Download A Single Amino Acid Change in the SPRY Domain of Human Trim5

Current Biology, Vol. 15, 73–78, January 11, 2005, ©2005 Elsevier Ltd. All rights reserved.
DOI 10.1016/j .c ub . 20 04 . 12 .0 4 2
A Single Amino Acid Change in the SPRY Domain of
Human Trim5␣ Leads to HIV-1 Restriction
Melvyn W. Yap, Sébastien Nisole,1
and Jonathan P. Stoye*
Division of Virology
National Institute for Medical Research
The Ridgeway
Mill Hill
London NW7 1AA
United Kingdom
Summary
Retroviral restriction factors are cellular proteins that
interfere with retrovirus replication at a postpenetration, preintegration stage in the viral life cycle [1–3].
The first restriction activity described was the mouse
Fv1 gene [4]. Three different alleles of Fv1, capable of
restricting different murine leukaemia viruses (MLV),
have been characterized at the molecular level [5, 6].
Two further activities, Ref1, which acts on MLV, and
Lv1, which acts on lentiviruses, have been identified
in other mammalian species [7–9]. Recently, it has
become clear that Ref1 and Lv1 are encoded by the
same gene, Trim5␣, which inhibits retrovirus replication in a species-specific manner [10–14]. A series of
chimeras between the human and rhesus monkey
Trim5 genes were created to map and identify these
specificity determinants. The Trim5␣ SPRY domain
was found to be responsible for targeting HIV-1 restriction. By contrast, N-MLV restriction appears dependent on both the coiled-coil domain and the SPRY
domain. A single amino acid substitution (R332P) in
the human Trim5␣ can confer the ability to restrict
HIV-1, suggesting that small changes during evolution
may have profound effects on our susceptibility to
cross-species infection.
Results and Discussion
Human Trim5␣ restricts N-tropic MLV, but not B-tropic
MLV or HIV-1, whereas rhesus monkey Trim5␣ restricts
N-tropic MLV and HIV-1, but not B-tropic MLV [11–14].
The human and rhesus forms of Trim5␣ can also be
distinguished by using the L117H mutation of N-MLV,
which is restricted by human, but not rhesus monkey
Trim5␣ [13]. A series of chimeras between the human
and rhesus gene was constructed with overlapping PCR
(Figure 1) to examine which domain of Trim5␣ was involved in determining the specificity of retroviral restriction. The structures of these chimeras (and all subsequent constructs) were confirmed by sequencing.
Trim5␣ has a modular design with a series of distinct
motifs. These include the RING motif, a B-Box, and a
coiled-coil domain, which together form the tripartite
RBCC domain characterizing Trim proteins, as well as
*Correspondence: [email protected]
1
Present address: CNRS UPR 9051, Centre Hayem, Hôpital SaintLouis, 16, rue de la Grange-aux-Belles, 75010 Paris, France.
a SPRY domain [15]. These different domains were exchanged between the two proteins. The chimeras HR3,
HR2, and HR1 contain the human Trim5␣ RING motif
with progressively longer substitutions of rhesus C-terminal residues. A reciprocal series was also made (RH3,
RH2, and RH1) where parts of the rhesus Trim5␣ are
substituted by human sequences. Restriction was measured by a previously described two-color FACS assay
[13, 16]. Briefly, nonrestricting cells were transduced
with an MLV-based vector carrying the Trim gene and
EYFP. Two days posttransduction, the cells were challenged with tester viruses incorporating various capsids
and encoding the EGFP marker. The percentage of Trim/
EYFP positive cells that were transduced with the EGFP
marker was then scored against that of Trim/EYFP negative cells. Restriction was expressed as a ratio of
EGFP⫹, EYFP⫹ cells to EGFP⫹, EYFP⫺ cells. A ratio
of less than or equal to 0.3 was taken as restriction,
whereas a ratio between 0.7 and 1 indicated the absence
of restriction. A typical result is shown in Figure 1A. As
observed previously, human Trim5␣ restricted N-MLV
and the L117H mutant of N-MLV, but not B-MLV or HIV-1
[13]. Rhesus Trim5␣ restricted HIV-1 as well as N-MLV,
but not B-MLV or N L117H. These restricting phenotypes
were used as standards for comparison with the restriction profile of the chimeric molecules.
All chimeras could restrict N-MLV, but not B-MLV,
indicating that the proteins were still functional after
exchanging domains (Figure 1B). Substitution of the
SPRY domain from the human Trim5␣ with the rhesus
SPRY domain in HR3 is sufficient to confer restriction
to HIV-1. The reciprocal chimera, where the SPRY of
rhesus Trim5␣ was replaced with the human form (RH3),
lost the ability to restrict HIV-1. This implied that the
SPRY domain of Trim5␣ was sufficient and necessary
for recognizing and restricting HIV-1. The determinants
for N-MLV restriction seemed to be more complex. Replacing the SPRY domain of rhesus Trim5␣ with the
human form (RH3) enabled it to restrict N L117H in addition to N-MLV, implying that the SPRY domain was also
involved in targeting the restriction of N-MLV. However,
substitution of the human SPRY domain with the corresponding rhesus sequences (HR3) did not abolish its
ability to restrict N L117H. It was only when the coiledcoil domain as well as the SPRY of human Trim5␣ was
replaced with that from rhesus (HR2) that restriction of
N L117H was lost. These results suggested that both
the coiled-coil and the SPRY domains play roles in determining the specificity of MLV restriction.
Two more sets of chimeras were constructed (Figure
2) to further delineate the regions of the SPRY domain
involved in restriction. HR4 to HR7 consisted of human
Trim5␣ with decreasing lengths of rhesus sequences
substituted within the SPRY domain, whereas RH4 to
RH7 formed a reciprocal series where the rhesus Trim5␣
was substituted with human SPRY sequences. As expected, all the chimeras could restrict N-MLV, but not
B-MLV, indicating that they were functional with respect
to restriction. HR4 was able to restrict HIV-1, but the
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74
Figure 1. Restriction of HIV-1 and MLV by
Human, Rhesus, and Chimeric Trim5␣
(A) Graphical representation of restriction by
human and rhesus Trim5␣. HT1080 and M.
dunni cells were transduced with MLV-based
vectors carrying Trim5␣ and EYFP at a multiplicity of infection of 1. Two days posttransduction, HT1080 cells were challenged with HIV-1
vectors carrying EGFP, whereas M. dunni cells
were challenged with either N-MLV, B-MLV,
or N L117H. The ratio of the percentage of
Trim5␣⫹ cells versus that of Trim5␣⫺ cells
that were infected was determined by FACS
analysis 2 days after challenging with tester
virus. A ratio of between 0.7 to 1 indicates
the absence of restriction, whereas a ratio of
less than 0.3 indicates the presence of restriction. Error bars represent the standard
error of the mean.
(B) Schematic representation of the human/
rhesus chimeric Trim5␣ constructs and their
ability to restrict HIV-1, N-MLV, B-MLV, and
N L117H. Restriction assays were performed
as described in (A). White boxes indicate human derived sequences; gray boxes show
rhesus monkey sequences. The amino acid
positions corresponding to the human Trim5␣
are indicated on the top of the figure, whereas
the ones corresponding to the rhesus sequence are indicated at the bottom.
restriction was absent when the rhesus sequences after
residue 374 were replaced with human ones from HR5
to HR7. This suggested that the region determining
specificity for HIV-1 restriction was between amino
acids 322 to 374 of the rhesus Trim5␣. RH4, which does
not restrict HIV-1, lacked the rhesus sequence in this
region. When the rhesus sequence was included in the
chimera RH5, restriction of HIV-1 was restored, confirming the requirement of this region in HIV-1 recognition
and restriction. The requirements for MLV restriction
were found to be somewhat different from that of HIV-1.
HR4 to HR7 could restrict N L117H. This was expected
as both human Trim5␣ and HR3 (Figure 1B) could restrict
this mutant. The chimeras RH4 to RH7 (Figure 2) contain
decreasing amount of human SPRY sequences compared to RH3 (Figure 1B). RH4 and RH5 could restrict
N L117H, but the restriction was abolished when human
residues before amino acid 401 were excluded, in RH6
and RH7. This suggested that the SPRY region in human
Trim5␣ influencing N-MLV restriction involved residues
up to amino acid 400. Taken with the previous data,
these results indicated that the specificity determinants
of MLV restriction encompassed a region somewhat
larger than that which governed HIV-1 restriction.
We next sought to identify individual residues between amino acids 322 and 374 in the SPRY domain
that affected restriction. Between the human and rhesus
sequence, there was a difference of seven amino acids,
two contiguous, the others scattered throughout the
region. In addition, a stretch of six residues starting from
amino acid 335 in the human sequence was replaced
by 8 different residues in the rhesus sequence. The residues at these positions in the human Trim5␣ were replaced with the corresponding residues from rhesus
monkey by site-directed mutagenesis. The mutants
were then assayed for restriction of HIV-1 and MLV (Figure 3). Only two positions in this region affected restriction to HIV-1. The arginine to proline change at residue
332 and the exchange of the six human residues at
position 335 for the eight amino acid rhesus sequence
conferred the ability to restrict HIV-1 to the human
Trim5␣. Conversely, changing each of these positions
in the rhesus Trim5␣ to the corresponding residues in
the human protein did not abolish restriction of HIV-1.
Indeed, when both positions of the human protein were
substituted by the rhesus residues, the resulting double
mutant had a restricting phenotype that was similar to
the two single mutants. This suggested that both of
Mapping Restriction Specificity Domains of Trim5␣
75
Figure 2. Restriction by Human/Rhesus
Monkey Trim5␣ SPRY Domain Chimeras
Structures of the chimeras made are shown
on the left; their restriction properties on the
right. The amino acid positions corresponding to the human Trim5␣ protein are indicated
above the constructs, whereas the ones corresponding to the rhesus sequence are indicated at the bottom. Restriction assays were
performed and scored as described for Figure 1.
these positions contributed to the restriction and that
they might be degenerate. Hence, the loss of one of
these positions could be compensated by the presence
of the other.
The corresponding change from proline to arginine in
position 334 in the rhesus protein did not confer restriction to N L117H, suggesting that unlike HIV-1 restriction,
this position was not involved in MLV restriction. Exchanging the eight residues at position 337 of the rhesus
protein with the six found in humans resulted in the restriction of N L117H, suggesting that the position was involved
in MLV restriction. However, corresponding changes in
the human sequence to the rhesus residues did not abolish
restriction of N L117H. This suggests that other regions
in the human Trim5␣ might be contributing degenerately
to restricting N L117H. Indeed, this could involve sequences found in the coiled-coil region (Figure 1B).
We have previously shown that Trim1 could also restrict N-MLV, but not HIV-1 [13]. Like Trim5, it consists
of an SPRY domain after the tripartite RBCC motif. Trim1
was first identified as a homolog of the Opitz syndrome
gene Trim18 (Mid1) to which it showed 83% similarity
at the amino acid level [17, 18]. However Trim18, unlike
Trim1, does not restrict N-MLV (Figure 4). Formally, this
might be due to a lack of protein expression. Alternatively, it provides a further opportunity to examine the
role of the SPRY domain in retroviral restriction. The
C-terminal portion of African green monkey Trim1,
Figure 3. Effects of Specific Amino Acid Changes within the SPRY Domain on the Restriction of HIV-1 and MLV
The altered sequences of the SPRY domain are shown on the left, whereas the restriction properties of the variants are shown on the right.
Restriction assays were performed and scored as described for Figure 1.
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76
Figure 4. Restriction of MLV by African
Green Monkey Trim1/Trim18 Chimeras
Residues 314 to 715 of Vero Trim1 were replaced with residues 314 to 667 of Vero Trim18
to form Vero Trim1/Trim18. Vero Trim18/Trim1
was constructed by replacing residues 314
to 667 of Vero Trim18 with residues 314 to
715 of Vero Trim1. Trim1 sequences are represented by white boxes, whereas Trim18 sequences are colored gray on the left. The ability to restrict is shown in the table on the
right. Restriction assays were performed and
scored as described for Figure 1.
cloned from Vero cells, was used to replace the corresponding region in Trim18 beginning from residue 314
(Figure 4). This chimera (Vero Trim18/Trim1) was active
against N-MLV as well as N-MLV containing the N82D
mutation, but not B-MLV, a restriction phenotype typical
of Trim1. The reciprocal chimera Vero Trim1/Trim18 did
not restrict N-MLV and N N82D. Taken together, these
results provided further evidence for the involvement
of the SPRY domain as the recognition domain in the
process of retroviral restriction.
In owl monkeys, a psuedogene encoding the cyclophilin A (CypA) protein is inserted within the seventh intron
of the Trim5␣ gene [19, 20]. It is thought that the cyclosporine A (CsA) sensitive Lv1 activity of OMK cells [21]
can be explained by the presence of a fusion protein in
which most of the Trim5␣ SPRY domain is replaced by
CypA [19, 20]. This implies that the function provided
by SPRY can be replaced by CypA; because CypA is
known to bind HIV-1 CA in a CsA sensitive manner [22],
this suggests that SPRY is providing binding as well as
specificity functions. To examine this idea further, we
replaced the SPRY domain of human and rhesus Trim5␣s
with the CypA insertion of the OMK Trim5␣ gene (Figure
5). Both novel chimeras restricted wild-type HIV-1 (Figure 5), but not a mutant form that is not bound by CypA,
CA G89V. In addition, the substitutions abolished activity
against N-MLV, which does not bind CypA [22]. Replacing the SPRY domain of Trim5␣ from African green monkey yielded identical results (data not shown). This confirmed that CypA was performing the function of CA
recognition and binding in OMK Trim5-CypA.
We have previously found that an artificially created
OMK Trim5␣ (V6), which contained the owl monkey
SPRY domain in place of CypA, was inactive against
both HIV-1 and MLV [20]. To further probe the role of
SPRY in CA recognition and binding, we asked if the
SPRY domain from rhesus Trim5␣ could rescue the restriction activity of OMK Trim5 V6. Replacing the SPRY
of OMK Trim5 V6 in OMKRhSPRY with that from rhesus
restored its activity against HIV-1 (Figure 5), suggesting
that this domain alone could account for the targeting
of HIV-1 CA. However, OMKRhSPRY was not active
against N-MLV. Intriguingly, the sequence of the coiledcoil domain of OMK Trim5 is very different from that of
the rhesus protein. In the light of the earlier result (Figure
1) suggesting the involvement of the coiled-coil domain
in MLV restriction, the failure of OMKRhSPRY to restrict
might be due to the lack of MLV CA recognition sequences in the coiled-coil domain of OMK Trim5.
Our results indicate that the SPRY domain is the primary determinant of restriction specificity and suggest
that this specificity might be mediated through SPRY
binding of capsid. The SPRY domain was originally characterized in proteins that regulated intracellular signaling [23]. It can occur as a subdomain within the B30.2
domain [24], which is also found in Trim1 and Trim18
[17, 18]. Although often associated with Trim proteins,
SPRY domains are also found in unrelated proteins such
as venom toxins and proteins with immunoglobulin-like
folds [24]. The functions of B30.2 and SPRY domains
are still not known but might include specific proteinprotein interactions. Our studies suggest that in Trim
proteins, they play a crucial role in the interaction with
infectious agents such as retroviruses to abolish infecFigure 5. Restriction Properties of Chimeric
Human, Rhesus, and Owl Monkey Trim5␣
A schematic representation of the chimeras
is presented on the left, whereas their ability
to restrict HIV-1 and MLV is shown on the
right. HuCypA and RhCypA were constructed
by replacing the human SPRY domain (residues 295 to 493) or rhesus SPRY domain (residues 297 to 497) with the cyclophilin A insertion in OMK Trim5 V4 (residues 296 to 474).
OMKRhSRPY was made by replacing the
OMK SPRY of V6 (residues 296 to 495) with
the rhesus SPRY domain (residues 297 to
497). Human sequences are represented by
white boxes, whereas rhesus and owl monkey sequences are represented by light and
dark gray boxes, respectively. Restriction
assays were performed and scored as described for Figure 1.
Mapping Restriction Specificity Domains of Trim5␣
77
tion. The SPRY domain seems to function independently
from the tripartite motif, because it can be exchanged
between different primate Trim5s and can even be replaced with CypA, with associated changes in restriction
specificity. It is tempting to speculate that these proteins, together with the other members of the Trim family, might be involved in innate immunity against different
infectious agents. Interestingly, mutations in the B30.2
domain in Trim18 (Mid1) [25, 26] and Trim20 (Pyrin) [27,
28] are associated with genetic defects. It has been
suggested [25–28] that these mutations are preserved
during evolution, because they offer enhanced protection against disease-causing organisms in a manner
analogous to the survival of the sickle cell alleles of
haemoglobin [29].
The region imparting restriction specificity appears to
lie between amino acids 332–340 of human Trim5␣. This
is the region showing greatest diversity between the
sequences of human, rhesus, and African green monkey
[11–13], and we are currently carrying out a detailed
phylogenetic analysis of this region to correlate sequence variation with changes in restriction function.
Our most provocative initial finding is the observation
that a single amino acid substitution (R332P) in the human Trim5␣ protein can confer the ability to restrict
HIV-1 replication. Although we do not know the sequence of the ancestral Trim5, we note that rhesus and
African green monkey sequences contain a proline at
this position, and these species are resistant to HIV-1.
This implies that during evolution, changes at this position affecting sensitivity to HIV-1 infection may have
occurred. Without such changes it seems unlikely that
the cross-species infection(s) leading to the current
AIDS epidemic [30] would have occurred. In this context,
it is tempting to speculate that the R332P and/or
RYQTFV335LFTFPSLT derivatives of human Trim5␣
might be suitable candidate(s) for gene therapy of HIV-1
infection, perhaps introduced in combination with
agents that target other steps in the viral life cycle such
as the Vif-insensitive, K128D derivative of human APOBEC3G [31–33] or HIV-1 siRNA [34].
Acknowledgments
This work was supported by the UK Medical Research Council.
Received: October 19, 2004
Revised: November 4, 2004
Accepted: November 5, 2004
Published: January 11, 2005
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