JOURNAL OF VIROLOGY, Sept. 2011, p. 8725–8737
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 17
Contribution of E3-Ubiquitin Ligase Activity to HIV-1 Restriction by
TRIM5?rh: Structure of the RING Domain of TRIM5??†
Maritza Lienlaf,1Fumiaki Hayashi,2Francesca Di Nunzio,5Naoya Tochio,2Takanori Kigawa,2,3
Shigeyuki Yokoyama,2,4and Felipe Diaz-Griffero1*
Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 104611; Systems and
Structural Biology Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan2;
Department of Computational Intelligence and Systems Science, Interdisciplinary Graduate School of Science and
Engineering, Tokyo Institute of Technology, 2-12-1 Ookayama Meguro-ku, Tokyo 152-8550, Japan3;
UT-RIKEN Cooperation Laboratory of Structural Biology, Graduate School of Science, University of
Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan4; and Institut Pasteur, Laboratoire de
Virologie Mole ´culaire et de Vaccinologie, 28 rue du Docteur Roux, 75015 Paris, France5
Received 10 March 2011/Accepted 23 June 2011
TRIM5?rhis a cytosolic protein that potently restricts HIV-1 before reverse transcription. TRIM5?rhis
composed of four different domains: RING, B-box 2, coiled coil, and B30.2(SPRY). The contribution of each of
these domains to restriction has been extensively studied, with the exception of the RING domain. The RING
domain of TRIM5? exhibits E3-ubiquitin ligase activity, but the contribution of this activity to the restriction
of HIV-1 is not known. To test the hypothesis that the E3-ubiquitin ligase activity of the RING domain
modulates TRIM5?rhrestriction of HIV-1, we correlated the E3-ubiquitin ligase activity of a panel of
TRIM5?rhRING domain variants with the ability of these mutant proteins to restrict HIV-1. For this purpose,
we first solved the nuclear magnetic resonance structure of the RING domain of TRIM5? and defined potential
functional regions of the RING domain by homology to other RING domains. With this structural information,
we performed a systematic mutagenesis of the RING domain regions and tested the TRIM5? RING domain
variants for the ability to undergo self-ubiquitylation. Several residues, particularly the ones on the E2-binding
region of the RING domain, were defective in their self-ubiquitylation ability. To correlate HIV-1 restriction to
self-ubiquitylation, we used RING domain mutant proteins that were defective in self-ubiquitylation but preserve
important properties required for potent restriction by TRIM5?rh, such as capsid binding and higher-order
self-association. From these investigations, we found a set of residues that when mutated results in TRIM5?
molecules that lost both the ability to potently restrict HIV-1 and their self-ubiquitylation activity. Remarkably, all
of these changes were in residues located in the E2-binding region of the RING domain. Overall, these results
demonstrate a role for TRIM5? self-ubiquitylation in the ability of TRIM5? to restrict HIV-1.
Several newly discovered proteins that are endogenously
expressed in primates show the ability to dominantly block
retroviral infection and cross-species transmission by interfer-
ing with the early phase of viral replication (3, 33, 57, 63). Of
particular interest are members of the tripartite motif (TRIM)
family of proteins. Splicing variant alpha of TRIM5 from rhe-
sus macaques (TRIM5?rh) is an ?53-kDa cytosolic protein
that potently restricts HIV-1 (28, 61). TRIM5?rhblocks HIV-1
and certain other retroviruses soon after viral entry but prior to
reverse transcription (28, 63). The retroviral capsid protein is
the viral determinant for susceptibility to restriction by
TRIM5? (48). Studies on the fate of the HIV-1 capsid in the
cytosol of infected cells have correlated restriction with a de-
crease amount of cytosolic particulate capsid (11, 51, 64).
TRIM5?rhis composed of four different domains: RING,
B-box 2, coiled coil, and B30.2(SPRY) (53). The RING do-
main of TRIM5?rhis an E3-ubiquitin ligase (13, 27, 37, 43, 68);
however, a role for the really interesting new gene (RING)
domain’s E3-ubiquitin ligase activity in HIV-1 restriction by
TRIM5?rhhas not been established. The B-box 2 domain of
TRIM5? and other TRIM proteins, such as TRIM63, self-
associates, forming dimeric complexes that are important for
TRIM5? higher-order self-association and capsid binding
avidity; these B-box 2 domain functions are essential for full
and potent restriction of HIV-1 (12, 15, 19, 25, 44, 49). The
coiled-coil domain enables TRIM5?rhdimerization (27, 37),
which is critical for the interaction of the B30.2(SPRY) domain
with the HIV-1 capsid (58, 64). The B30.2(SPRY) domain,
which provides the capsid recognition motif, dictates the spec-
ificity of restriction (45, 56, 62, 65, 70).
The specific interaction of substrates with other TRIM pro-
teins, such as TRIM8 and TRIM11, results in RING domain-
dependent ubiquitylation and proteasomal degradation of the
target protein (24, 46, 66). The RING domain, originally
termed the A-box domain, is involved in protein-protein inter-
actions (17). This domain binds two Zn2?atoms tetrahedrally
in a cross-brace conformation (5, 21). RING domains in other
proteins play the role of molecular scaffolds, allowing the for-
mation of supramolecular complexes by self-association of the
RING domain, which in some cases improves E3-ubiquitin ligase
activity (4, 29, 30, 36, 52). Interestingly, RING-RING interactions
* Corresponding author. Mailing address: Albert Einstein College
of Medicine, 1301 Morris Park-Price Center 501, New York, NY
10461. Phone: (718) 678-1191. Fax: (718) 632-4338. E-mail: Felipe
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 6 July 2011.
have been reported to be functionally important for genomic
stability, as in the heterodimer formed by the RING domain of
BRCA1 with the RING domain of BARD-1 (6).
TRIM5?rhexhibits an intrinsic rapid turnover of 50 to 60
min that is dependent on an intact RING domain, but this
property is apparently not important for restriction (11, 13,
68). However, TRIM5?rhdegrades at a higher rate than its
normal turnover when in the presence of the HIV-1 capsid
(54). Interestingly, this capsid-dependent degradation is inhib-
ited by the use of proteasome inhibitors. Disruption of protea-
some function alters TRIM5?rhlocalization and allows the
completion of HIV-1 late reverse transcription during infec-
tion (1, 13, 67); however, inhibitors of the proteasome do not
alleviate restriction (1, 50, 54, 64, 67). Altogether these results
suggest a role for the RING domain and proteasome in re-
We present a nuclear magnetic resonance (NMR) structure
of the TRIM5 RING domain. Alteration of the different func-
tional regions of the RING domain revealed structures impor-
tant for TRIM5 self-ubiquitylation, HIV-1 restriction, higher-
order self-association, and capsid binding. To understand
which RING functions contribute to retroviral restriction, the
relationship among these TRIM5 properties was investigated.
We found that alteration of the RING domain self-ubiquity-
lation activity correlated with a loss of restriction potency.
These results suggested a contribution of the RING domain
self-ubiquitylation activity to the restriction of HIV-1 by
MATERIALS AND METHODS
Sample preparation of the TRIM5 RING domain. The DNA fragment encod-
ing the RING domain of TRIM5?hu(amino acid residues 1 to 78, Swiss-Prot
accession no. Q9C035) was amplified via PCR from Invitrogen Japan K. K. clone
IOH 14670 and cloned into the plasmid vector pCR2.1 (Invitrogen, Carlsbad,
CA) as a fusion with an N-terminal His tag and a tobacco etch virus protease
cleavage site. The13C- and15N-labeled protein was synthesized by a cell-free
protein expression system (32). Purification was performed by a standard pro-
cedure (39). For structure determination, a single 1.25 mM uniformly13C- and
15N-labeled sample was prepared in a mixture of 20 mM Tris-HCl buffer at pH
7.0, 100 mM NaCl, 1 mM dithiothreitol (DTT), 0.02% NaN3, 0.05 mM ZnCl2,
and 1 mM iminodiacetic acid, with the addition of D2O to 10% (vol/vol). The
engineered protein sample used for the NMR measurements includes seven
additional residues (GSSGSSG) as a tag linker.
NMR spectroscopy, structure determination, and analysis. All of the NMR
spectra for structure determination were recorded at 23°C on Bruker AVANCE
600 and 800 spectrometers equipped with a pulse-field gradient triple-resonance
probe. Sequence-specific resonance assignments were made using the standard
triple-resonance techniques. The backbone assignment was achieved by the com-
bined analysis of HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB, and
CBCA(CO)NH spectra. The side chain resonances were identified by the com-
bined use of HBHA(CO)NH, (H)CC(CO)NH, HCCH correlation spectroscopy,
HCCH total correlation spectroscopy (TOCSY), (H)CCH TOCSY, and two-
dimensional1H and15N heteronuclear single quantum coherence (HSQC) and
1H-13C-HSQC spectra. Nuclear Overhauser effect (NOE) data for structure
determination were extracted from three-dimensional15N- and13C-edited NOE
spectra recorded with a mixing time of 150 ms. The stereospecific assignments for
prochiral b-methylene protons were carried out with HN(CO)HB and HNHB.
The1H-15N-HSQC spectra for concentration-dependent experiments were re-
corded at 25°C on a Varian INOVA 800 spectrometer equipped with a pulse-
field gradient triple-resonance probe. The NMRpipe software package (8) and
the program KUJIRA (34), created on the basis of NMRView (26), was em-
ployed for optimal visualization and spectral analysis. Automated NOE cross-
peak assignments (22) and structure calculations with torsion angle dynamics
were performed using the software package CYANA 2.0.17. Dihedral angle
restrains were derived using the program TALOS (7). A total of 100 conformers
were calculated independently. The 20 conformers with the lowest final CYANA
target function values were finally selected. The structures were validated using
PROCHECK-NMR (38). The program MOLMOL (35) was used to analyze the
resulting 20 conformers and to prepare drawings of the structures, unless noted
otherwise in the figure legends. The 20 selected conformers have been deposited
in the Protein Data Bank (PDB; entry 2ECV).
Creation of cells stably expressing TRIM5? variants. Retroviral vectors en-
coding wild-type or mutant rhesus monkey TRIM5?rhproteins were created
using the pLPCX vector. The TRIM5?rhproteins contained an influenza hem-
agglutinin (HA) epitope tag at the C terminus or a FLAG epitope tag at the N
terminus. Recombinant viruses were produced in 293T cells by cotransfecting the
pLPCX plasmids with the pVPack-GP and pVPack-VSV-G packaging plasmids
(Stratagene). The pVPack-VSV-G plasmid encodes the vesicular stomatitis virus
(VSV) G envelope glycoprotein, which allows efficient entry into a wide range of
vertebrate cells. Cf2Th canine thymocytes were transduced and selected in 5
?g/ml puromycin (Sigma).
Infection with viruses expressing green fluorescent protein (GFP). Recombi-
nant HIV-1 and equine infectious anemia virus (EIAV) expressing GFP were
prepared as described previously (14). All recombinant viruses were pseudotyped
with the VSV G glycoprotein. For infections, 3 ? 104Cf2Th cells seeded in
24-well plates were incubated at 37°C with virus for 24 h. Cells were washed and
returned to culture for 48 h and then subjected to fluorescence-activated cell
sorter (FACS) analysis with a FACScan (BD). HIV-1 and EIAV stocks were
titrated by serial dilution on Cf2Th cells to determine the concentration of
Protein analysis. Cellular proteins were extracted with radioimmunoprecipi-
tation assay buffer (10 mM Tris [pH 7.4], 100 mM NaCl, 1% sodium deoxy-
cholate, 0.1% sodium dodecyl sulfate [SDS], 1% NP-40, 2 mg/ml aprotinin, 2
mg/ml leupeptin, 1 mg/ml pepstatin A, 100 mg/ml phenylmethylsulfonyl fluo-
ride). The cell lysates were analyzed by SDS-PAGE (10% acrylamide), followed
by blotting onto nitrocellulose membranes (Amersham Pharmacia Biotech).
Detection of protein by Western blotting utilized monoclonal antibodies directed
against the HA epitope tags (Roche) and FLAG epitope tags (Sigma) and
monoclonal antibodies to ?-actin (Sigma) directly conjugated to horseradish
peroxidase (HRP). Proteins were detected by enhanced chemiluminescence
(NEN Life Science Products) using the FluorChem FC2 detection system (Alpha
Innotech). Signals were acquired as an image (TIFF) file and quantified by the
Quantity One software (Bio-Rad Laboratories).
TRIM5? self-ubiquitylation. Human 293T cells were transfected with plas-
mids encoding FLAG-tagged mutant and wild-type TRIM5?rhproteins. Forty-
eight hours later, the cells expressing each TRIM5?rhvariant were lysed in 1 ml
of whole-cell extract buffer (50 mM Tris [pH 8.0], 280 mM NaCl, 0.5% octyl-
phenoxypolyethoxyethanol [IGEPAL]–10% glycerol, 1 mM DTT, protease in-
hibitor cocktail [Roche]). Lysates were centrifuged at 14,000 rpm for 1 h at 4°C.
Postspin lysates were then precleared using protein A-agarose (Sigma) for 1 h at
4°C. Precleared lysates were incubated with anti-FLAG-agarose beads (Sigma)
for 2 h at 4°C to precipitate the FLAG-tagged proteins. Beads containing the
immunoprecipitate were washed four times in whole-cell extract buffer. Subse-
quently, immune complexes were eluted using 200 ?g/ml FLAG tripeptide in
whole-cell extract buffer. The eluted samples were separated by SDS-PAGE and
analyzed by Western blotting using anti-FLAG antibodies conjugated to HRP.
Subsequently, similar amounts of mutant and wild-type TRIM5? were supple-
mented with 5 ?M ubiquitin aldehyde, a potent inhibitor of all ubiquitin C-ter-
minal hydrolases, ubiquitin-specific proteases, and deubiquitylating enzymes
(BostonBiochem). The inhibitor-treated fractions containing mutant and wild-
type TRIM5?rhwere incubated in a final reaction mixture containing 200 nM E1
(human recombinant UBE1; BostonBiochem), 100 nM E2 (human recombinant
UbcH5b; BostonBiochem), 200 ?M ubiquitin tagged with a myc epitope (human
recombinant ubiquitin), and ATP (energy regeneration solution containing
MgCl2, ATP, and ATP-regenerating enzymes to recycle hydrolyzed ATP;
BostonBiochem). The reaction mixture was incubated at 37°C for 1 h, and
collected fractions were analyzed by Western blotting using HRP-conjugated
antibodies against FLAG and myc. Similar reactions were performed in the
absence of recombinant E1 and E2 enzymes to determine the contribution of
endogenous E1 and E2 enzymes to TRIM5?rhubiquitylation.
Higher-order self-association of TRIM5?. Human 293T cells were indepen-
dently transfected with plasmids encoding FLAG-tagged and HA-tagged mutant
or wild-type TRIM5?rhproteins. Forty-eight hours later, the cells expressing
each TRIM5?rhvariant were lysed in 1 ml of whole-cell extract buffer (50 mM
Tris [pH 8.0], 280 mM NaCl, 0.5% octylphenoxypolyethoxyethanol–10% glyc-
erol, 1 mM DTT, protease inhibitor cocktail [Roche]). Lysates were centrifuged
at 14,000 rpm for 1 h at 4°C. Postspin lysates were then precleared using protein
A-agarose (Sigma) for 1 h at 4°C; a small aliquot of each of these lysates was
stored as an input sample. Precleared lysates containing the differently tagged
8726 LIENLAF ET AL.J. VIROL.
proteins were mixed at a 1:1 ratio and incubated with anti-FLAG–agarose beads
(Sigma) for 2 h at 4°C to precipitate the FLAG-tagged proteins. Beads contain-
ing the immunoprecipitate were washed four times in whole-cell extract buffer.
Subsequently, immune complexes were eluted using 200 ?g/ml FLAG tripeptide
in whole-cell extract buffer. The eluted samples were separated by SDS-PAGE
and analyzed by Western blotting using anti-HA or anti-FLAG antibodies con-
jugated to HRP.
HIV-1 capsid-nucleocapsid (CA-NC) expression and purification. The HIV-1
CA-NC protein was expressed, purified, and assembled as previously described
(18, 20). The pET11a expression vector (Novagen) expressing the CA-NC pro-
tein of HIV-1 was used to transform Escherichia coli BL-21(DE3). CA-NC
expression was induced with 1 mM isopropyl-?-D-thiogalactopyranoside (IPTG)
when the culture reached an optical density at 600 nm of 0.6. After 4 h of
induction, the cells were harvested and resuspended in a mixture of 20 mM
Tris-HCl (pH 7.5), 1 ?M ZnCl2, 10 mM 2-mercaptoethanol, and protease in-
hibitors (Roche). Lysis was performed by sonication, and debris was pelleted for
30 min at 35,000 ? g. Nucleic acids were stripped from the solution by using 0.11
equivalent of 2 M (NH4)2SO4and the same volume of 10% polyethylenimine.
Nucleic acids were removed by stirring and centrifugation at 29,500 ? g for 15
min. The protein was recovered by addition of 0.35 equivalent of saturated
(NH4)2SO4. The protein was centrifuged at 9,820 ? g for 15 min and resus-
pended in a mixture of 100 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 ?M ZnCl2,
and 10 mM 2-mercaptoethanol. The CA-NC protein was dialyzed against a
mixture of 50 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 ?M ZnCl2, and 10 mM
2-mercaptoethanol and stored at ?80°C.
In vitro assembly of CA-NC complexes. HIV-1 CA-NC particles were assem-
bled in vitro by diluting the CA-NC protein to a concentration of 0.3 mM in a
mixture of 50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, and 2 mg/ml DNA oligo-
(TG)50. The mixture was incubated at 4°C overnight and centrifuged at 8,600 ?
g for 5 min. The pellet was resuspended in assembly buffer (50 mM Tris-HCl [pH
8.0], 0.5 M NaCl) at a final protein concentration of 0.15 mM (18, 20) and stored
at 4°C until needed.
Binding of TRIM5?rhvariants to HIV-1 capsid complexes. 293T cells were
transfected with plasmids expressing wild-type or mutant TRIM5?rhproteins.
Forty-eight hours after transfection, cell lysates were prepared as follows. Pre-
viously washed cells were resuspended in hypotonic lysis buffer (10 mM Tris [pH
7.4], 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT). The cell suspension was frozen,
thawed, and incubated on ice for 10 min. Afterwards, the lysate was centrifuged
at full speed in a refrigerated Eppendorf microcentrifuge (?14,000 ? g) for 5
min. The supernatant was supplemented with 1/10 volume of 10? phosphate-
buffered saline (PBS) and then used in the binding assay. In some cases, samples
containing the TRIM5?rhvariants were diluted with extracts prepared in parallel
from untransfected cells. To test binding, 5 ?l of CA-NC particles assembled in
vitro was incubated with 200 ?l of cell lysate at room temperature for 1 h. A
fraction of this mixture was stored (input). The mixture was spun through a 70%
sucrose cushion (70% sucrose, 1? PBS, 0.5 mM DTT) at 100,000 ? g in an SW55
rotor (Beckman) for 1 h at 4°C. After centrifugation, the supernatant was care-
fully removed and the pellet was resuspended in 1? SDS-PAGE loading buffer
(pellet). The level of TRIM5?rhproteins was determined by Western blotting
with an anti-HA antibody as described above. The level of HIV-1 CA-NC protein
in the pellet was assessed by Western blotting with an anti-p24 CA antibody.
Quantitative real-time PCR. Cf2Th cells expressing wild-type and mutant
TRIM5?rhproteins were challenged with HIV-1–GFP at a multiplicity of infec-
tion (MOI) of 0.2. Viruses were pretreated with DNase to prevent contamination
from carryover plasmid DNA. An infection using a virus that was heat inactivated
(60°C for 30 min) was performed as a control for carryover plasmid DNA in the
PCR. After 6 h, the cells were lysed and DNA was extracted using a Qiagen
Blood Tissue DNA extraction kit. PCRs were prepared using the QuantiTect
probe PCR kit. Each sample contained 100 ng of total cellular DNA. PCR was
carried out using two primers that amplify a 263-bp fragment of GFP (GFP-fwd,
5?-GAC GTA AAC GGC CAC AAG-3?; GFP-rev, 5?-GGT CTT GTA GTT
GCC GTC GT-3?; GFP-Probe, 5?-56-FAM-CCT ACG GCA AGC TGA CCC
TGA-36-TAMRA-3?). The calibration curve was prepared using an HIV-1–GFP
Protein structure accession number. The coordinates and structure factors for
the RING domain of human TRIM5? have been deposit in the PDB under
accession number 2ECV.
Solution structure of the TRIM5? RING domain. A frag-
ment of human TRIM5? that encompasses the RING domain
(residues 1 to 78) was expressed and labeled in a cell-free
system. The solution structure of the RING domain was solved
using multidimensional NMR spectroscopy (PDB entry
2ECV). The solution structures were well defined from resi-
dues 10 to 70 (Fig. 1). Residues 10 to 61 form a core structure
that interacts with the C-terminal region composed of residues
62 to 70; however, the interaction between the core and C-ter-
minal regions did not form a single conformation. The root
mean square deviations (RMSD) from the mean structure
were 0.70 ? 0.18 Å for backbone atoms and 1.05 ? 0.15 Å for
heavy atoms in the defined region of the domain. The struc-
tural statistics are summarized in Table 1. The RING domain
of TRIM5? adopts a ??? RING fold; however, it contains
shorter ?-strands and a longer ?-helix than typical RING do-
main folds (9). Most hydrophobic residues are packed in the
core region of the RING domain between the ?-hairpin and
?-helix. The hydrophobic core is partly exposed to solvent and
forms two hydrophobic patches (Fig. 1).
Mutational analysis of the TRIM5?rhRING surface. Sev-
eral studies suggest that RING domains exhibit two functional
surfaces (9). One surface is destined to interact with an E2
enzyme, as has been shown for a large number of RING
domains (9). The ability of the RING domain (E3) to interact
with E2 facilitates the transfer of ubiquitin from E2 to the
target substrate, which might be a protein interacting with the
E3-containing protein or the E3-containing protein itself (9).
In the case of the RING domain of TRIM5?rh, we named this
surface the E2-binding region (Fig. 2A). Opposite to the E2-
binding region, a second functional surface is destined to either
self-associate, as shown for the RING domain of RAG1 (2), or
associate with a related RING domain, as in the case of
BRCA1-BARD1 interaction (6). We named this surface the
RING-RING interaction region of the RING domain of
TRIM5?rhbecause of the structural similarity to the RING-
RING interaction region between BRCA-1 and BARD1 (Fig.
2B). We created an extensive panel of mutant proteins that
included both the E2-binding and RING-RING interaction
regions based on the NMR structure of the RING domain
(Table 2). Additionally, we mutagenized residues that were
located neither in the E2-binding region nor in the RING-
RING interaction region.
Assay of the E3-ubiquitin ligase activity of TRIM5?rhRING
domain variants. The RING domain of TRIM5?rhpresents
self-ubiquitylation activity and could use UbcH5b as an E2-
conjugating enzyme (13, 27, 37, 43, 68). Self-ubiquitylation
assays are widely used as a sensitive indicator of potential E3
activity of RING domain proteins, particularly in vitro, and
when the nature of the substrate is not established (69), as
in the case of TRIM5?rh. To measure the ability of the
TRIM5?rhRING domain variants to undergo RING domain-
dependent self-ubiquitylation, we established an in vitro assay
using purified, FLAG-tagged TRIM5?rhby immunoprecipita-
tion. Purified RING domain variants and wild-type TRIM5?rh
proteins were incubated with recombinant UBE-1 (E1), re-
combinant UbcH5b (E2), an energy regeneration system, and
myc-tagged ubiquitin. TRIM5?rhself-ubiquitylation is ob-
served only when E1 and E2 enzymes are added to the reaction
mixture (Fig. 3). The amount of ubiquitylated TRIM5? protein
was determined by subtracting the amount of nonubiquitylated
TRIM5? protein remaining in the reaction mixture that was
VOL. 85, 2011ROLE OF TRIM5?’s E3-LIGASE ACTIVITY IN RESTRICTION8727
incubated with E1 and E2 enzymes from the amount of
TRIM5? protein in the control reaction mixture, which was
not incubated with E1 and E2 enzymes. The value of nonubiq-
uitylated TRIM5 in the reaction mixtures was quantified by
using the Quantity-One software from Bio-Rad. TRIM5? self-
ubiquitylation was expressed as the percentage of the total
TRIM5? variant input protein (Table 2).
On the E2-binding region of the RING domain, we identi-
fied residues that when mutated led to a loss of self-ubiquity-
lation activity: I17, L19, E20, L21, A41/N42, S46, L48, Y49/
K50, S55, P57, V58, and R60 (Fig. 3; see Fig. S1 in the
supplemental material). Among all of the mutations that af-
fected self-ubiquitylation (Table 2), we found several residues
in the E2-binding region that when mutated dramatically af-
fected self-ubiquitylation, such as I17, L19, E20, L21, A41/N42,
L48, V58, and R60, as shown by the purple filled circles in Fig.
3. These results were in agreement with those of other studies
where mutation of the E2-binding region of the RING domain
affected the self-ubiquitylation or ubiquitylation of a specific
substrate (23, 42, 59, 71).
Several residues located in the RING-RING interaction re-
gion of the RING domain also affected the self-ubiquitylation
activity of TRIM5?rh, i.e., N67, I68, Q69, P70, N71, R72, and
V74 (Fig. 3; see Fig. S1 in the supplemental material). Inter-
estingly, residues I68 and N71, represented by green filled
circles in Fig. 3, were affected the most in their self-ubiquity-
lation ability. These results suggested that the RING-RING
interaction region might also be important for self-ubiquityla-
FIG. 1. Solution structure of the human TRIM5? RING domain. (A) Superposition of 20 NMR structures showing the ?-helix in red, ?-strands
in blue, and Zn2?in green. (B) Ribbon diagram of the NMR structure shown in panel A from the same perspective. The Zn2?coordinating
residues are also shown in green. (C) Electrostatic mapping of the RING domain surface highlighting the positions of positive (blue), negative
(red), and neutral (white) charges. Dotted circles indicate the location of the putative E2-binding site for the RING domain of TRIM5?. (D) A
Corey-Pauling-Koltun model of the RING domain of human TRIM5? is shown with labels of the visualized residues. Acidic and basic residues
are shown in red and blue, respectively. Hydrophobic residues are shown in green.
TABLE 1. Summary of conformational constraints and statistics of
the final 20 structures of the RING domain of TRIM5?
NOE upper distance restraints
Dihedral angle restraints (? and ?)......................................26
CYANA target function value (Å2)......................................0.20 ? 0.01
No. of violations
Distance violations (?0.30 Å)............................................
Dihedral angle violations (?5.0°)......................................
RMSD from averaged coordinates (Å)d
Backbone atoms...................................................................0.70 ? 0.18
Heavy atom...........................................................................1.05 ? 0.15
Ramachandran plot (%)d........................................................
Residues in most-favored regions......................................
Residues in additional allowed regions.............................
Residues in generously allowed regions ...........................
Residues in disallowed regions ..........................................
a?i ? j? ? 0.
b1 ? ?i ? j? ? 4.
c?i ? j? ? 4.
dValues calculated for the region encompassing residues 8 to 63.
8728LIENLAF ET AL. J. VIROL.
FIG. 2. The putative E2-binding and RING-RING interaction regions of the TRIM5?rhRING domain. The structure of the TRIM5?rhRING
domain is based on the human TRIM5? RING domain (PDB 2ECV) and was assembled by using the SWISS-MODEL protein homology modeling
program. (A) The putative E2-binding region (magenta) was identified by fitting the model structure to the Cbl-UBCH7 complex structure and
by modeling the interaction between the RING domain and UBCH7. (B) The putative RING-RING interaction region (green) was identified in
the same way by fitting it to the BRCA1-BARD1 RING structure for the RING domain region. Since conformational prediction of the N-terminal
and C-terminal regions was difficult due to poor sequence homology, these regions were not included. Residues that interact with Zn2?atoms are
indicated by black asterisks above the sequence alignment.
VOL. 85, 2011 ROLE OF TRIM5?’s E3-LIGASE ACTIVITY IN RESTRICTION8729
TABLE 2. Phenotypes of TRIM5? RING variantsa
potency ? SDcagainst:
Mean % TRIM5
Mean binding to
complexes ? SDe
Mean % reverse
Mean % higher-order
100.00 100.0010010 100
101.00 ? 0.24
100.98 ? 0.02
101.23 ? 0.45
102.20 ? 0.33
93.00 ? 3.70
91.23 ? 1.73
89.79 ? 7.38
73.23 ? 6.75
73.18 ? 2.57
68.99 ? 5.64
67.71 ? 3.83
63.08 ? 9.79
62.99 ? 2.84
58.10 ? 7.21
54.18 ? 4.50
53.17 ? 2.60
53.04 ? 5.71
49.11 ? 8.33
47.48 ? 4.51
45.50 ? 6.36
45.08 ? 5.55
43.35 ? 5.17
40.81 ? 9.63
40.51 ? 3.54
39.67 ? 3.30
39.52 ? 3.51
38.94 ? 9.81
34.99 ? 4.08
32.18 ? 5.30
29.94 ? 4.16
29.29 ? 3.23
26.23 ? 7.39
25.54 ? 5.00
23.54 ? 3.59
23.13 ? 7.25
21.31 ? 4.67
19.21 ? 5.36
15.65 ? 5.16
14.71 ? 3.83
14.70 ? 3.81
14.03 ? 7.03
12.24 ? 2.49
10.47 ? 4.43
10.46 ? 2.18
8.27 ? 1.80
101.44 ? 2.10
102.66 ? 0.87
103.55 ? 0.45
99.75 ? 1.70
99.34 ? 2.30
101.34 ? 5.67
104.45 ? 0.27
22.45 ? 2.34
50.39 ? 3.70
95.00 ? 0.56
49.92 ? 0.98
21.12 ? 1.78
37.86 ? 2.51
66.52 ? 3.67
40.00 ? 0.97
92.70 ? 3.48
43.43 ? 1.52
74.24 ? 1.33
35.20 ? 4.71
66.75 ? 2.89
93.00 ? 3.21
29.69 ? 1.76
30.62 ? 1.44
59.50 ? 2.59
83.69 ? 2.33
31.95 ? 3.72
22.60 ? 0.79
48.49 ? 3.21
58.75 ? 2.19
36.50 ? 3.66
63.09 ? 1.76
22.74 ? 1.79
20.11 ? 1.21
71.67 ? 3.66
20.68 ? 1.22
50.39 ? 1.99
61.80 ? 2.51
14.92 ? 1.24
15.98 ? 0.83
45.33 ? 3.88
46.25 ? 2.13
31.20 ? 2.45
35.30 ? 1.66
33.25 ? 3.11
15.40 ? 2.61
99.56 ? 3.24
96.79 ? 4.77
98.22 ? 9.82
97.37 ? 9.34
100.32 ? 6.55
105.26 ? 4.24
97.05 ? 5.99
102.41 ? 11.12
105.26 ? 5.78
40.33 ? 7.65
101.23 ? 3.15
111.58 ? 9.73
114.63 ? 5.99
102.26 ? 4.32
32.42 ? 2.39
101.33 ? 8.4
74.32 ? 6.39
99.39 ? 4.51
27.05 ? 8.22
45.79 ? 5.99
105.26 ? 5.11
49.63 ? 3.65
44.26 ? 4.21
21.02 ? 4.88
71.25 ? 2.84
103.47 ? 6.22
44.74 ? 8.7
49.13 ? 5.33
48.69 ? 4.59
107.88 ? 9.41
49.63 ? 6.16
14.77 ? 3.98
2.11 ? 1.45
31.99 ? 3.45
23.94 ? 3.11
102.34 ? 6.71
101.45 ? 8.16
1.1 ? 0.03
0.98 ? 0.08
0.81 ? 0.20
1.02 ? 0.30
0.69 ? 0.20
0.91 ? 0.32
1.1 ? 0.05
1.15 ? 0.09
0.5 ? 0.15
0.65 ? 0.10
0.72 ? 0.06
0.91 ? 0.35
0.67 ? 0.07
0.57 ? 0.14
0.62 ? 0.15
0.81 ? 0.11
0.94 ? 0.10
0.89 ? 0.08
0.54 ? 0.07
0.4 ? 0.03
0.71 ? 0.19
0.67 ? 0.05
0.87 ? 0.27
0.89 ? 0.04
0.98 ? 0.10
1.15 ? 0.20
0.97 ? 0.05
1.03 ? 0.10
1.1 ? 0.02
0.56 ? 0.20
0.84 ? 0.18
1.05 ? 0.25
0.94 ? 0.30
0.95 ? 0.09
0.65 ? 0.30
1.02 ? 0.14
0.89 ? 0.04
0.96 ? 0.11
1.1 ? 0.06
0.32 ? 0.12
0.62 ? 0.02
0.51 ? 0.05
1.03 ? 0.03
1.12 ? 0.04
0.36 ? 0.09
2.63 ? 0.35
4.95 ? 3.79
0.33 ? 0.05
5.89 ? 1.33
0 ? 0.15
1.96 ? 0.19
5.67 ? 0.64
2.92 ? 0.45
8.74 ? 3.05
0.74 ? 0.15
1.96 ? 2.49
0.41 ? 0.23
4.5 ? 1.59
2.98 ? 0.55
2.84 ? 1.43
4.62 ? 2.72
2.35 ? 0.16
6.24 ? 0.08
5.89 ? 0.07
0.43 ? 0.07
5.86 ? 0.061
0 ? 0.09
1.98 ? 1.74
1.96 ? 0.66
3.23 ? 1.19
15.72 ? 4.93
14.82 ? 2.87
6.46 ? 0.11
19.65 ? 1.12
18.73 ? 12.68
2.98 ? 0.23
1.96 ? 0.53
3.25 ? 1.3
19.73 ? 0.58
3.93 ? 2.02
2.81 ? 0.1
19.38 ? 0.91
4.95 ? 2.59
8.09 ? 2.53
3.93 ? 0.05
15.56 ? 0.57
5.68 ? 0.55
14.97 ? 1.35
4.11 ? 0.53
1.96 ? 0.05
105.32 ? 3.56
103.09 ? 5.61
40.23 ? 3.29
99.81 ? 4.77
45.21 ? 7.51
102.26 ? 2.91
100.33 ? 3.59
75.32 ? 2.56
78.36 ? 7.8
100.19 ? 5.6
101.25 ? 6.72
55.04 ? 9.11
100.04 ? 3.11
30.37 ? 4.62
48.98 ? 7.21
55 ? 5.71
97.51 ? 5.17
70.16 ? 2.57
101.11 ? 3.18
51.38 ? 7.15
56.74 ? 0.85
59.73 ? 3.19
9.32 ? 4.73
11.3 ? 4.55
89.56 ? 2.5
62.11 ? 0.34
91.46 ? 3.77
100.09 ? 3.87
98.51 ? 6.33
7.83 ? 1.86
60.82 ? 4.75
55.29 ? 7.19
90.41 ? 3.11
10.95 ? 5.29
5.18 ? 2.77
30.42 ? 3.69
90.48 ? 7.92
88.23 ? 4.93
5.7 ? 2.11
97 ? 6.49
101.25 ? 4.93
74.23 ? 0.45
98.26 ? 3.72
aShaded variants retain wild-type binding to HIV-1 capsid and higher-order self-association. These variants were used to establish a correlation between TRIM5?rh
self-ubiquitylation and anti-HIV-1 activity (see Fig. 7).
bLocation of each TRIM5?rhvariant on the NMR structure of the RING domain. E2 binding means that the residue is located in the E2-binding region of the RING
domain. Similarly, RING-RING represents a variant located in the RING-RING interaction region. NA means not applicable. Other means location on a surface
different from the E2-binding and RING-RING interaction regions.
cRestriction was measured by infecting cells expressing the indicated TRIM5?rhvariants with HIV-1 and EIAV expressing GFP. After 48 h, the percentage of GFP-positive
cells (infected cells) was determined by flow cytometry. Restriction potency was defined here as the fraction of TRIM5?rh’s RING domain variant restriction fold relative to
the wild-type’s restriction fold when 50% of the control cells are infected. Experiments were performed at least three times; typical results are shown.
dTo assay the E3-ubiquitin ligase activity of TRIM5?rhRING domain variants, the ability of these TRIM5? variants to undergo self-ubiquitylation was assayed.
FLAG-tagged TRIM5? variants from transfected 293T cells semipurified by immunoprecipitation were incubated with E1, E2, myc-tagged ubiquitin, and ATP for 1 h
at 37°C. Samples resulting from the incubation were analyzed by Western blotting using antibodies against FLAG and myc for the detection of TRIM5? and ubiquitin,
respectively. The amount of ubiquitylated TRIM5? protein was determined by subtracting the amount of nonubiquitylated TRIM5? protein remaining in the reaction
mixture that was incubated with E1 and E2 from the TRIM5? protein in the control reaction mixture, which was not incubated with E1 and E2 enzymes. The amount
of nonubiquitylated TRIM5? in the reaction mixtures was quantified by using the Quantity-One software from Bio-Rad. TRIM5? self-ubiquitylation was expressed as
the percentage of the total TRIM5? variant input protein. Experiments were performed at least three times.
eBinding to the HIV-1 capsid complexes was determined for each TRIM5?rhvariant as described in Materials and Methods. Binding is expressed as the amount
of the variant TRIM5?rhbound to HIV-1 capsid complexes divided by the amount of bound wild-type TRIM5?rhat a similar input level. Experiments were repeated
at least three times. Note that because the binding ratios are calculated at input levels at which some binding of the mutant TRIM5?rhprotein to the HIV-1 capsid
complexes can be detected, these ratios overestimate the relative capsid-binding affinities of the mutant proteins.
fHIV-1 reverse transcription was measured by real-time PCR 7 h after infection as described in Materials and Methods. The value shown represents the percentage
of late reverse transcripts observed in cells expressing the indicated ????5?rhvariant relative to the level of late reverse transcripts in control HIV-1-infected cells
transduced with the empty LPCX vector. Experiments were performed at least two times.
gEach TRIM5?rhvariant was assayed for higher-order self-association as described in Materials and Methods. The percentage represents the fraction of the
TRIM5?rhvariant coprecipitated with itself relative to the coprecipitation of wild-type TRIM5?rhwith itself.
hA high background level was observed for the TRIM5?rhvariant in the control sample without a TRIM5? target protein; the reported values may be less reliable
as a result of this background level.
8730LIENLAF ET AL.J. VIROL.
tion activity, as has been shown for other RING domains (4,
30, 36, 52). Altogether, these results demonstrated that the
self-ubiquitylation activity of TRIM5?rhcould be eliminated
by mutating single residues in the E2-binding or the RING-
RING interaction region.
Effect of TRIM5?rhRING changes on HIV-1 capsid binding.
The capacity of TRIM5?rhto bind the HIV-1 capsid is a
property essential to its ability to block HIV-1 infection (16,
64). To identify RING domain variants that lost self-ubiquity-
lation but bind the HIV-1 capsid, we measured the abilities of
these mutant proteins to bind in vitro-assembled HIV-1
CA-NC complexes. To measure the binding of the different
TRIM5?rhRING domain variants to in vitro-assembled HIV-1
CA-NC complexes, we used our previously described quanti-
tative binding assay (15), which adjusts the input levels of
TRIM5? variants to compare capsid-binding abilities more
accurately. Of all of the TRIM5?rhRING domain variants that
were defective in self-ubiquitylation, those that retained wild-
type binding to HIV-1 CA-NC (Fig. 4) were the I17E, L19K,
E20K, L21K, A41E/N42E, S46A, L48A, L48D, Y49A/K50A,
V58A, R60A, R60K, and R60E mutant proteins. The normal
binding of these RING domain variants to capsid implied that
the defect in self-ubiquitylation is likely to be a defect in the
E2-binding region. The majority of the mutations in the E2-
binding regions of different RING domains result in a defect in
the recruitment of the corresponding E2 to the RING domain
(9). Remarkably, most of the TRIM5?rhRING domain vari-
ants that lost self-ubiquitylation but bound HIV-1 CA-NC
complexes at wild-type levels were located in the E2-binding
region of the RING domain (Fig. 2A). However, a few muta-
tions in the RING-RING interaction region, including H29A,
N67E, and I68E, retained wild-type binding to capsid but were
deficient in self-ubiquitylation (Fig. 4; see Fig. S2 in the sup-
Higher-order association of TRIM5?rhRING mutant pro-
teins. Higher-order self-association is important for the ability
of TRIM5?rhto restrict HIV-1 (12, 15, 19, 40). The B-box 2
domain is critical to the formation of higher-order complexes,
FIG. 3. E3-ubiquitin ligase activities of TRIM5?rhRING domain variants. Human 293T cells were transfected with plasmids encoding
FLAG-tagged mutant and wild-type TRIM5?rhproteins. Forty-eight hours later, the cells expressing each TRIM5?rhvariant were lysed in
whole-cell extract and immunoprecipitated using anti-FLAG–agarose beads as described in Materials and Methods. Beads containing the
immunoprecipitated TRIM5?rhvariants were washed and eluted using 200 ?g/ml FLAG tripeptide in whole-cell extract buffer as described in
Materials and Methods. Samples were supplemented with 5 ?M ubiquitin aldehyde, a potent inhibitor of all ubiquitin C-terminal hydrolases,
ubiquitin-specific proteases, and deubiquitinating enzymes. Similar amounts of inhibitor-treated samples containing mutant and wild-type
TRIM5?rhwere incubated with or without 200 nM enzyme E1 (human recombinant UBE1) and 100 nM enzyme E2 (human recombinant UbcH5a)
as indicated. Reaction mixtures were supplemented with 200 ?M ubiquitin tagged with a myc epitope (human recombinant ubiquitin) and an
energy regeneration solution containing MgCl2, ATP, and ATP-regenerating enzymes to recycle hydrolyzed ATP. The reaction mixture was
incubated at 37°C for 1 h, and collected fractions were analyzed by Western blotting using HRP-conjugated antibodies against FLAG to detect
the levels of TRIM5?rhvariants. To detect ubiquitylated forms of TRIM5?rhvariants, membranes were blotted using HRP-conjugated antibodies
against myc. Purple circles and green circles indicate TRIM5?rhvariants with defective self-ubiquitylation activity located on the E2-binding and
RING-RING interaction region, respectively. The results of three independent experiments were similar; the result of a single experiment is shown.
VOL. 85, 2011 ROLE OF TRIM5?’s E3-LIGASE ACTIVITY IN RESTRICTION8731
which have been shown to increase the avidity of TRIM5?rh
for the HIV-1 capsid (15, 40); residue R121 in the B-box 2
domain of TRIM5?rhis essential for the ability of TRIM5?rh
to form hexagonal structures on the surface of the HIV-1
capsid (12, 15, 19). Even though the B-box domain is in close
contact with the RING domain, the role of the RING domain
in higher-order self-association has not been tested. To more
stringently analyze our mutant proteins that are deficient in
self-ubiquitylation but bind the HIV-1 capsid, we measured
their capacity to undergo higher-order self-association, an im-
portant property of TRIM5?rhrequired for potent restriction
of HIV-1 (12, 15, 40). We tested higher-order self-association
in the mutant proteins that lost their self-ubiquitylation activity
but retained binding to the HIV-1 capsid (Fig. 5). To test the
abilities of the RING mutant proteins to form higher-order
complexes, we mixed cell lysates containing FLAG-tagged
RING domain variants with lysates containing the same RING
domain variant tagged with HA. After precipitation with an
anti-FLAG antibody, the precipitates were Western blotted
with antibodies directed against the FLAG and HA epitope
tags. The wild-type TRIM5?rh-HA protein was efficiently co-
precipitated with TRIM5?rh-FLAG using anti-FLAG antibod-
ies (Fig. 5), consistent with the ability of wild-type TRIM5?rh
to self-associate in higher-order complexes (15, 40). After
studying the higher-order self-association capabilities of the
RING domain mutant proteins that were defective in self-
ubiquitylation but bind the HIV-1 capsid at wild-type levels, we
narrowed our study down to the following 9 RING domain
variants (Fig. 5 and Table 2): I17E, H29A, A41E/E42E, S46A,
L48A, R60A, R60K, R60E, and N67E. These 9 RING domain
variants showed wild-type capsid binding and higher-order
self-association, but they were defective in self-ubiquitylation.
Several TRIM5?rhRING domain variants lost the ability to
form higher-order complexes (Fig. 5 and Table 2; see Fig. S3 in
the supplemental material). These results demonstrated that,
besides the B-box 2 domain (15), an intact RING domain is
also necessary for higher-order self-association. Apparently,
the ability of TRIM5?rhto form higher-order complexes is
sensitive to changes in different surface residues of the B-box
2 and RING domains.
Next we analyzed the abilities of this selected group of mu-
tant proteins, which exhibit deficient self-ubiquitylation but
normal capsid binding and higher-order self-association, to
block HIV-1 infection.
Retroviral restriction by TRIM5?rhRING domain variants.
To examine the abilities of TRIM5?rhRING mutant proteins
to inhibit retroviral infection, dog Cf2Th cells stably expressing
these mutant proteins (see Fig. S4 in the supplemental mate-
rial) were challenged with recombinant HIV and equine infec-
tious anemia virus expressing GFP as a reporter abbreviated
here as HIV-1–GFP and EIAV-GFP, respectively (Fig. 6; see
Fig. S5 in the supplemental material) (47, 55). The TRIM5?rh
RING variants exhibited a range of HIV-1-restricting abilities
and are rank ordered in Table 2 according to restriction po-
tency—defined here as the fraction of restriction by the mutant
relative to the wild-type TRIM5?rhrestriction when 50% of
the control cells are infected.
Infection of cells stably expressing the different RING do-
main variants by HIV-1–GFP and EIAV-GFP identified resi-
dues essential for restriction. Changing arginine 60 in the
FIG. 4. Binding of TRIM5?rhRING mutant proteins to assembled
HIV-1 capsids. 293T cells were transfected with plasmids expressing
the indicated wild-type and mutant TRIM5?rhproteins tagged with
HA epitopes. Thirty-six hours after transfection, cells were lysed. The
lysates were incubated at room temperature for 1 h with HIV-1
CA-NC complexes that had been assembled in vitro. The mixtures were
applied to a 70% sucrose cushion and centrifuged. INPUT represents
the lysates analyzed by Western blotting (WB) before being applied to
the 70% cushion. The input mixtures were Western blotted for the HA
tag. The pellet from the 70% cushion (PELLET) was analyzed by
Western blotting using antibodies against the HA tag and HIV-1
CA-NC protein. The Western blots were quantitated as described in
Materials and Methods, and binding values are shown in Table 2. The
results of three independent experiments were similar; the result of a
single experiment is shown.
FIG. 5. Higher-order self-association of TRIM5?rhRING mutant proteins. 293T cells were transfected with plasmids expressing the indicated
wild-type or mutant TRIM5? proteins with a FLAG or an HA epitope tag. Cells expressing wild-type and mutant TRIM5?rhproteins were lysed
48 h after transfection. The cell lysates containing similar inputs were mixed, and the indicated mixtures were used for immunoprecipitation (IP)
with an antibody directed against the FLAG epitope, as described in Materials and Methods. Elution of the immunocomplexes was performed with
a FLAG tripeptide and analyzed by Western blotting (WB) using anti-HA and anti-FLAG antibodies. The results of three independent
experiments were similar; the result of a single experiment is shown.
8732LIENLAF ET AL.J. VIROL.
RING domain of TRIM5?rhto lysine, glutamic acid, or alanine
drastically reduced the potency of TRIM5?rhagainst HIV-1 by
5- to 10-fold (Fig. 6A). Similarly, the I17E, A41E/N42E, N67E,
and L48A variants showed reduced potency against HIV-1
(Fig. 6B). Remarkably, all of these residues were located in the
E2-binding region of the RING domain, with the exception of
N67E, which is located in the RING-RING interaction region.
When cells expressing these RING domain variants were chal-
lenged with EIAV, we observed a consistent decrease in re-
striction (Fig. 6C and D). These results suggested a role for
ubiquitylation in HIV-1 restriction by TRIM5?rh.
TRIM5?rhself-ubiquitylation correlates with HIV-1 restric-
tion. Using this selected group of variants that exhibit deficient
self-ubiquitylation but normal capsid binding and higher-order
self-association (shaded residues in Table 2), we tested the
hypothesis that TRIM5? self-ubiquitylation correlates with re-
striction. To do so, we graphically represented HIV-1 restric-
tion versus self-ubiquitylation for this selected group of vari-
ants. Remarkably, a strong correlation (rs? 0.9090, P ? 0.001)
between TRIM5? self-ubiquitylation and HIV-1 restriction by
this panel of mutant proteins was observed (Fig. 7). These
results support the hypothesis that the E3-ligase activity of the
RING domain represents a major contribution of the RING
domain to HIV-1 restriction by TRIM5?.
Inhibition of HIV-1 reverse transcription by TRIM5?rhmu-
tant proteins. HIV-1 restriction by TRIM5?rhoccurs prior to
FIG. 6. Restriction of HIV-1 and EIAV infection by TRIM5?rhmutant proteins. Cf2Th cells were transduced with the LPCX vector
expressing HA-tagged wild-type and mutant TRIM5?rhproteins. Stable cell lines were selected with 5 ?g/ml puromycin, and the expression
levels of mutant and wild-type TRIM5?rhproteins were assayed by Western blotting using HRP-conjugated antibodies against HA (see Fig.
S4 in the supplemental material). The cells were challenged with different amounts of HIV-1–GFP (A, B) or EIAV-GFP (C, D). The
percentage of GFP-positive cells was measured 48 h later by FACS. The results of three independent experiments were similar; the results
of a single experiment are shown.
VOL. 85, 2011 ROLE OF TRIM5?’s E3-LIGASE ACTIVITY IN RESTRICTION 8733
the initiation of reverse transcription (28, 63). However, the
use of proteasome inhibitors during restriction allows the oc-
currence of reverse transcription without affecting the block-
age of infection (1, 64, 67). To examine the ability of the
TRIM5?rhRING domain variants to block HIV-1 reverse
transcription, we assayed the level of late reverse transcripts in
mutant-expressing cells challenged with HIV-1 (Fig. 8; see Fig.
S6 in the supplemental material). HIV-1 reverse transcript
levels were low in cells expressing potently restricting
TRIM5?rhRING domain variants (Fig. 8 and Table 2). In
contrast, in cells expressing RING domain variants that did not
restrict HIV-1 infection potently, the levels of HIV-1 reverse
transcripts were higher (Fig. 8 and Table 2).
Half-lives of TRIM5?rhRING domain variants. We mea-
sured the half-lives of the TRIM5?rhRING domain variants
that lost self-ubiquitylation but preserved capsid binding and
higher-order self-association (HOSA) (see Fig. S7 in the sup-
plemental material), as previously described (12, 15). Some of
the RING domain variants exhibited a longer half-life than
wild-type TRIM5?rh, which is ?50 min (13). For example, the
A41E/N42E, N67E, and S46A variants exhibited a half-life
slightly longer than the wild-type half-life of ?70 min; the I17E
variant exhibited a half-life of ?105 min. Other variants, such
as the R60E variant, exhibit a half-life similar to that of the
wild-type protein of ?55 min. Interestingly, the R60A and
R60K variants exhibited half-lives shorter than that of the
wild-type protein at ?15 min and ?45 min, respectively. These
results did not correlate self-ubiquitylation with degradation
since we found mutant proteins that did not self-ubiquitylate
but have shorter half-lives than wild-type TRIM5?rh. These
results are in agreement with previous observations suggesting
that the degradation of TRIM5?rhis modestly affected by the
FIG. 7. TRIM5? self-ubiquitylation activity correlates with anti-
HIV-1 activity. The abilities of RING domain variants to self-
ubiquitylate (TRIM5? self-ubiquitylation) and restrict HIV-1 were
assessed as described in the footnotes to Table 2 and in Materials
and Methods. TRIM5? RING domain variants that were not de-
fective in binding to the HIV-1 capsid and higher-order self-asso-
ciation were analyzed. The Spearman rank correlation coefficient,
rs, is 0.9090, with a 95% confidence interval of 0.8623 to 0.9847
(two-sided P value of ? 0.0001).
FIG. 8. Blockade of HIV-1 reverse transcription by TRIM5?rhRING mutant proteins. Cf2Th cells expressing the indicated wild-type and
mutant TRIM5?rhproteins or containing the empty LPCX vector were challenged at an MOI of 0.4 with DNase-pretreated HIV-1–GFP. After
7 h, cells were lysed and total DNA was extracted. The levels of viral DNA were measured by quantitative real-time PCR using a probe against
GFP as described in Materials and Methods. Similar results were obtained in three independent experiments.
8734LIENLAF ET AL. J. VIROL.
use of proteasome inhibitors in the absence of restricted vi-
The RING domain of TRIM5? exhibits E3-ubiquitin ligase
activity, but the contribution of this activity to the restriction of
HIV-1 is not understood. Here we present the structure of the
RING domain of human TRIM5? and use this information to
direct a mutational analysis of the functional surfaces of the
RING domain of TRIM5?rh. To explore the role of the E3-
ubiquitin ligase in restriction, we correlated the E3-ubiquitin
ligase activity of TRIM5? with the different properties of the
restriction factor TRIM5?, including HIV-1 restriction, bind-
ing to the HIV-1 capsid, inhibition of reverse transcription, and
the ability to form higher-order complexes. We found a distinct
set of TRIM5? variants located on the E2-binding surface of
the RING domain, where the loss of E3-ubiquitin ligase activ-
ity correlated with a defect in HIV-1 restriction ability. Our
results demonstrate that E3-ubiquitin ligase activity has a role
in HIV-1 restriction by TRIM5?, as has been previously sug-
gested by others (1, 50, 54, 64, 67).
The RING domain of TRIM5? adopts a ??? RING fold
containing shorter ?-strands and a longer ?-helix than the
typical fold observed in RING domains. Comparison of the
RING domain of TRIM5? with other RING domains revealed
that this structure has two regions that are potentially impor-
tant for RING domain function (Fig. 2). Similar to the RING
domains of Cbl, CHIP, and cIAP2 (9), the RING domain of
TRIM5? presents a distinct E2-binding region composed of
similar amino acids (Fig. 2A). Opposite to the E2-binding
region is the RING-RING interaction region, which is similar
to the interaction region that allows BRCA1 and BARD1
RING domains to form a heterodimer (6) (Fig. 2B). The
construct used to solve the NMR structure of the RING do-
main of TRIM5? did not include the last 10 amino acids; these
residues are part of the association helix used by BRCA1 and
BARD1 RING domains to heterodimerize. Longer constructs
of the TRIM5? RING domain resulted in a poor HSQC spec-
The E2-binding region of RING domains is essential for
docking of the E2 protein and allows the transfer of ubiquitin
from E2 to the target protein. In order to disrupt this docking
site in the RING domain of TRIM5?, we generated a series of
mutations on the different surfaces of the RING domain (Ta-
ble 2); these variants were tested for the different properties of
the restriction factor TRIM5?. These experiments revealed
that TRIM5? self-ubiquitylation requires an intact E2-binding
region, which suggested that an intact E2 docking site in the
RING domain is required for the self-ubiquitylation property
of TRIM5?. Mutations in all of the residues of the E2-binding
site of the RING domain affected self-ubiquitylation to a cer-
tain extent; in some cases, complete loss of self-ubiquitylation
was observed. Mutations in the E2-binding region that resulted
in a partial effect on self-ubiquitylation could be explained by
the existence of complementation by a different RING do-
main. Hetero-oligomerization with a related RING domain
could rescue ubiquitylation activity in a defective RING do-
main, as has been shown for ubiquitylation-deficient mutant
proteins of the Mdm2 RING domain that can be rescued by
hetero-oligomerization with the RING domain of MdmX (60).
To exclude RING mutations that had effects on other prop-
erties that are important for HIV-1 restriction by TRIM5?, we
quantitatively measure the binding of these variants to the
HIV-1 capsid, as previously shown (15). Similarly, we also
measured the ability of the RING variants to undergo higher-
order self-association, which is also required for potent restric-
tion of HIV-1. Remarkably, TRIM5? self-ubiquitylation activ-
ity correlates with restriction activity on mutant proteins where
binding to the HIV-1 capsid and higher-order self-association
were not affected. This correlation supports the hypothesis that
the E3-ubiquitin ligase activity of the RING domain is re-
quired for potent restriction of HIV-1 by TRIM5?.
Several observations have linked the restriction of HIV-1 by
TRIM5? with the proteasome. The observation that protea-
somal inhibitors allow the occurrence of reverse transcription
without affecting restriction suggests that TRIM5? blocks re-
striction before and after reverse transcription (1, 64, 67). The
use of proteasome inhibitors in the fate of the capsid assay
showed an increase in particulate capsid during infection in the
presence of TRIM5?, which also suggests a role for the pro-
teasome in restriction and uncoating (11, 14). The Aiken lab-
oratory has demonstrated that TRIM5? is degraded in a pro-
teasome-dependent manner in the presence of a restricted
capsid, which links the proteasome with the HIV-1 restriction
by TRIM5? (54). The present work attempted to connect
ubiquitylation, a process preceding proteasomal degradation,
with the ability of TRIM5? to block HIV-1. Similar to what we
observed for a panel of B-box 2 mutant proteins (15), the levels
of HIV-1 late reverse transcripts for this panel of RING mu-
tant proteins inversely correlated with the degree of restriction.
Mutations in the RING-RING interaction region that re-
moved the ability to undergo self-ubiquitylation might cause a
defect in RING oligomerization, which is different from affect-
ing the docking site of the E2 enzyme. Several mutations in the
RING-RING interaction region also affected the self-ubiqui-
tylation activity of TRIM5? without affecting folding measured
by HIV-1 capsid binding and higher-order self-association.
This is in agreement with findings suggesting that RING do-
main oligomerization enhances ubiquitylation (30, 31, 52).
Loss of RING domain oligomerization could account for the
partial defect in self-ubiquitylation observed in some of the
RING-RING interaction region variants. In some cases, loss of
RING dimerization could result in complete loss of E3-ubiq-
uitin ligase activity, as has been shown for the RING domain of
RNF4 (41). Even though concentration dependence experi-
ments to test RING domain dimerization failed to prove ho-
modimerization (data not shown), these experiments did not
exclude the possibility that the RING domain hetero-oli-
gomerizes with a different RING domain, as shown for
BRCA-1 and BARD (6).
Our results demonstrated that potent restriction of HIV-1
by TRIM5?rhrequires intact self-ubiquitylation activity. One
could conceive a model in which the self-ubiquitylation of
TRIM5? is required to remove TRIM5? when it is forming
hexagonal structures on the surface of the capsid (19, 54);
removal of TRIM5? from the surface of the capsid will allow
a decrease on the amount of particulate capsid during infec-
tion, assisting a rapid uncoating process (10). Further analysis
VOL. 85, 2011ROLE OF TRIM5?’s E3-LIGASE ACTIVITY IN RESTRICTION8735
destined to understand the nature of the endogenous E2 en-
zyme and the ubiquitylation substrate of TRIM5? will give new
mechanistic insights into restriction.
We thank Steve Porcelli for critical reading of the manuscript. We
thank Joe Sodroski for the initial support of this project. We thank
Takashi Umehara for analytical ultracentrifugation measurements and
Xu-rong Qin for concentration dependency measurements in1H-15N-
HSQC experiments. We also thank Satoru Watanabe, Takushi
Harada, Takeshi Nagira, Yasuko Tomo, Masaomi Ikari, Kazuharu
Hanada, Yukiko Fujikura, and Akiko Tanaka for sample preparation
and help with the screening data of the human TRIM5 RING domain.
The work of the structure determination was supported by the
RIKEN Structural Genomics/Proteomics Initiative (RSGI) of the
National Project on Protein Structural and Functional Analyses,
Ministry of Education, Culture, Sports, Science and Technology of
Japan. This work has also been supported by a K99/R00 Pathway to
Independence Award to F.D.-G. (4R00MH086162-02) and grant
R01AI7930231 from the National Institutes of Health, an American
Foundation for AIDS Research Mathilde Krim fellowship phase II
in basic biomedical research (amfAR research grant 107787-47-
RKHF), and a Claudia Adams Barr award from the Dana-Farber
Cancer Institute to F.D.-G.
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