Replacement of a small segment of huTRIM5α in the B30.2/SPRY domain confers potent anti-HIV-1 activity.
Of the TRIM5α variants in primates characterized thus far, several have broad antiretroviral activity, and only the human protein lacks the ability to inhibit HIV-1. We adopted a gain-of-function approach to determine which sequences in huTRIM5α are responsible for its inability to inhibit HIV-1. Like other members of the tripartite motif family of proteins, TRIM5α contains RING, B-box, and coiled-coil domains (25
). In addition, TRIM5α contains a carboxy-terminal B30.2/SPRY domain. The rhesus monkey and human TRIM5α proteins share 87% amino acid identity (28
), and many of the differences cluster in the B30.2/SPRY domain (Fig. ). In assays where TRIM5α is overexpressed by using retroviral vectors, both proteins restrict N-MLV, but only rhTRIM5α inhibits HIV-1 (13
). Therefore, we generated a series of chimeras of human and rhesus TRIM5α proteins (see Fig. ). The chimeric proteins were appended with a C-terminal epitope tag from influenza virus HA and were stably expressed by using retrovirus vectors in MDTF cells. As a control, the parental wild-type huTRIM5α and rhTRIM5α proteins, also containing a C-terminal HA tag, were stably expressed in MDTF cells. The addition of an HA tag to the C terminus of TRIM5α did not affect the efficiency with which the proteins inhibited infection (28
; data not shown). Cells expressing the intact and chimeric TRIM5α proteins were then infected with a GFP-expressing HIV-1-based vector. The vector was packaged by using either an intact HIV-1 Gag-Pol expression plasmid or an otherwise identical Gag-Pol expression plasmid encoding a capsid domain from SIVMAC
, termed HIV(SIVCA), which is not sensitive to rhTRIM5α (13
As can be seen in Fig. , a chimera that contained the C-terminal half (residues 237 to 497) from rhTRIM5α and the N-terminal half of huTRIM5α, termed hu(rh237-497), conferred resistance to infection by the HIV-1 vector to the same extent as did the intact rhTRIM5α protein. Neither protein affected the infectivity of the HIV(SIVCA) vector. Conversely, the reciprocal rh(hu235-493) chimera, like huTRIM5α and unlike rhTRIM5α, did not confer resistance to HIV-1 infection (Fig. ). Western blot analysis showed that each of these proteins was expressed at similar levels (Fig. ), and minor variations did not correlate with restriction activity. Thus, the C-terminal half of the rhTRIM5α contains determinants required for restriction of HIV-1 infection that are lacking in huTRIM5α. Therefore, further huTRIM5α-based chimeras containing smaller portions of the C-terminal half of the rhTRIM5α protein were generated (Fig. ). Cells expressing the rh(hu372-493) chimera were as susceptible to HIV-1 vectors as cells expressing huTRIM5α, whereas cells expressing the hu(rh237-376) chimera behaved similarly to cells expressing rhTRIM5α in that they were highly resistant to HIV-1 but susceptible to HIV(SIVCA) (Fig. ). All of the aforementioned chimeras conferred resistance to N-MLV but not B-MLV (data not shown), as do both the intact rhesus and human TRIM5α proteins. In contrast, two reciprocal chimeras, hu(rh376-497) and rh(hu235-372), although efficiently expressed, did not confer resistance to any retrovirus tested, including N-MLV. Because we cannot be certain that hu(rh376-497) and rh(hu235-372) were inactive for irrelevant reasons, such as misfolding, they were considered uninformative (data not shown). Nonetheless, these results show that huTRIM5α amino acids 235 to 372 harbor the defect that confers inactivity against HIV-1 infection, which can be rescued by replacement with their rhTRIM5α (amino acid 237 to 376) equivalents.
To determine more precisely the amino acids within this region responsible for the differential ability of huTRIM5α and rhTRIM5α to inhibit HIV-1, we generated further chimeras (see Fig. ). In contrast to the chimeras shown in Fig. , some of these additional chimeras exhibited intermediate HIV-1 restriction activity, and in several cases it was difficult to distinguish whether the cells that became infected did so because of partial restriction activity or because there exists a small fraction of the transduced pool of MDTF cells that did not express the chimeric TRIM5α proteins. Indeed, immunofluorescence analysis with an α-HA antibody revealed that a few cells in the TRIM5α-expressing cell pools expressed undetectable levels of TRIM5α, and these were preferentially infected by the HIV-1 vector (data not shown). Therefore, to ensure that any differences in the apparent degree of restriction activity among TRIM5α chimeras was not due to variable frequencies of cells that did not express the chimeras, single-cell clones of MDTF cells expressing each chimera were isolated and tested. The results from these experiments are shown in Fig. . MDTF clones expressing intact huTRIM5α and rhTRIM5α were also derived for control purposes and, as expected, clones in which 100% of cells expressed the rhTRIM5α proteins were more resistant to HIV-1 infection than were rhTRIM5α-expressing pools (compare Fig. and Fig. ). For each chimera, at least two representative single-cell clones were used, and similar levels of chimeric TRIM5α expression in each of these clones were verified by Western blot analysis (Fig. ). Each pair of clones behave similarly, and for simplicity results from one clone of each pair is shown in Fig. .
Within the 237- to 376-amino-acid region of rhTRIM5α, residues that were different to the huTRIM5α cluster between amino acids 325 and 344. This coincides with a position in the TRIM5α protein where the AGM TRIM5α carries a 20-amino-acid insertion relative to huTRIM5α. We reasoned that this apparently hypervariable segment of TRIM5α was a likely candidate for the determinant of the inability of huTRIM5α to inhibit HIV-1 and therefore replaced a short 18-amino-acid stretch in huTRIM5α with the corresponding rhTRIM5α sequence to generate the hu(rh325-344) chimera (Fig. ). The level of resistance to HIV-1 induced by hu(rh325-344) was almost 100-fold and comparable to that observed in single cell clones expressing rhTRIM5α (Fig. ). Thus, rhTRIM5α amino acids 325 to 344 contain determinants that are able to confer on huTRIM5α the ability to potently inhibit HIV-1 infection.
We also tested the degree to which chimeras induced resistance to N-MLV because this property differed between huTRIM5α, which restricted N-MLV by >200-fold, and rhTRIM5α, which restricted N-MLV by ~20-fold (Fig. ). Interestingly, the hu(rh325-344) chimera inhibited N-MLV infection as potently as huTRIM5α and more potently than rhTRIM5α, suggesting that residues outside the hypervariable segment contribute to the potency with which huTRIM5α restricts N-MLV. In addition, clones expressing the hu(rh325-344) chimera exhibited a modest degree of resistance to infection by the HIV(SIVCA) vector (Fig. ). These cells were equivalently susceptible to B-MLV, demonstrating that the observed differences in N-MLV, HIV-1, and HIV(SIVCA) sensitivity were not due to a generalized loss of susceptibility to retroviral infection in the cell clones expressing this chimera (Fig. ). This finding indicates that, at least when overexpressed, chimeric TRIM5α proteins can also inhibit retroviruses that are not targeted by either parental TRIM5α variant. Thus, the specificity with which incoming retroviral capsids are recognized is likely determined in a combinatorial manner by more than one sequence motif in TRIM5α.
Within the 323-340/325-344 hypervariable segment of huTRIM5α/rhTRIM5α, 11 of 18 residues are different and 2 residues are inserted in rhTRIM5α compared to huTRIM5α (Fig. ). To determine whether the entire rhTRIM5α hypervariable segment was required for HIV-1 inhibition, we generated additional huTRIM5α-based chimeras containing the N-terminal (residues 325 to 335) or C-terminal (residues 336 to 344) halves of the rhTRIM5α segment (Fig. ). The hu(rh325-335) chimera indeed induced resistance to HIV-1, but the degree to which this occurred (12-fold resistance) was less than that induced by hu(rh325-344) or rhTRIM5α. The hu(rh336-344) chimera also induced modest resistance to HIV-1 (four- to fivefold). Both of these chimeras strongly restricted N-MLV but did not affect B-MLV infection (Fig. ). Two further chimeras were generated that contained larger segments of rhTRIM5α in addition to the N-terminal or C-terminal halves of the hypervariable segment, namely, hu(rh237-335) and hu(rh336-376) (Fig. ). Both chimeras conferred levels of resistance to HIV-1 comparable to those encoding only the respective halves of the hypervariable segment (Fig. ), suggesting differences between huTRIM5α and rhTRIM5α outside the 325 to 344 segment do not contribute in a major way to HIV-1 specific restriction. Again, there were only minor variations in the expression level of each of the chimeras that did not correlate with restriction activity (Fig. ). Therefore, transfer of the entire 325 to 344 segment from rhTRIM5α to huTRIM5α was both necessary and sufficient to attain the levels of anti-HIV-1 activity induced using the wild-type rhTRIM5α protein.
huTRIM5α domains required for anti-N-MLV activity.
Although sequence differences in the B30.2/SPRY domain account for the inability of huTRIM5α to restrict HIV-1, huTRIM5α is clearly able to confer resistance to other retroviruses such as N-MLV and equine infectious anemia virus (13
). Therefore, we next sought to determine which TRIM5α domains are required for N-MLV restriction activity. We generated a series of truncation mutants of huTRIM5α, removing entire domains but retaining the HA tag at the C terminus of each protein. For simplicity, the mutants are named according to which of the four domains, namely, RING (R), B-box (B), coiled-coil (CC), and SPRY, were retained in the truncation mutant (Fig. ). These mutants were stably expressed in MDTF cells, and their restriction activity was tested against N-MLV, which is highly sensitive to full-length huTRIM5α, and B-MLV, which is resistant (13
FIG. 3. TRIM5 domains required for retrovirus restriction. (A) Schematic representation of truncation mutants of huTRIM5α (see the text for details). (B) Expression of TRIM5α truncation mutants in MDTF cells. Lysates from each pool of transduced (more ...)
The truncated RBCC, BCCSPRY, and CCSPRY proteins were expressed as efficiently as full-length huTRIM5α (Fig. ). Indeed, the BCCSPRY mutant, which lacks the RING domain did exhibit a weak but significant (twofold) inhibitory activity against N-MLV (Fig. ). Conversely, the CCSPRY mutant, which lacks both the RING and the B-box domains mutant was devoid of restriction activity (Fig. ), as was the RBCC mutant, which lacks the SPRY domain. (Fig. ). Of the remaining mutants, the RB protein was expressed at very low levels and was detectable only after overexposure of the Western blot, and expression of the isolated SPRY protein was undetectable (data not shown). Not surprisingly, therefore, these truncated proteins did not exhibit any N-MLV restriction activity. The CC protein was not tested in this assay because, based on the fact that it lacks two domains (RB and SPRY) that appeared necessary for activity necessary, (Fig. ) it was not expected to be active.
Truncated huTRIM5α proteins that multimerize are dominant inhibitors of restriction activity.
It has been previously shown that expression of rhTRIM5γ, a form of TRIM5 that lacks the SPRY domain and anti-HIV-1 activity, relieves the block to HIV-1 infection when expressed in rhesus cells but that RING domain mutants lack this activity (28
). We sought to determine whether the huTRIM5α truncation mutants depicted in Fig. could exhibit dominant-negative activity when expressed in HeLa cells. N-MLV is strongly restricted in HeLa cells, and this phenotype is accentuated by transduction with a retrovirus vector expressing huTRIM5α (Fig. ). However, expression of either the RBCC or the CCSPRY proteins completely abolished restriction, and HeLa cells expressing these truncated proteins were approximately equivalently sensitive to N-MLV and B-MLV (Fig. ). The BCCSPRY protein was also efficiently expressed in HeLa cells (Fig. ) and also significantly enhanced N-MLV titers therein. However, it did not completely abolish restriction (Fig. ), probably because it has weak restricting activity itself (Fig. ). Notably, the CC protein, which encodes only the TRIM5 coiled-coil domain also suppressed restriction activity in HeLa cells. Incomplete suppression by the CC protein may be due to the fact that it is expressed at very low levels compared to the RBCC and CCSPRY proteins (Fig. ). Thus, each truncation mutant that retained the coiled-coil domain of huTRIM5α acted as a dominant inhibitor of the activity of the full-length protein endogenously expressed by HeLa cells.
FIG. 4. TRIM5α domains required for dominant-negative activity and multimerization. (A) Inhibition of restriction by huTRIM5α truncation mutants expressed in HeLa cells. Unmodified HeLa cells (control) or HeLa cells stably expressing full-length (more ...)
As was the case in MDTF cells, neither the RB protein nor the SPRY protein were well expressed in HeLa cells, and neither suppressed N-MLV restriction therein (Fig. and data not shown). Nevertheless, we could not exclude the possibility that these proteins could also have dominant-negative activity if it were possible to overexpress them. Consequently, these proteins were fused to the N terminus of GST in an attempt to generate fusion proteins with improved expression levels. This strategy was successful in the case of RB-GST fusion protein, which was expressed in transduced HeLa cells at high levels (Fig. ). However, RB-GST overexpression did not relieve restriction (Fig. ). As a control, an RBCC-GST fusion was also generated and restored N-MLV infectivity as efficiently as the original, HA-tagged RBCC protein (Fig. ). In fact, this protein was quite a potent inhibitor of endogenous TRIM5α restriction activity because we were unable to detect RBCC-GST protein expression (Fig. ). It should be noted that the GST antibody is less efficient than the HA antibody (data not shown); thus, the expression levels of HA versus GST fusion proteins cannot be meaningfully compared. Nonetheless, the experiments in Fig. demonstrate that expression of the huTRIM5α coiled-coil domain in the context of a truncated protein is necessary and sufficient to inhibit the activity of the full-length protein.
Coiled-coil domains often promote protein multimerization and, like many TRIM proteins, TRIM5α has been reported to homomultimerize (25
). Thus, it seemed possible that the dominant-negative activity of the truncation mutants was due to their ability to multimerize with the full-length protein. To test this, the truncation mutants depicted in Fig. were also tested for their ability to multimerize with the full-length protein in a yeast two-hybrid assay. In this assay, huTRIM5α exhibits quite strong homomultimerization activity (Fig. ). All of the truncated proteins retaining the coiled-coil domain, namely, BCCSPRY, CCSPRY, and CC, bound to full-length huTRIM5α in the yeast two-hybrid assay. The exception was the RBCC truncation mutant which, surprisingly, was not expressed in yeast cells (Fig. ). Proteins that lacked the coil-coil domain, namely, RB and SPRY, failed to multimerize with full-length huTRIM5α even though they were efficiently expressed in yeast cells (Fig. ).
To confirm these findings in mammalian cells, each HA-tagged TRIM5α truncation mutant was transiently expressed in 293T cells together with a GST-huTRIM5α fusion protein or, as a control, an unfused GST protein. Protein complexes were then precipitated by using glutathione-Sepharose and analyzed by Western blotting with an α-HA antibody. Full-length huTRIM5α and all of the truncation mutants retaining the coiled-coil domain coprecipitated with GST-huTRIM5α (Fig. ). The expression of the RB protein was easily detected in transiently transfected 293T cells, albeit at lower levels than the other truncation mutants, but coimmunoprecipitation with GST-huTRIM5α was not detected even after overexposure of the Western blot (Fig. and data not shown). The CC protein appeared to interact at least as strongly with GST-huTRIM5α as did any other truncation mutant or full-length TRIM5α, based on their relative expression levels (Fig. ). Overall, the ability of each huTRIM5α truncation mutant to multimerize with the full-length protein in mammalian cells correlated with their ability to do so in the yeast two-hybrid assays and with their ability to inhibit the activity of endogenous huTRIM5α in HeLa cells. Thus, the coiled-coil domain is necessary and sufficient for huTRIM5α multimerization and for dominant inhibition of the restriction activity of the full-length huTRIM5α protein.
Subcellular localization of restricting and nonrestricting, intact and truncated TRIM5α proteins.
Previously, TRIM5 has been reported to concentrate in large and discrete structures in the cytoplasm, termed cytoplasmic bodies (25
). To examine whether this is a property of TRIM5α that is gained or lost in active and inactive TRIM5α truncation mutants and chimeras, we analyzed their localization when stably expressed in MDTF and/or HeLa cells by immunofluorescence with the α-HA antibody. In MDTF cell clones that strongly restrict N-MLV or HIV-1 infection (for example, see Fig. ), huTRIM5α and rhTRIM5α did not accumulate in large cytoplasmic bodies but were distributed throughout the cell cytoplasm (Fig. ). High-resolution deconvolution imaging revealed that both huTRIM5α and rhTRIM5α were present in small puncta and, upon close examination, TRIM5α seemed to form a three-dimensional irregular net-like structure with α-HA reactive linkages between the puncta (Fig. , upper-panel insets).
FIG. 5. Subcellular localization of TRIM5α variants, chimeras and deletion mutants. MDTF or HeLa cells, as indicated, were transduced with LNCX2-based retrovirus vectors expressing the indicated TRIM5-derived proteins bearing a C-terminal HA epitope tag. (more ...)
It is likely that the differences in localization between this and previously published data using transiently transfected GFP-TRIM5 forms (25
) are due to lower expression levels of the tagged TRIM5α proteins in our study. Indeed, we were able to reproduce the previous findings of large cytoplasmic bodies in 293T cells transiently transfected with huTRIM5α-HA and rhTRIM5α-HA expression plasmids (data not shown). Thus, distributions observed in Fig. should be interpreted with caution in the absence of a definitive phenotype for endogenously expressed TRIM5α because, even in the context of the stable cell lines shown in Fig. , TRIM5α is overexpressed. Nonetheless, the active and inactive TRIM5α chimeras and mutants did not appear to differ significantly in their subcellular distribution (Fig. and data not shown). The exceptions to this was the CC and, to a lesser extent, the CC-SPRY truncation mutants that were observed in the nucleus and in the cytoplasm, presumably because they are small enough to permit passive diffusion through nuclear pores.