- Open Access
Both TRIM5α and TRIMCyp have only weak antiviral activity in canine D17 cells
© Bérubé et al; licensee BioMed Central Ltd. 2007
- Received: 19 June 2007
- Accepted: 24 September 2007
- Published: 24 September 2007
TRIM5α, which is expressed in most primates and the related TRIMCyp, which has been found in one of the New World monkey species, are antiviral proteins of the TRIM5 family that are able to intercept incoming retroviruses early after their entry into cells. The mechanism of action has been partially elucidated for TRIM5α, which seems to promote premature decapsidation of the restricted retroviruses. In addition, through its N-terminal RING domain, TRIM5α may sensitize retroviruses to proteasome-mediated degradation. TRIM5α-mediated restriction requires a physical interaction with the capsid protein of targeted retroviruses. It is unclear whether other cellular proteins are involved in the inhibition mediated by TRIM5α and TRIMCyp. A previous report suggested that the inhibition of HIV-1 by the rhesus macaque orthologue of TRIM5α was inefficient in the D17a canine cell line, suggesting that the cellular environment was important for the restriction mechanism. Here we investigated further the behavior of TRIM5α and TRIMCyp in the D17 cells.
We found that the various TRIM5α orthologues studied (human, rhesus macaque, African green monkey) as well as TRIMCyp had poor antiviral activity in the D17 cells, despite seemingly normal expression levels and subcellular distribution. Restriction of both HIV-1 and the distantly related N-tropic murine leukemia virus (N-MLV) was low in D17 cells. Both TRIM5αrh and TRIMCyp promoted early HIV-1 decapsidation in murine cells, but weak levels of restriction in D17 cells correlated with the absence of accelerated decapsidation in these cells and also correlated with normal levels of cDNA synthesis. Fv1, a murine restriction factor structurally unrelated to TRIM5α, was fully functional in D17 cells, showing that the loss of activity was specific to TRIM5α/TRIMCyp.
We show that D17 cells provide a poor environment for the inhibition of retroviral replication by proteins of the TRIM5 family. Because both TRIM5α and TRIMCyp are poorly active in these cells, despite having quite different viral target recognition domains, we conclude that a step either upstream or downstream of target recognition is impaired. We speculate that an unknown factor required for TRIM5α and TRIMCyp activity is missing or inadequately expressed in D17 cells.
- Feline Immunodeficiency Virus
- African Green Monkey
- Equine Infectious Anemia Virus
- Canine Osteosarcoma
- Canine Osteosarcoma Cell Line
TRIM5α is a primate protein expressed in the cytoplasm of many cell types that is able to inhibit ("restrict") the replication of selected retroviruses [1–3]. Individual TRIM5α alleles are able to restrict a few or many retroviruses (although never all of them). The specificity of the restriction, i.e. the viral targets for each particular TRIM5α allele, is species-dependent more than it is cell type-dependent. The specific recognition of viral targets is determined by the SPRY/B30.2 region at the C-terminus of TRIM5α [4–9]. On the virus side, capsid (CA) proteins seem to be the only determinant of sensitivity to TRIM5α [10–12], and a physical interaction takes place between TRIM5α and capsid, as evidenced by pull-down assays [13, 14]. It is worth noting, however, that the interaction has not yet been documented using purified TRIM5α, and thus it is possible that other cellular factors are relevant to this step. TRIM5α forms trimers and possibly multimers of higher orders of complexity [15, 16]. TRIM5α multimerization is linked to its restriction activity . In addition, TRIM5α targets multimers of properly maturated and assembled retroviral CA constituting the capsid core of incoming viral particles [18, 19]. Thus, the initial TRIM5α-retrovirus interaction might involve the assembly of a multimer of TRIM5α around the capsid core of incoming retroviruses very early after entry.
Following this initial interaction, replication of the restricted retrovirus can be impaired in several ways. First, TRIM5αrh and TRIM5αhu seem to promote premature decapsidation of HIV-1 and N-tropic murine leukemia virus (N-MLV), respectively [14, 20]. More specifically, TRIM5α causes post-entry disappearance of CA in its particulate form, which is assumed to belong to not-yet-disassembled viruses. Second, replication is inhibited by a mechanism involving the proteasome. This is evidenced by the partial rescue of retroviral replication from TRIM5α restriction in the presence of the proteasome inhibitor MG132 [21, 22]. In addition, the ubiquitin ligase activity associated with the RING domain of TRIM5α is important for full restriction activity . It has also been proposed recently that TRIM5αrh might promote the degradation of HIV-1 CA through a non-proteasomal, non-lysosomal pathway . Thirdly, TRIM5α interferes with the nuclear transport of retroviral pre-integration complexes [21, 22]. TRIM5α from the squirrel monkey seems to restrict the mac251 strain of simian immunodeficiency virus (SIVmac251) mostly, if not only, by inhibiting this nuclear transport step .
Interestingly, a recent report pointed to late steps (i.e. assembly and release) of retroviral replication as possibly targeted by TRIM5α, although the molecular basis for late-stage restriction specificity is distinct from that of early stages .
In the owl monkey, a New World species, the SPRY/B30.2 domain of TRIM5α is replaced by the full coding sequence of the highly conserved, ubiquitously expressed peptidyl-prolyl isomerase Cyclophilin A (CypA), yielding a protein called TRIMCyp or TRIM5-CypA [26, 27]. TRIMCyp inhibits HIV-1, the African green monkey strain of SIV (SIVAGM), feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV) [28–30]. CypA was isolated fifteen years ago as a cellular protein interacting with HIV-1 CA  and TRIMCyp binds CA through its CypA domain [27, 28]. CypA-CA interaction and TRIMCyp-mediated restriction are abrogated in the presence of cyclosporine (CsA), a drug that targets the same structural motif in CypA to which CA binds [27, 32]. Like TRIM5αrh, TRIMCyp causes an early block to HIV-1 replication, preventing the accumulation of retroviral cDNA in the infected cells [16, 28, 33]. Prior to the present work, however, it was not known whether TRIMCyp promoted HIV-1 premature decapsidation.
Are other cellular factors important for the restriction mediated by TRIM5α? Efficient inhibition of HIV-1 by TRIM5α in several Old World monkey cell lines requires the presence of CypA, as seen by gene knock-down [34, 35]. The proposed model  is that CypA catalyzes the cis-trans isomerization of HIV-1 CA at proline 90 , thus turning it into a target for some simian TRIM5α orthologues. However, the impact of CypA on the restriction of HIV-1 is much less significant when TRIM5α is over-expressed in non-primate cells [34, 35, 37]. It is not clear whether other cellular proteins are important in the steps leading to the initial viral recognition step. Downstream of this TRIM5α-target interaction, it is expected that cellular proteins take part in the targeting of restricted viruses to proteasome-dependent degradation, although the exact mechanism has not been elucidated yet. Whether cellular proteins other than TRIM5α are also required for CA premature decapsidation and the inhibition of nuclear transport is totally unknown.
The restriction phenotype stemming from TRIM5α and TRIMCyp activity is retained upon expression of these proteins in non-primate cells such as murine and feline cells, suggesting that if cellular factors other than TRIM5α are required, they must be widely conserved among mammals. However, the Poeschla group recently reported that restriction of HIV-1 by the rhesus macaque TRIM5α orthologue was inefficient in D17 cells, a canine osteosarcoma cell line . As a first step toward the isolation of additional factors involved in the restriction by TRIM5α, we decided to characterize further the restriction phenotype in the D17 cells.
We then challenged the cell lines generated with N-MLV and B-MLV vectors expressing GFP. Upon infection with multiple virus doses, we found as expected that N-MLV was 10- to 12-fold less infectious in the MDTF cells expressing the human or African green monkey orthologues of TRIM5α, compared with the control cells (Fig. 2A). In the D17 cells, however, the magnitude of restriction by TRIM5αhu or TRIM5αAGM was only to 2- to 3-fold. As expected, B-tropic MLV replication was not affected by any of the TRIM5α orthologues. In an independent experiment, we infected all the MDTF and D17 cell lines generated with N-MLVGFP and B-MLVGFP at a single virus dose. TRIM5αAGM and TRIM5αrh each inhibited N-MLV infection by about 100-fold in the MDTF cells, and TRIM5αhu had an even greater inhibitory effect (Fig. 2B). In contrast, restriction in the D17 cells was much smaller (about 10-fold) (Fig. 2B). As expected, N-MLV was not inhibited by TRIMCyp and B-MLV was not inhibited by either TRIM5α or TRIMCyp.
Both TRIMCyp-mediated restriction of HIV-1 and enhancement of HIV-1 replication by CsA are more efficient in the MDTF cells compared with the D17 cells (Fig. 3 and 4). Thus, we examined the effect of CsA on the levels of particulate CA in MDTF-TRIMCyp and D17-TRIMCyp cells (Fig. 6). Like before, the decrease in particulate CA caused by TRIMCyp was more acute in the MDTF cells compared with the D17 cells (5-fold versus 1.6-fold). In addition, CsA restored wild-type levels of particulate CA in both cell types, although, as expected, the magnitude of this effect was greater in the MDTF cells.
The mechanism by which TRIM5α and TRIMCyp intercept and inhibit incoming retroviruses is incompletely understood. TRIM5α is able to trimerize in cells, and it is probably in this form (or as a multimer of higher complexity) that it recognizes its viral target [15, 17]. This initial interaction is followed by the disappearance of particulate CA but not soluble CA. The loss of particulate CA has been attributed to an acceleration of viral uncoating in restrictive conditions [14, 20]. However, as observed here and by others , the decrease in particulate HIV-1 CA in restrictive conditions is not necessarily accompanied by an increase in soluble CA. Thus, it remains possible that incoming retroviral cores are not disassembled faster under TRIM5α/TRIMCyp restriction but instead are specifically targeted to a degradation pathway. Accordingly, pharmacological approaches have revealed a role for the proteasome in the restriction mediated by TRIM5α [21, 22]. Of course, the two models are not mutually exclusive, as proteasome-mediated degradation might well follow premature decapsidation.
We find retroviral restrictions mediated by either TRIM5α or TRIMCyp (but not Fv1) to be poorly efficient in the canine cells D17. These results confirm and extend previous findings by Saez and colleagues . The restriction defect did not appear to be caused by poor expression or mislocalization of TRIM5α or TRIMCyp. Consistent with the HIV-1GFP transduction data, TRIM5α and TRIMCyp had little effect on the accumulation of HIV-1 cDNA in D17 cells. In addition, TRIM5α and TRIMCyp induced the disappearance of HIV-1 particulate CA at relatively low rates in D17 cells, compared with the MDTF cells. Therefore, D17 cells provided a poor environment for the restriction. We hypothesize that a cellular factor important for the activity of TRIM5α and TRIMCyp is not functional or is expressed at low levels in these cells. The missing factor might be important for TRIM5 multimerization or for its interaction with the proteasome. Conversely, a dominant negative factor might be expressed in the D17 cells. That both N-MLV and HIV-1 were less restricted in D17 cells implies that CypA is not relevant to the observed phenotype. Reciprocally, it is unlikely that the SPRY/B30.2 domain of TRIM5α is relevant to its loss of function in the D17 cells, since a similar effect was observed with TRIMCyp.
The canine D17 cells offer a cellular context that is unfavorable to the restriction mechanism mediated by TRIM5α and TRIMCyp. This cell line may thus represent a unique opportunity to isolate and characterize cellular genes regulating retroviral restrictions.
pMIP-TRIM5αrh-FLAG, pMIP-TRIM5αAGM-FLAG, pMIP-TRIM5αhu-FLAG, and pMIP-TRIMCyp-FLAG express C-terminal FLAG tagged versions of cDNAs amplified respectively from rhesus macaque FRhK4 cells, African green monkey Vero cells, human TE671 cells, or owl monkey OMK cells, and were generous gifts from Jeremy Luban . pCLNCX-Fv1b , which encodes both Fv1b and the red fluorescent protein (RFP), was a kind gift of Greg Towers (University College, London). pMD-G, pΔR8.9, pTRIP-CMV-GFP, pCL-Eco, pCIG3N, pCIG3B and pCNCG have all been extensively described before [44–49].
Cells and virus production
Human embryonic kidney 293T, human cervical epithelial carcinoma cells HeLa, mus dunni tail fibroblasts (MDTF; a gift from Jeremy Luban) and canine osteosarcoma D17 cells (a kind gift from Monica Roth) were all grown in DMEM medium supplemented with 10% fetal bovine serum and antibiotics. All viruses used in this study were produced through transient transfection of 293T cells using polyethylenimine. For that, a mixture of the appropriate DNAs diluted in 1 ml of DMEM without serum or antibiotics was mixed with 45 μl of a 1 mg/ml solution of polyethylenimine (Polysciences). This transfection mix was then added to 70% confluent 293T cells in a 10-cm tissue culture dish. The next day, cells were PBS-washed once and put back in culture in fresh medium. 2 days after transfection, virus-containing supernatants were collected, clarified by low-speed centrifugation and stored in 1-ml aliquots at -80°C.
To produce the CLNCX and MIP vectors used to transduce fv1b and the various TRIM5 alleles, the transfection mix included 10 μg of pCL-Eco, 5 μg of pMD-G, and 10 μg of the appropriate pMIP or pCLNCX construct. To produce the N-MLVGFP and B-MLVGFP vectors, the transfection mix included 10 μg of pCIG3 N or B, 5 μg of pMD-G, and 10 μg of pCNCG. To produce the HIV-1GFP vector, cells were transfected with 10 μg of pΔR8.9, 5 μg of pMD-G, and 10 μg of pTRIP-CMV-GFP.
TRIM5-expressing cell lines
HeLa and D17 cells were plated at 300,000 cells per well and MDTF cells were plated at 140,000 cells per well in 6-well plates. The next day, supernatants were aspirated and replaced with MIP-TRIM5α or MIP-TRIMCyp vector preparations (2 ml per well). 2 days later, cells were placed in medium containing 1 μg/ml (HeLa, D17) or 3 μg/ml (MDTF) of puromycin (EMD Biosciences). These puromycin concentrations were determined to kill all sensitive cells after one or two days of treatment. Puromycin selection was allowed to proceed for 4 days, and then again periodically during the course of this work. Expression of the transduced TRIM5 cDNAs was analyzed by western blotting, using antibodies directed against the FLAG epitope (mouse monoclonal; Sigma) or actin (goat polyclonal; Santa Cruz).
Cells were plated at 25,000 cells (HeLa, D17) or 10,000 cells (MDTF) in 0.4 ml per well of 24-well plates. Cells were infected the next day with HIV-1GFP, N-MLVGFP, or B-MLVGFP vectors. When CsA (Sigma) or nevirapine were used, they were added 15 min prior to the virus. Cell supernatants were replaced with fresh medium without drugs 16 h after infection. 2 days after infection, cells were trypsinized and fixed in 2% formaldehyde-PBS. Flow cytometry was done on a FC500 MPL instrument (Beckman Coulter) using the CXP software for analysis. Intact cells were identified based on light scatter profiles, and only those cells were included in the analysis. Ten thousand cells per sample were processed, and cells positive for GFP expression were gated and counted as a percentage of total intact cells. Cells expressing Fv1b and RFP were first gated for RFP expression and infected cells were computed as % of cells expressing both RFP and GFP among all RFP-positive cells. False-positive results were insignificant, as shown by controls corresponding to uninfected cells (not shown).
Cells were plated at 24,000 (MDTF) or 50,000 (D17) on LabTek II four-chamber slides (LabTek). The next day, cells were washed with PBS, fixed for 30 min in 4% formaldehyde-PBS, washed three times in PBS and permeabilized with 0.1% Triton X-100 for 2 min on ice. Cells were then washed again with PBS and treated with 50 mM NH4Cl (in PBS) for 10 min at RT. Then, cells were washed 3 times in PBS and treated with 10% normal goat serum (Vector laboratories) for 30 min at RT. This saturation step was followed by incubation with an antibody against FLAG (M2 mouse monoclonal; Sigma) at a 1:400 dilution in PBS with 10% normal goat serum. Fluorescent staining was done using an Alexa488-conjugated goat anti-mouse antibody (Molecular Probes) at a 1:500 dilution. Cells were washed 4 times in PBS before mounting in Vectashield (Vector Laboratories). Hoechst33342 (0.8 μg/ml; Molecular Probes) was added along with the penultimate PBS wash to reveal DNA. Pictures were generated using a Olympus BX-60 microscope with the Image-Pro Express software.
The protocol used was adapted from Stremlau et al . Cells were plated at 80% confluence in 10-cm culture dishes. 12 hours later, they were layered with 8 ml of HIV-1 VLPs, which is a high MOI (equivalent to 50–80% infected control cells by HIV-1GFP). VLP infections were performed in the presence or absence of nevirapine (80 μM) or CsA (5 μM). 4 hours later, supernatants were replaced with fresh media containing the appropriate drugs and the cells were put back in culture for an additional 2 hours. Cells were then lysed in 1.5 ml of a hypotonic lysis buffer (100 μM Tris-Cl pH8.0, 0.4 mM KCl, 2 μM EDTA) containing a protease inhibitor mix (Sigma). After Dounce homogenization (15 strokes) and clarification by low-speed centrifugation, 50 μl of the lysate were saved ("whole lysate"), and 1 ml was layered on top of a 50% sucrose cushion prepared in PBS. Particulate CA was sedimented by ultracentrifugation using a Beckman SW41Ti rotor. The centrifugation was carried in Beckman Ultraclear tubes for 2 hours at 32,000 rpm and at 4°C. Following this step, 200 μl of the supernatants were carefully transferred to a fresh tube and lysed in SDS sample buffer. Remaining supernatant and sucrose cushions were discarded by carefully inverting the tubes, and pellets were resuspended in 50 μl of SDS sample buffer. Equal volumes of whole cell lysate, supernatant, and pellet fractions were processed for western blotting using a anti-CA mouse monoclonal antibody (clone 183; a gift of Jeremy Luban)
Monitoring HIV-1 cDNA synthesis
50,000 cells (D17) or 20,000 cells (MDTF) were plated in 0.4 ml per well in 24-well plates. 12 hours later, cells were infected with 10 μl HIV-1GFP that had been treated with DNase I (NEB; 23 U/ml of virus preparation) for 10 min at 25°C. Cells were washed with PBS and trypsinized after 12 hours of infection. Total cellular DNA was extracted using the DNeasy kit (Qiagen) and digested for one hour at 37°C with Dpn1 to further reduce contamination of the samples with plasmid DNA. Aliquots (5 μl out of 200 μl) of each sample were submitted to a 30-cycle PCR analysis using the following oligodeoxynucleotides: GFPs, 5'-GACGACGGCAACTACAAGAC and GFPas, 5'-TCGTCCATGCCGAGAGTGAT. PCR products were separated on a 2% agarose-TAE gel, and revealed with ethidium bromide staining. For real-time PCR analysis, 2 μl of each DNA preparation were subjected to a 45-cycle PCR in 20 μl total volume containing 10 μl of QuantiTect SYBR Green PCR master mix (Qiagen). Amplification curves were analyzed with Light Cycler relative quantification software v1.0, and quantifications were determined relative to dilutions of pTRIP-CMV-GFP.
We thank Jeremy Luban, Greg Towers and Monica Roth for the generous gift of reagents. We also thank Valérie Leblanc and Marie-Claude Déry for their help with real-time PCR analysis and IF microscopy. Nevirapine was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. This work was supported by the Canadian Institutes for Health Research, Institute of Infection and Immunity.
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