonication buffer plus inhibitors, and five 1-ml elutions of elution buffer (50 mM Tris, pH 8.0, 20 mM reduced glutathi-one) were eluted. Elutions were desalted by dialysis in two 3-h steps at 4°C with 4 liters of storage buffer (50 mM sodium phosphate [pH 7.0], 150 mM NaCl, 1 mM-mercaptoethanol [BME]) and analyzed for purity by SDS-PAGE plus staining and immunoblotting with anti-GST primary antibody (SC-138; at 1:1,000 dilution; Santa Cruz Biotech). GST purifi-cations yielded protein stocks that were⬎90% pure and were stored fro-zen (⫺80°C) in aliquots at 1 to 2 mg/ml. GST-CT purifications yielded samples that corresponded to 50% GST-CT and 50% processed GST and were stored frozen as described above.
RNA binding assays.Fluorescence anisotropy (FA) binding assays were performed as described previously (11,13). Briefly, 10 nM fluores-cein isothiocyanate (FITC)-labeled Sel25 RNA oligomer (5= FITC-G GACA GGAAU UAAUA GUAGC UGUCC-3=; Invitrogen) samples were incubated in 25 mM sodium phosphate (pH 6.0), 50 mM NaCl with successive additions of 30M protein to achieve final concentrations of 0 to 5,120 nM protein. Incubations were performed in 12- by 75-mm dis-posable borosilicate glass tubes, and measurements were obtained on a PanVera Beacon 2000 fluorescence polarizer (Invitrogen) at an excitation wavelength of 490 nm. Readings were made in triplicate at room temper-ature, polarization values were calculated from emitted light intensities according to the ratio of parallel minus perpendicular to parallel plus perpendicular (18–20), and binding isotherms were fitted using Prism.
Electrophoretic mobility shift assays (EMSAs) were performed using the chemically synthesized untagged Sel25 RNA (Invitrogen) as described previously (11,13). EMSA binding reactions were performed for 30 min at 4°C in 25 mM sodium phosphate (pH 6.0), 50 mM NaCl, and employed 15M Sel25 plus 0 to 37.5M MA. Bound and free RNAs were separated by electrophoresis on 12% native polyacrylamide gels in 0.5⫻Tris-borate buffer (44.5 mM Tris base, 44.5 mM boric acid [pH 8.0]) and visualized by staining with Stains-all (Sigma) (11,13).
FIG 1Alternative MA assemblies. (A) Atomic model of the HIV-1 MA trimer (PDB entry1HIW) is shown with glutamine 62 (Q62) at the trimer interface in red, and residues that affect Env protein incorporation (12L, 16E, 30L, 34V, and 98E) are in purple. (B) Observed (56) organization of MA proteins on PI(4,5)P2-containing membranes as a hexamer of trimers, with a large pro-tein-free hexamer hole in green. (C) Observed (55) organization of MA pro-teins on membranes with no PI(4,5)P2, where trimer contacts are not evident, and hexamer holes (green) are smaller in diameter. In both panels B and C, Q62 residue locations are colored red, and other residues that affect Env pro-tein incorporation are depicted in dark blue/purple.
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[image:2.585.38.287.66.146.2]Membrane binding.Liposome bead binding assays were performed as described previously (9,13) with WT or 62QR MA beads and fluores-cently tagged liposomes. MA-coated beads were prepared by following our standard protocol (9,13), and the estimated bead-bound MA used in each assay was 300 nM. Liposomes were prepared from stock solutions in chloroform of cholesterol (Sigma), 1,2-dioleoyl-sn -glycerol-3-phospho-choline (PC; Avanti), 1,2-dioleoyl-sn
-glycero-3-phospho-ethanolamine-N-(7-nitro-2-1,3benzoxadiazol-4-yl) (NBD-DOPE; Avanti), and brain PI(4,5)P2 Avanti) by sonication (9,11,13). PC liposomes were composed of 20% cholesterol, 79.8% PC, and 0.2% NBD-DOPE. PIP(4,5)P2-con-taining liposomes were composed of 20% cholesterol, 69.8% PC, 0.2% NBD-DOPE, and 10% PI(4,5)P2. Binding reactions were performed for 16 to 18 h at 4°C with 60l MA beads plus 2l of 2 mg/ml liposomes in wash buffer (25 mM sodium phosphate [pH 7.8], 50 mM NaCl, 0.1 mg/ml bovine serum albumin [BSA]). After binding incubations, beads were pelleted, washed twice with 0.3 ml wash buffer, and suspended in 50l of wash buffer on ice. For viewing, 10-l samples were applied to microscope slides, covered with 22-mm by 22-mm coverslips, and imaged on a Zeiss Axioplan fluorescence microscope using a 20⫻(LDA-Plan) objective with Zeiss filter set 10 (excitation band pass, 450 to 490 nm; beam splitter Fourier transform, 510 nm; emission band pass, 515 to 565 nm). Bead brightness values were calculated and averaged as described previously (9,
13) and were normalized to values obtained with WT MA-coated bead plus PI(4,5)P2-containing liposome incubations.
CT binding assays.Beads for pulldown assays were prepared by incu-bation of 100l of packed glutathione agarose resin (Sigma, Pierce) in 0.25 ml of assay buffer (25 mM sodium phosphate [pH 7.0], 150 mM NaCl, 10% glycerol, 0.1 mg/ml BSA) plus 5g GST or GST-CT at 4°C for 1 h. Beads then were pelleted, washed with assay buffer, and resuspended in assay buffer. Binding reactions included 33l resin in a total of 0.1 ml assay buffer containing 1.5M MA or CA protein, and they were per-formed at 4°C for 16 to 18 h on a rotator. After binding reactions, beads were pelleted and unbound (total) samples were collected. Beads were then washed three times for 1 min each with 0.5 ml assay buffer (minus BSA), and bound material was eluted in 15-min incubations with elution buffer (50 mM Tris [pH 8.0], 20 mM glutathione). Total and bound samples were fractionated by SDS-PAGE and immunoblotted (17,20,31) with primary antibodies to MA (01848170; 1:2,000; Capricorn), CA (Hy183; 1:30 from hybridoma media), and anti-GST primary antibody (SC-138; 1:1,000 dilution; Santa Cruz Biotech) for protein detection. To-tal and bound band levels were quantified by densitometric scanning and analysis with Image J software (46) and normalized as percent bound levels. Fluorescent protein GST binding assays were performed with GST and GST-CT beads prepared as described above in incubations with 1.5
M WT or 62QR MAGFP protein. Binding reactions and washes were performed as described above. After washing, beads with bound MAGFP proteins were imaged and analyzed as described for membrane binding assays.
Cross-link analysis.Protein cross-linking was performed by UV light exposure, which has been reported to cross-link proteins at tyrosine, cys-teine, and histidine residues (47–49). For UV cross-linking, 20-l drops of 50M protein in 50 mM sodium phosphate (pH 7.0), 150 mM NaCl, 1 mM BME were placed in the center rows of 96-well plates (vinyl; Costar) at 25°C. UV cross-linking reactions were performed with a UV Stratalinker (Stratagene) using the auto-cross-link parameter (1,200J [⫻100]) for 0, 1, 3, or 10 min. At each time point, samples were removed from the plate and processed for electrophoresis and immunoblotting (17,20,31). Monomers, dimers, and trimers were determined by com-parison of mobilities with known size standards (161-0374; Bio-Rad) and were quantified by densitometric scanning and analysis with Image J soft-ware (46). For each 10-min UV reaction, the amounts of monomer and trimer signals were normalized to the monomer signals to obtain esti-mates of trimer formation.
EM.Assembly of MA proteins on lipid monolayers, electron micros-copy (EM), and image analysis proceeded by following methods described
previously (38,39). WT and 62QR MA proteins (250M) were assembled in 25 mM sodium phosphate (pH 7.8), 5 mM sodium acetate, 300 mM NaCl, 20% glycerol, 1.25 mM BME beneath lipid monolayers of 60% egg PC (Avanti), 20% cholesterol (Sigma), and 20% brain PI(4,5)P2 (Avanti). After incubations, samples were lifted onto lacey EM grids (Ted Pella), rinsed for 30 s on drops of distilled water, stained for 45 to 60 s with 1.33% (wt/vol) uranyl acetate, wicked, and dried. Samples were imaged under low-dose conditions at 120 keV on the OHSU FEI Tecnai transmission electron microscope (TEM) equipped with an Eagle 4 megapixel charge-coupled device (CCD) at 4.37 Å/pixel and defocus values of ⫺200 to⫺1,500 nm to give a contrast transfer function (CTF) first zeroes of beyond 2 nm. Ordered areas in images were boxed, Fourier transformed, indexed, and unbent using the 2dx image processing package (50). Result-ing amplitudes and phases (aph) files were subjected to space group anal-ysis (38,39), yielding best fits based of p6 symmetry for both WT and 62QR crystals, with real space dimensions for both proteins ofa⫽b⫽
87.6 Å and␥⫽120°. Merged WT and 62QR aph files were complete to 25 Å, were filtered to that resolution limit, and were back-transformed as-suming p6 symmetry to yield Fourier filtered images in which proteins appear white and protein-free areas are dark.
RESULTS
Matrix protein oligomerization.
Because the HIV-1 MA 62QR
mutation at the matrix trimer interface strongly suppressed the
defects imposed by mutations located at MA trimer spokes (
22–
25
) (
Fig. 1
), it was of interest to compare the activities of the 62QR
protein relative to the WT protein. Initially, we compared the
sedimentation profiles of purified 62QR and WT myristoylated
MA (here referred to as MA) proteins. Our results indicated that
50
M WT MA (in 50 mM sodium phosphate, pH 6.0, 100 mM
NaCl, 2 mM Tris-2-carboxyethyl phosphine) sedimented with an
apparent molecular mass of 21,606 Da, slightly higher than the
predicted 16,812 Da, whereas the 62QR mutant sedimented with
an apparent mass of 33,532 Da (data not shown). These results
were consistent with the notion that the proteins oligomerize as
dimers and/or trimers, but we were unable to fit data clearly into
monomer-dimer, monomer-trimer, or monomer-dimer-trimer
equilibrium models.
As an alternative approach, we subjected WT and 62QR MA
proteins to UV cross-link analysis. To do so, purified proteins
were mock or UV treated, separated by electrophoresis, and
sub-jected to immunoblot detection. As shown in
Fig. 2
(upper left),
while UV treatment increased the proportion of WT MA dimers,
levels of trimers were low. In contrast, with 62QR MA, UV
treat-ment yielded both dimers and trimers (
Fig. 2
, upper right). Profile
plots of samples receiving UV treatment for 10 min clearly showed
a significant trimer band for 62QR MA, but the band was less clear
for WT MA (
Fig. 2
, lower left). Quantitation of trimers versus
monomers after 10 min of UV treatment in 10 independent
ex-periments indicated that WT MA protein trimers were observed at
2% monomer levels, while 62QR trimers were observed at 11%
monomer levels (
Fig. 2
, bottom right). These results indicate that
the 62QR trimer interface mutation stabilized MA trimers.
As noted in the Introduction, one hypothesis as to how 62QR
proteins suppress Env incorporation defects posits that
mem-brane-bound lattices of 62QR proteins better accommodate Env
CT tails than do WT lattices as a consequence of MA trimerization
(
Fig. 1B
and
C
). We examined this possibility via analysis of how
WT and 62QR proteins organize on membranes. For this, WT and
62QR MA proteins were assembled on membranes containing
cholesterol and PI(4,5)P2 and analyzed by transmission EM. As
observed previously (
39
), the MA proteins assembled into small
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ordered two-dimensional (2D) crystalline patches that were
ame-nable to image analysis. Consequently, ordered regions were
boxed and Fourier transformed, and lattice reflections were
in-dexed to give unit cell parameters. Interestingly, both WT and
62QR MA proteins yielded unit cells with hexagonal (p6)
symme-try and identical real space unit cell sizes (
a
⫽
b
⫽
87.6 Å,
␥ ⫽
120°). To visualize the MA-membrane lattices, Fourier transforms
were filtered and back-transformed, and assemblies are shown in
Fig. 3
, viewed from the membrane side with protein regions in
white. Of particular note is that the assemblies appear nearly
iden-tical, with trimer units surrounding equally sized hexamer holes.
These results do not lend support to the hypothesis that WT and
62QR proteins organize on membranes with differently sized
hex-amer holes (
22–25
), but it is possible that potential differences
were masked by the 25-Å resolution of our reconstructions.
RNA and membrane binding.
The WT HIV-1 MA protein
binds to RNAs and PI(4,5)P2-containing membranes at
overlap-ping binding sites (
11
,
13
). To examine whether the 62QR
muta-tion affects this binding, we initially tested myristoylated and
myr-istoylation-minus [Myr(⫺)MA] WT and 62QR proteins for
binding to the previously identified (
11
,
13
,
51
) Sel25 RNA ligand.
Fluorescence anisotropy (FA) assays were performed with
fluores-cently tagged Sel25 ligand and increasing concentrations of WT
and 62QR MA and Myr(⫺)MA (
Fig. 4A
). For both the MA and
Myr(
⫺
)MA proteins, we observed slightly higher affinities of the
62QR than the WT variants to Sel25: for the myristoylated
pro-teins the calculated binding constants were 496 nM (WT) and 180
nM (62QR), while the unmyristoylated protein constants were
334 nM (WT) and 130 nM (62QR). These trends also were
ob-served in gel shift (EMSA) assays, in which bound (B) and free (F)
Sel25 RNAs were detected after incubation with protein, and
elec-trophoretic separation (
Fig. 4B
). Interestingly, we did not see
sig-nificant differences in bound RNA mobilities between the WT and
62QR proteins, consistent with the previously observed 1:1
bind-ing of WT MA and Sel15 RNA (
11
) and the low levels of observed
oligomers for both WT and 62QR proteins in our cross-link
anal-ysis (
Fig. 2
).
As for membrane binding to WT and 62QR MA, we addressed
this using liposome bead-binding assays, as we have in the past (
9
,
11
). Here, fluorescent PC-cholesterol liposomes, prepared with or
without PI(4,5)P2, were assayed for binding to beads coated with
WT or 62QR MA proteins. As shown in
Fig. 6
(left), the WT and
62QR beads both bound PI(4,5)P2-containing liposomes at much
higher levels than the PC-cholesterol liposomes. Thus, selectivity
for binding to PI(4,5)P2 membranes was retained for the 62QR
proteins. However, in multiple measurements, we did observe
that PI(4,5)P2 liposomes bound 30 to 40% less well to the 62QR
than the WT MA beads (
Fig. 5
, right). Because MA residue 62
maps away from the PI(4,5)P2 binding site (
3
,
14
,
52
), this binding
difference would appear to be due to an as-yet undetermined
ef-fect.
Matrix-Env CT binding.
Since the 62QR mutation was shown
to affect Env incorporation into virions in a CT-dependent
fash-ion (
22–25
), it was of interest to analyze WT and 62QR MA-CT
binding
in vitro
. Although different previous investigations have
resulted in some uncertainty concerning the nature of MA-CT
interactions (
22–25
,
30–33
,
52
,
53
), several studies have reported
MA-CT binding (
52
,
53
). Previous work indicated that the HIV-1
Env CT could be purified from bacteria as a GST fusion protein
(GST-CT) and, when prebound to glutathione resin, could be
used successfully in MA binding assays (
52
). Because of this, we
expressed and purified GST-CT (and GST) from bacteria under
nondenaturing conditions. In this regard, it is pertinent to note
that our efforts to purify the CT as a His-tagged or maltose binding
protein-tagged protein under nondenaturing conditions were
un-successful (data not shown).
For binding reactions, GST- or GST-CT-coated beads were
incubated with WT or 62QR MA proteins (or CA as a control),
FIG 3Membrane-bound organization of MA proteins. WT and 62QR MA proteins were assembled onto lipid monolayers containing PI(4,5)P2, lifted onto EM grids, stained, and imaged. Fourier transforms of ordered regions in micrographs were indexed, Fourier filtered, and back transformed as described in Materials and Methods. In each case, assemblies are shown perpendicular to the membrane, protein regions are in white, protein-free regions are dark, and real space unit cell dimensions werea⫽b⫽87.6 Å and␥ ⫽120°. Note that at this resolution (25 Å), matrix proteins appear as interconnected trimers (one trimer indicated in each panel) around hexamer holes, and that the two assem-blies are nearly indistinguishable.
FIG 2Matrix protein trimerization. Purified WT or 62QR MA proteins at 50
M concentrations were UV cross-linked for 0, 1, 3, or 10 min (left to right) as described in Materials and Methods, separated by electrophoresis, and immu-noblotted with an anti-MA antibody. Monomer (1), dimer (2), and trimer (3) sizes were determined by the mobilities of size standards run in parallel. The bottom left shows the band intensity profile plots of the 10-min-time-point lanes from the immunoblot images above, with the WT plot as a dotted line and the 62QR plot as a solid line; note the intensity difference of the trimer bands. The bottom right shows the percentage of trimers (relative to mono-mers) from the 10-min time point averaged from 10 separate experiments, with standard deviations as indicated. At the bottom, the amounts of mono-mers (light gray), dimono-mers (dark gray), and trimono-mers (black) from the 10-min
time point were quantified as a percentage of the MA monomer signals.
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[image:4.585.62.267.62.288.2] [image:4.585.299.544.66.139.2]after which unbound (total) and bound fractions were isolated,
separated by electrophoresis, and detected by immunoblotting. As
shown in
Fig. 6
(top), none of the proteins bound well to GST
beads, and the CA and WT MA proteins also bound poorly to
GST-CT beads. In contrast, the 62QR MA proteins clearly bound
to the GST-CT beads. From multiple experiments, we found that
approximately 30% of the input 62QR MA protein bound to the
GST-CT beads (
Fig. 6
, bottom). In contrast, our results indicated
that GST-CT binding levels were close to background levels for the
WT MA and CA proteins. Thus, GST-CT binding levels appeared
to parallel trimerization levels observed in cross-linking
experi-ments (
Fig. 2
) for the MA proteins.
As a second approach to assay MA-CT binding, we bound WT
or 62QR MAGFP proteins to GST or GST-CT beads and tracked
binding via fluorescent detection of GFP. Our results (
Fig. 7
) were
consistent with results shown in
Fig. 6
. As shown in
Fig. 7
(left),
while neither MAGFP protein bound well to GST beads, the WT
variant bound slightly to GST-CT beads, and the 62QR variant
bound considerably better. Quantitation of fluorescence levels
from multiple beads indicated that 62QR binding to GST-CT was
higher than WT binding (
Fig. 7
, right).
Because the 62QR mutation was selected as one that
sup-pressed the Env incorporation defects of MA variants such as the
12LE mutation (
22–25
), it was of interest to examine how 62QR
affects the oligomerization and CT binding capabilities of 12LE
mutant proteins. To do so, 12LE and 12LE/62QR MA proteins
were purified and subjected to UV cross-linking and CT binding
FIG 4Matrix binding to 25mer RNA ligands. Binding of WT or 62QR MA (Myr⫹) or unmyristoylated MA (Myr⫺) to the Sel25 RNA ligand was tracked by fluorescence anisotropy (A) or electrophoretic mobility shift assay (B). In panel A, 10 nM fluorescent Sel25 RNA samples were incubated with increasing concentrations of protein, as indicated. Fluorescence millipolarization values were measured and plotted as percentages of the maximum polarization values obtained for each sample. The curves represent averages from triplicate measurements, andKds (dissociation constants) were calculated from half-maximal
polarization values. In panel B, 15M samples of Sel25 were incubated with 0, 7.5, 15, 22.5, 30, and 37.5M protein, after which free (F) and bound (B) RNAs were separated by electrophoresis and stained. At the bottom of each panel, densitometrically determined percent bound (%B) values are given. Note that M indicates mock lanes containing bromphenol blue dye that was excluded from RNA-containing lanes, and that results are representative of three independent experiments.
FIG 5Membrane binding of WT and 62QR MA. Liposome binding assays were performed with beads coated with WT or 62QR (62) MA proteins and fluorescently labeled liposomes containing PC plus cholesterol (PC) or PC plus cholesterol plus 10% PI(4,5)P2 (PIP). Examples of liposome-bound beads are shown on the left. Liposome binding levels were determined by measuring bead fluorescence brightness levels from at least 17 beads of each type imaged in two separate experiments. Values were normalized to the over-all average of PI(4,5)P2-containing liposomes bound to WT MA beads.
FIG 6MA binding to the HIV-1 Env CT. The indicated WT MA, 62QR MA, or CA proteins at 1.5M were incubated with beads coated with glutathione
S-transferase (GST) (CT⫺) or GST-CT (CT⫹), after which total unbound proteins were collected (total) and beads were washed and eluted to collect bound proteins. Total and bound samples were electrophoretically separated and immunoblotted in parallel for protein detection. Immunoblot bands were quantified densitometrically, and binding levels are expressed as percentages of bound versus total protein. Calculated percentages derive from five (62QR MA), four (WT MA), or three (CA) independent experiments.
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[image:5.585.115.470.65.241.2] [image:5.585.360.481.493.640.2] [image:5.585.79.249.544.648.2]analysis. As shown in
Fig. 8
(top), while 12LE dimers were
detected, trimers were present at only 2% monomer levels, similar
to that observed for the WT MA protein (
Fig. 2
). In contrast, UV
treatment clearly yielded 12LE/62QR trimers (top center), and
trimers were observed at 12% monomer levels (top right), similar
to the 62QR protein (
Fig. 2
). These results indicate that the 62QR
mutation fosters MA trimerization on both WT and 12LE
back-grounds. Importantly, the 62QR mutation also conferred an
in-creased capacity for CT binding to 12LE MA proteins. As shown in
Fig. 8
(bottom left), 12LE binding to either GST or GST-CT resin
appeared minimal. In contrast, while hardly any 12LE/62QR MA
bound to GST resin, approximately 20% of the protein bound to
GST-CT, two thirds of the level observed for the 62QR MA protein
itself (
Fig. 6
). These results indicate that the 62QR mutation allows
12LE MA proteins to bind to the HIV-1 Env CT.
DISCUSSION
The 62QR mutation, located at the hub of MA trimer structures
(
Fig. 1
), is a unique mutation in that it suppresses the envelope
incorporation defects of an Env CT mutation and of multiple MA
mutations (including 12L, 16E, 30L, 34V, and 98E) that locate
toward the ends of MA trimer spokes. Our data favor a model
(
20–22
) in which the 62QR change promotes MA trimerization
(
Fig. 2
). The mutation also slightly increased the affinity of MA for
the small Sel25 RNA ligand (
Fig. 4
), but our gel shift data (
Fig. 4
)
are consistent with our previous observation (
11
) of 1:1 MA-RNA
binding for both the WT and 62QR variants. Thus, we hypothesize
that the addition of a basic residue near other residues that have
been implicated in MA-RNA binding (
11
) accounts for the
ob-served differences in WT and 62QR Sel25 binding. We also noted
that the WT MA protein bound slightly better to
PI(4,5)P2-con-taining membranes than did the 62QR protein (
Fig. 5
). It will be of
interest to extend these observations in the future.
Given that the 62QR mutation impacted CT-dependent Env
incorporation into virions in cell culture (
22–25
), we tested its
effects relative to the WT protein in
in vitro
MA-CT binding
as-says. To do so, we adapted the previously described MA-CT
bind-ing assay (
52
) and found that 62QR MA proteins bound better to
GST-CT beads than their WT counterparts (
Fig. 6
and
7
).
Addi-tionally, we observed that the 62QR mutation in the context of the
12LE Env incorporation mutation conferred the ability to bind
GST-CT beads (
Fig. 8
). Of particular interest was that GST-CT
binding of MA proteins correlated with their abilities to trimerize
(
Fig. 2
and
8
). What might explain this behavior? One pertinent
consideration is the oligomerization status of the glutathione
res-in-bound GST-CT. Because the
Schistosoma japonicum
GST
ex-pressed from pGEX plasmids preferentially forms dimers (
54
), it is
reasonable to expect that resin-bound GST-CT packs at least pairs
of CTs in close proximity. However, the packing density of
gluta-thione agarose (
⬎
40 mg GST per ml of settled resin) is such that
GST-CT monomers may be 8 to 9 nm apart from each other on
average, assuming that the entire volume of each bead can bind
GST-CT ligand. This GST-CT packing density permits at least two
possibilities. One possibility is that some percentage of CTs
assem-ble into trimer structures, and these trimers bind to MA trimers in
a 1:1 stoichiometry. An alternative is that the spokes of MA trimers
(which are 6 to 7 nm apart) bind independently to separate CTs,
and MA-CT binding avidity depends on relatively weak MA
spoke-CT interactions and MA trimerization. In this case, we
en-vision that MA trimers may align beneath Env trimers at
mem-branes, as shown in
Fig. 9
, and that separate spokes of an MA
FIG 8Analysis of 12LE and 12LE/62QR MA proteins. The 12LE and 12LE/ 62QR proteins were analyzed for trimerization (top) as described in the legend toFig. 2and for Env CT binding as described in the legend toFig. 6. In the top left, 50M concentrations of the indicated proteins were UV cross-linked for 0, 1, 3, or 10 min (left to right) and detected as monomers (1), dimers (2), or trimers (3) after electrophoresis and immunoblotting. At the top right, the percentages of trimers relative to monomers derive from eight independent experiments. The bottom left shows immunoblots of bound and total un-bound 12LE and 12LE/62QR proteins from 1.5M MA incubations with GST-CT (CT⫹) or GST (CT⫺) beads. At the bottom right, percentages of bound versus total protein were derived from four independent experiments.
FIG 7MAGFP binding to the HIV-1 Env CT. WT or 62QR MAGFP proteins (30M) were incubated with beads coated with glutathioneS-transferase (GST) or GST-CT, after which beads were washed and imaged for fluorescence detection. Examples of WT and 62QR protein binding to GST-CT and GST beads are shown on the left. On the right, MAGFP binding levels were quan-tified by measuring fluorescence brightness values from 8 to 10 beads of each class. Values were normalized to the bead brightness average of WT MAGFP bound to GST-CT beads.
FIG 9Overlay of Env and MA trimers. The HIV-1 Env SU plus TM (gp120⫹gp41; left) and observed TM residues (right) from PDB entry3J5M
are overlaid onto MA trimer structures (PDB entry1HIW) as backbone and side-chain traces or space-filling models. The CT structures are not known and not depicted but are envisioned in this overlay to bind to separate members of the MA. In such a model, CT tails of a single Env trimer bind to separate monomers of the MA trimer at separate hexamer holes of the MA lattices depicted inFig. 1.
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[image:6.585.336.507.65.215.2] [image:6.585.42.286.66.157.2] [image:6.585.362.483.569.645.2]trimer may bind to the separate CTs of Env trimers at three
dif-ferent hexameric lattice cage holes (
Fig. 1
).
It is worthwhile to note that the modes of MA-CT binding
proposed above do not argue against the roles of cellular factors in
fostering Env protein transport to assembly sites or in facilitating
virion incorporation (
32
,
33
). The trimer binding models also do
not contradict the possibility that hexamer hole sizes in MA
lat-tices contribute to the regulation of Env assembly into virus
par-ticles (
22–25
). Although we did not observe significant differences
between membrane-bound WT and 62QR MA lattices (
Fig. 3
),
the resolution of our reconstructions was such that potentially
important distinctions might be masked. In this regard, whereas
MA lattices have not as yet been observed in EM reconstructions
of HIV-1 virions (
37–41
), observations concerning viral
pseu-dotyping, Env fluidity in virus particles, and CT cleavage support
models that infer close interactions between Env CT tails and MA
lattices (
22–25
,
52
,
53
,
55
,
56
). We believe that further study of
62QR and other MA mutations will help elucidate the complex
interplay between the HIV-1 envelope and matrix proteins.
ACKNOWLEDGMENTS
We are grateful to Rachel Sloan and Claudia Lopez for assistance and advice and to the NIH for support through grants R01 GM060170 and R01 GM101983.
FUNDING INFORMATION
This work, including the efforts of Eric Barklis, was funded by HHS | NIH | National Institute of General Medical Sciences (NIGMS) (R01 GM060170 and R01 GM101983).
REFERENCES
1. Chukkapalli V, Ono A. 2011. Molecular determinants that regulate plasma membrane association of HIV-1 Gag. J Mol Biol410:512–524.
http://dx.doi.org/10.1016/j.jmb.2011.04.015.
2.Holm K, Weclewicz K, Hewson R, Suomalainen M.2003. HIV-1 assem-bly and lipid rafts: Pr55Gag complexes associate with membrane-domains that are largely resistant to Brij 98, but sensitive to Triton X-100. J Virol
77:4805– 4817.http://dx.doi.org/10.1128/JVI.77.8.4805-4817.2003. 3.Lindwasser O, Resh M.2001. Multimerization of human
immunodefi-ciency virus type 1 Gag promotes its localization to barges, raft-like mem-brane microdomains. J Virol 75:7913–7924.http://dx.doi.org/10.1128 /JVI.75.17.7913-7924.2001.
4.Nguyen D, Hildreth J.2000. Evidence for budding of human immuno-deficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts. J Virol74:3264 –3272.http://dx.doi.org/10.1128/JVI.74.7.3264 -3272.2000.
5.Graham D, Chertova E, Hilburn J, Arthur L, Hildreth J.2003. Choles-terol depletion of human immunodeficiency virus type 1 and simian im-munodeficiency virus with b-cyclodextrin inactivates and permeabilizes the virions: evidence for virion-associated lipid rafts. J Virol77:8237– 8248.http://dx.doi.org/10.1128/JVI.77.15.8237-8248.2003.
6.Brugger B, Glass B, Haberkant P, Leibrecht I, Wieland F, Krausslich H-G.2006. The HIV lipidome: a raft with an unusual composition. Proc Natl Acad Sci U S A103:2641–2646.http://dx.doi.org/10.1073/pnas .0511136103.
7.Ono A, Freed EO.2001. Plasma membrane rafts play a critical role in HIV-1 assembly and release. Proc Natl Acad Sci U S A98:13925–13030.
http://dx.doi.org/10.1073/pnas.241320298.
8.Ono A, Ablan S, Lockett S, Nagashima K, Freed EO.2004. Phosphati-dylinositol (4,5) bisphosphate regulates HIV-1 Gag targeting to the plasma membrane. Proc Natl Acad Sci U S A101:14889 –14894.http://dx .doi.org/10.1073/pnas.0405596101.
9.Alfadhli A, Still A, Barklis E.2009. Analysis of human immunodeficiency virus type 1 matrix binding to membranes and nucleic acids. J Virol83:
12196 –12203.http://dx.doi.org/10.1128/JVI.01197-09.
10. Chukkapalli V, Oh S, Ono A.2010. Opposing mechanisms involving RNA and lipids regulate HIV-1 Gag membrane binding through the
highly basic region of the matrix domain. Proc Natl Acad Sci U S A107:
1600 –1605.http://dx.doi.org/10.1073/pnas.0908661107.
11. Alfadhli A, McNett H, Tsagli S, Bachinger HP, Peyton D, Barklis E.
2011. HIV-1 matrix binding to RNA. J Mol Biol410:653– 666.http://dx .doi.org/10.1016/j.jmb.2011.04.063.
12. Jones C, Datta S, Rein A, Rouzina I, Musier-Forsyth K.2011. Matrix domain modulates HIV-1 Gag’s nucleic acid chaperone activity via inosi-tol phosphate binding. J Virol85:1594 –1603.http://dx.doi.org/10.1128 /JVI.01809-10.
13. Alfadhli A, McNett H, Eccles J, Tsagli S, Noviello C, Sloan R, Lopez C, Peyton D, Barklis E.2013. Analysis of small molecule ligands targeting the HIV-1 matrix protein-RNA binding site. J Biol Chem288:666 – 676.
http://dx.doi.org/10.1074/jbc.M112.399865.
14. Chukkapalli V, Inlora J, Todd G, Ono A.2013. Evidence in support of RNA-mediated inhibition of phosphatidylserine-dependent HIV-1 Gag membrane binding in cells. J Virol87:7155–7159.http://dx.doi.org/10 .1128/JVI.00075-13.
15. Inlora J, Collins D, Trubin M, Chung J, Ono A. 2014. Membrane binding and subcellular localization of retroviral Gag proteins are differ-entially regulated by MA interactions with phosphatidylinositol-(4,5)-bisphosphate and RNA. mBio5:e02202.
16. Kutluay S, Zang T, Blanco-Melo D, Powell C, Jannain D, Errando M, Bieniasz P.2014. Global changes in the RNA binding specificity of HIV-1 Gag regulate virion genesis. Cell 159:1096 –1109. http://dx.doi.org/10 .1016/j.cell.2014.09.057.
17. Wang C, Zhang Y, McDermott J, Barklis E.1993. Conditional infectivity of a human immunodeficiency virus matrix domain deletion mutant. J Virol67:7067–7076.
18. Reil H, Bukovsky A, Gelderblom H, Gottlinger H.1998. Efficient HIV-1 replication can occur in the absence of the viral matrix protein. EMBO J
17:2699 –2708.http://dx.doi.org/10.1093/emboj/17.9.2699.
19. Jouvenet N, Neil S, Bess C, Johnson M, Virgen C, Simon S, Bieniasz P.
2006. Plasma membrane is the site of productive HIV-1 particle assembly. PLoS Biol4(12):3435.
20. Scholz I, Still A, Dhenub T, Coday K, Webb M, Barklis E.2008. Analysis of human immunodeficiency virus matrix domain replacements. Virol-ogy371:322–335.http://dx.doi.org/10.1016/j.virol.2007.10.010. 21. Urano E, Aoki T, Futahashi Y, Murakami T, Morikawam Y, Yamamoto
N, Komano J.2008. Substitution of the myristoylation signal of human immunodeficiency virus type 1 Pr55Gag with the phospholipase C-␦1 pleckstrin homology domain results in infectious pseudovirion produc-tion. J Gen Virol89:3144 –3149.http://dx.doi.org/10.1099/vir.0.2008 /004820-0.
22. Tedbury PR, Ablan SD, Freed EO.2013. Global rescue of defects in HIV-1 envelope glycoprotein incorporation: implications for matrix structure. PLoS Pathog 9(11):e1003739.http://dx.doi.org/10.1371 /journal.ppat.1003739.
23. Tedbury PR, Mercredi PY, Gaines CR, Summers MF, Freed EO.2015. Elucidating the mechanism by which compensatory mutations rescue an HIV-1 matrix mutant defective for gag membrane targeting and envelope glycoprotein incorporation. J Mol Biol427(Part B):1413–1427. 24. Tedbury PR, Freed EO.2014. The role of matrix in HIV-1 envelope
glycoprotein incorporation. Trends Microbiol22:372–378.http://dx.doi .org/10.1016/j.tim.2014.04.012.
25. Tedbury PR, Novikova M, Ablan S, Freed EO.2016. Biochemical evi-dence of a role for matrix trimerization in HIV-1 envelope glycoprotein incorporation. Proc Natl Acad Sci U S A113:E182–E190.http://dx.doi.org /10.1073/pnas.1516618113.
26. Ott D.2002. Potential roles of cellular proteins in HIV-1. Rev Med Virol
12:359 –374.http://dx.doi.org/10.1002/rmv.367.
27. Burns J, Friedmann T, Driever W, Burrascano M, Yee J-K. 1993. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mamma-lian and nonmammamamma-lian cells. Proc Natl Acad Sci U S A90:8033– 8037.
http://dx.doi.org/10.1073/pnas.90.17.8033.
28. Sanders D.2002. No false start for novel pseudotyped vectors. Curr Opin Biotechnol13:437– 442.http://dx.doi.org/10.1016/S0958-1669(02)00374-9. 29. Jorgenson R, Vogt V, Johnson M.2009. Foreign glycoproteins can be
actively recruited to virus assembly sites during pseudotyping. J Virol83:
4060 – 4067.http://dx.doi.org/10.1128/JVI.02425-08.
30. Johnson M.2011. Mechanisms for Env Glycoprotein Acquisition by Ret-roviruses. AIDS Res Hum Retrovir27:239.http://dx.doi.org/10.1089/aid .2010.0350.
on November 7, 2019 by guest
http://jvi.asm.org/
31. Ritchie C, Cylinder I, Platt E, Barklis E.2015. Analysis of HIV-1 Gag protein interactions via biotin ligase tagging. J Virol89:3988 – 4001.http: //dx.doi.org/10.1128/JVI.03584-14.
32. Murakami T, Freed EO.2000. The long cytoplasmic tail of gp41 is re-quired in a cell type-dependent manner for HIV-1 envelope glycoprotein incorporation into virions. Proc Natl Acad Sci U S A97:343–348.http://dx .doi.org/10.1073/pnas.97.1.343.
33. Qi M, Chu H, Chen X, Choi J, Wen X, Hammonds J, Ding L, Hunter E, Spearman P.2015. A tyrosine-based motif in the HIV-1 envelope glycoprotein tail mediates cell-type- and Rab11-FIP1C-dependent incor-poration into virions. Proc Natl Acad Sci U S A112:7575–7580.http://dx .doi.org/10.1073/pnas.1504174112.
34. Lyumkis D, Julien JP, de Val N, Cupo A, Potter CS, Klasse PJ, Burton DR, Sanders RW, Moore JP, Carragher B, Wilson IA, Ward AB.2013. Cryo-EM structure of a fully glycosylated soluble cleaved HIV-1 envelope trimer. Science342:1484 –1490.http://dx.doi.org/10 .1126/science.1245627.
35. Hill C, Worthylake D, Bancroft D, Christensen A, Sundquist W.1996. Crystal structures of the trimeric human immunodeficiency virus type 1 matrix protein: implications for membrane association and assembly. Proc Natl Acad Sci U S A93:3099 –3104.http://dx.doi.org/10.1073/pnas .93.7.3099.
36. Morikawa Y, Zhang W, Hockley D, Nermut M, Jones I.1998. Detection of a trimeric human immunodeficiency virus type 1 Gag intermediate is dependent on sequences in the matrix domain, p17. J Virol72:7659 –7663. 37. Freed EO, Martin M.1995. Virion incorporation of envelope glycopro-teins with long but not short cytoplasmic tails is blocked by specific, single amino acid substitutions in the human immunodeficiency virus type 1 matrix. J Virol69:1984 –1989.
38. Alfadhli A, Huseby D, Kapit E, Colman D, Barklis E.2007. The HIV-1 matrix protein assembles on membranes as a hexamer. J Virol81:1472– 1478.http://dx.doi.org/10.1128/JVI.02122-06.
39. Alfadhli A, Barklis R, Barklis E. 2009. HIV-1 matrix organizes as a hexamer of trimers on membranes containing phosphatidylinositol-(4,5)P2. Virology387:466 – 472.http://dx.doi.org/10.1016/j.virol.2009.02 .048.
40. Fuller SD, Wilk T, Gowen B, Krausslich H, Vogt V.1997. Cryo-electron microscopy reveals ordered domains in the immature HIV-1 particle. Curr Biol7:729 –738.http://dx.doi.org/10.1016/S0960-9822(06)00331-9. 41. Wilk T, Gross I, Gowen B, Rutten T, de Haas F, Welker R, Krausslich HG, Boulanger P, Fuller SD.2001. Organization of immature human immunodeficiency virus type 1. J Virol75:759 –771.http://dx.doi.org/10 .1128/JVI.75.2.759-771.2001.
42. Wright E, Schooler J, Ding H, Kieffer C, Fillmore C, Sundquist W, Jensen G.2007. Electron cryotomography of immature HIV-1 virions reveals the structure of the CA and SP1 Gag shells. EMBO J26:2218 –2226.
http://dx.doi.org/10.1038/sj.emboj.7601664.
43. Bharat T, Castillo Menendez L, Hagen W, Lux V, Igonet S, Schorb M, Schur F, Kräusslich HG, Briggs JA.2014. Cryo-electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature
virus assembly. Proc Natl Acad Sci U S A111:8233– 8238.http://dx.doi .org/10.1073/pnas.1401455111.
44. Schur F, Hagen W, Rumlová M, Ruml T, Müller B, Kräusslich HG, Briggs JA.2015. Structure of the immature HIV-1 capsid in intact virus particles at 8.8 Å resolution. Nature517:505–508.
45. Adachi A, Gendelman H, Koenig S, Folks T, Willey R, Rabson A, Martin M.1986. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol59:284 –291.
46. Schneider C, Rasband W, Eliceiri K.2012. NIH Image to ImageJ: 25 years of image analysis. Nat Methods9:671– 675.http://dx.doi.org/10.1038 /nmeth.2089.
47. Guptasarma P, Balasubramanian D, Matsugo S, Saito I.1992. Hydroxyl radical mediated damage to proteins, with special reference to the crystallins. Biochemistry31:4296 – 4303.http://dx.doi.org/10.1021/bi00132a021. 48. Davies M.2003. Singlet oxygen-mediated damage to proteins and its
consequences. Biochem Biophys Res Commun305:761–770.http://dx .doi.org/10.1016/S0006-291X(03)00817-9.
49. Leo G, Altucci C, Bourgoin-Voillard S, Gravagnuolo A, Esposito R, Marino G, Costello C, Velotta R, Birolo L. 2013. UV laser-induced cross-linking of proteins. Rapid Commun Mass Spectrom27(14).http: //dx.doi.org/10.1002/rcm.6610.
50. Gipson B, Zheng X, Zhang ZY, Stahlberg H.2007. 2dx– user-friendly image processing for 2D crystals. J Struct Biol157:64 –72.http://dx.doi .org/10.1016/j.jsb.2006.07.020.
51. Purohit P, Dupont S, Stevenson M, Green M.2001. Sequence-specific interaction between HIV-1 matrix protein and viral genomic RNA re-vealed by in vitro selection. RNA7:576 –584.http://dx.doi.org/10.1017 /S1355838201002023.
52. Cosson P.1996. Direct interaction between the envelope and matrix pro-teins of HIV-1. EMBO J15:5783–5788.
53. Hourioux C, Brand D, Sizaret P, Lemiale F, Lebigot S, Barin F, Ro-ingeard P.2000. Identification of the glycoprotein 41 (TM) cytoplasmic tail domains of HIV-1 that interact with Pr55Gag particles. AIDS Res Hum Retrovir16:1141–1147.http://dx.doi.org/10.1089/088922200414983. 54. McTigue M, Williams D, Tainer J.1995. Crystal structures of
schisto-somal drug and vaccine target: glutathione S-transferase fromSchistosoma japonicaand its complex with the leading antischistosomal drug praziqu-antel. J Mol Biol246:21–27.http://dx.doi.org/10.1006/jmbi.1994.0061. 55. Chojnacki J, Staudt T, Glass B, Bingen P, Engelhardt J, Anders M,
Schneider J, Muller B, Hell S, Krausslich H-G. 2012. Maturation-dependent HIV-1 surface protein redistribution revealed by fluorescence nanoscopy. Science338:524 –528.http://dx.doi.org/10.1126/science .1226359.
56. Muranyi W, Malkusch S, Müller B, Heilemann M, Kräusslich H-G.
2013. Super-resolution microscopy reveals specific recruitment of HIV-1 envelope proteins to viral assembly sites dependent on the envelope C-ter-minal tail. PLoS Pathog9(2):e1003198.http://dx.doi.org/10.1371/journal .ppat.1003198.
on November 7, 2019 by guest
http://jvi.asm.org/
