ABSTRACT
Membrane-associated RING-CH (MARCH) family members are cellular transmembrane proteins that downregulate several host membrane proteins. We have recently reported that one family member, MARCH8, inhibits infection with both HIV-1 and vesicular stomatitis virus G-glycoprotein (VSV-G)-pseudotyped viruses by reducing virion incorporation of envelope glycoproteins. The molecular mechanisms by which MARCH8 targets envelope glycoproteins remain unknown. Here, we show two different mechanisms by which MARCH8 inhibits viral infection. Viruses pseudotyped with the VSV-G mutant, in which cytoplasmic lysine residues were mutated, were insensitive to the inhibitory effect of MARCH8, whereas those with a similar lysine mutant of HIV-1 Env remained sensitive to it. Ubiquitination assays showed that wild-type VSV-G, but not its lysine mutant, was ubiquitinated by MARCH8. Furthermore, the MARCH8 mutant, which had a disrupted cytoplasmic tyrosine motif that is critical for intracellular protein sorting, did not inhibit HIV-1 Env-mediated infection, while it still impaired infection by VSV-G-pseudotyped viruses. Overall, we conclude that MARCH8 impairs viral infectivity by downregulating envelope glycoproteins through two different mechanisms mediated by a ubiquitination-dependent or tyrosine motif-dependent pathway.
INTRODUCTION
Membrane-associated RING-CH (MARCH) 8 is one of 11 members of the MARCH family of RING-finger E3 ubiquitin ligases, which consist of an N-terminal cytoplasmic tail (CT) domain containing a C4HC3 RING finger (RING-CH finger) motif, two transmembrane (TM) domains, between which a short ectodomain is located, and a C-terminal CT domain1,2. MARCH8 downregulates a variety of cellular transmembrane proteins, such as MHC-II3, CD864, CD815, CD446, TRAIL receptor 17, CD986, IL-1 receptor accessory protein8, and transferrin receptor9. We have recently reported that MARCH8 reduces HIV-1 infectivity by downregulating HIV-1 envelope glycoproteins (Env) from the cell surface, resulting in a reduced incorporation of Env into virions10. Intriguingly, vesicular stomatitis virus G-glycoprotein (VSV-G) was even more sensitive to the inhibitory effect of MARCH8. In the case of HIV-1 Env, it is retained intracellularly without degradation after cell-surface downregulation. In contrast, VSV-G is not only downregulated from the cell surface but also undergoes lysosomal degradation by MARCH810. In this regard, we hypothesized that VSV-G, whose cytoplasmic tail is lysine-rich (5 out of 29 amino acids), could be readily ubiquitinated by the E3 ubiquitin ligase MARCH8 and therefore undergo lysosomal degradation, whereas HIV-1 Env carries only two lysines (out of 151 amino acids) in its cytoplasmic tail and may rarely undergo degradation after being trapped by MARCH8. In this study, we created lysine mutants of both HIV-1 Env and VSV-G, together with newly generated MARCH8 mutants to explore the hypothesis described above. The results with these mutants show that MARCH8 targets HIV-1 Env and VSV-G by two different inhibitory mechanisms.
RESULTS AND DISCUSSION
We have recently reported that MARCH8 inhibits lentiviral infection by reducing virion incorporation of both HIV-1 Env and VSV-G in a RING-CH domain-dependent manner. Because the RING-CH domain is known to be essential for the E3 ubiquitin ligase activity of MARCH8, we asked whether these envelope glycoproteins are susceptible to MARCH8-mediated ubiquitination. To investigate this, we first created the VSV-G mutant CT5K/R in which five arginine residues were introduced in place of cytoplasmic lysine residues that could be ubiquitination targets (Fig. 1A, upper). We also generated the HIV-1 Env gp41 mutant CT2K/R harboring two arginines in place of the cytoplasmic lysines (Fig. 1A, lower). Then, we prepared HIV-1 luciferase reporter viruses pseudotyped with the mutant envelope glycoproteins (VSV-G CT5K/R and HIV-1 Env CT2K/R) from 293T cells transiently expressing MARCH8, and compared their viral infectivity with that of control viruses pseudotyped with wild-type (WT) envelope glycoproteins. The infectivity of viruses harboring either VSV-G CT5K/R or HIV-1 gp41 Env CT2K/R was almost comparable to WT-enveloped viruses (Figs. 1B and 1C). As expected, the virus pseudotyped with VSV-G CT5K/R was completely resistant to MARCH8 (Fig. 1B). In contrast, the HIV-1 Env CT2K/R-pseudotyped virus was still susceptible to the inhibitory effect of MARCH8 (Fig. 1C). Consistent with these results, immunofluorescence staining showed that the five lysine mutations in the CT domain of VSV-G conferred resistance to MARCH8-mediated intracellular degradation (Fig. 1D), whereas the two lysine mutations in the CT domain of HIV-1 Env had no effect on its cell-surface downregulation by MARCH8 (Fig. 1E). It should be noted that MARCH8-resistant VSV-G CT5K/R colocalized with MARCH8 (Fig. 1D). We thus speculated that MARCH8 ubiquitinates lysine residues of VSV-G but not of HIV-1 Env at their CT domains.
To investigate this possibility, we performed immunoprecipitation (IP)/Western-based ubiquitination assays. In cells coexpressing WT HA-MARCH8, VSV-G was efficiently ubiquitinated, whereas in cells coexpressing the RING-CH mutant of HA-MARCH8, the ubiquitination of VSV-G was lost. More importantly, the VSV-G lysine mutant CT5K/R did not undergo MARCH8-mediated ubiquitination, as expected (Fig. 1F), suggesting that the lysine residues at the CT domain of VSV-G are specifically ubiquitinated by MARCH8. These findings are consistent with the immunofluorescence results (Figs. 1D and 1E). We therefore conclude that lysine residues at the CT domain of VSV-G are ubiquitinated by MARCH8, which determines the difference in the MARCH8-mediated intracellular fate between these viral glycoproteins.
Because unlike VSV-G, HIV-1 Env was retained intracellularly without degradation, as we previously reported10, we hypothesized that these viral envelope glycoproteins might undergo endocytosis with different mechanisms of action. It has been reported that another MARCH family member, MARCH11, has a conserved tyrosine-based motif, YXXϕ, which is known to be recognized by the adaptor protein (AP) μ-subunits in the C-terminal CT domain11. We therefore looked for the same motif(s) in MARCH8 and found the 222YxxL225 and 232YxxV235 sequences in the CT domain at the C-terminus (Fig. 2A). Based on this finding, we generated tyrosine motif mutants of MARCH8, in which either 222Y or 232Y was mutated to alanine (designated 222AxxL225 or 232AxxV235). The protein expression in cells transfected with each MARCH8 plasmid was confirmed by immunoblotting using an anti-hemagglutinin (HA) antibody (Fig. 2B). Then, we examined whether these YXXϕ motifs are important for the antiviral activity of MARCH8. The infectivity of VSV-G–pseudotyped viruses was still inhibited by the expression of both 222AxxL225 and 232AxxV235 MARCH8 mutants (Fig. 2C). In contrast, the infectivity of HIV-1 Env– pseudotyped viruses was not impaired by 222AxxL225 MARCH8 expression but was reduced by that of 232AxxV235 and WT MARCH8 (Fig. 2D), suggesting that the first tyrosine motif (222YxxL225) is involved in the antiviral activity of MARCH8 against HIV-1 Env but not VSV-G.
We previously reported that the MARCH8-mediated reduction in viral infectivity was due to reduced entry efficiency, resulting from a decreased virion incorporation of envelope glycoproteins through their downregulation from the cell surface10. Therefore, by performing a β-lactamase (BlaM)-fused viral protein R (Vpr)-based entry assay, we first focused on whether the loss of function of 222AxxL225 MARCH8 against HIV-1 Env but not VSV-G would indeed be due to the loss of its inhibitory activity on viral entry. Whereas the entry of VSV-G–pseudotyped HIV-1 prepared from cells expressing either WT or 222AxxL225 MARCH8 was reduced compared with that of the control virus (Fig. 2E), the inhibition of the entry of whole HIV-1 virions was abrogated in viruses produced from cells expressing 222AxxL225, as expected (Fig. 2D). We further analyzed whether this motif of MARCH8 is indeed involved in the reduced virion incorporation of HIV-1 Env, which results from its cell-surface downregulation. To address this, we conducted flow cytometric analysis and quantified the levels of cell-surface and intracellular expression of Env glycoproteins. In accordance with the results obtained in infectivity assays (Fig. 2C), 222AxxL225 MARCH8 still reduced intracellular VSV-G expression as well as WT MARCH8 did (Fig. 2G). On the other hand, the mutant MARCH8 had a completely abrogated ability to downregulate cell-surface HIV-1 Env, whereas WT expression led to the downregulation of HIV-1 Env from the cell surface and its intracellular retention (Fig. 2H), as we previously observed12. The results were consistent with those of the inhibitory activity of MARCH8 against the virion incorporation of HIV-1 Env (Fig. 2I). We therefore conclude that 222YxxL225 is critical for the MARCH8-mediated downregulation of HIV-1 Env but not VSV-G, which results in its reduced virion incorporation leading to impaired viral entry.
In summary, we first show the two different mechanisms by which MARCH8 inhibits viral infections, one being a ubiquitin-dependent downregulation that mediates lysosomal degradation of VSV-G whose cytoplasmic lysine residues are recognized by the RING-CH domain of MARCH8 (Fig. 3, left), and the other, a YxxΦ motif-dependent downregulation that could explain the intracellular retention of HIV-1 Env without degradation after cell-surface downregulation (Fig. 3, right). In terms of the latter mechanism, although this could be attributed to the AP-dependent trafficking that requires the YxxΦ motif, which binds to AP μ-subunits, we were unable to prove the interaction between these subunits and MARCH8 (data not shown). We have recently reported that MARCH1 and MARCH2 are also antiviral MARCH family members that inhibit HIV-1 infection, although their antiviral activity is less robust, which is probably due to the lower protein expression and/or stability than that of MARCH812. Because MARCH1 and MARCH2 also harbor the Yxxϕ motif in their C-terminal CT domains, it would be intriguing to verify the importance of the motif in these proteins. Overall, our present findings are consistent with our previous studies showing that MARCH8-induced downregulation of VSV-G leads to lysosomal degradation, while that of HIV-1 Env results in intracellular retention without degradation. Thus, we conclude that MARCH8 targets HIV-1 Env and VSV-G by two different inhibitory mechanisms (either ubiquitin-dependent or YxxΦ motif-dependent downregulation). Further investigations will clarify the more detailed host defense mechanisms of this protein.
METHODS
DNA constructs
The Env-deficient HIV-1 proviral indicator construct pNL-Luc2-E(-), HIV-1 Gag-Pol expression plasmid pC-GagPol-RRE, HIV-1 Env-expression vector pC-NLenv, HIV-1 Rev expression plasmid pCa-Rev, HIV-1 Tat expression plasmid pLTR-Tat, lentiviral packaging plasmid pMDLg/pRRE, VSV-G expression plasmid pC-VSVg and its C-terminally T7-epitope-tagged version pVSVg-T7E, GFP expression plasmid pCa-EGFP, Vpr/β-lactamase (BlaM) expression plasmid pMM310, and MARCH8 expression plasmid either pC-MARCH8 or pC-HA-MARCH8 and its RING-CH mutant pC-HA-MARCH8-CS have previously been described elsewhere10. The HIV-1 Env gp41 mutant CT2K/R, in which cytoplasmic lysine residues at positions 792 and 806 were mutated to arginine, and the VSV-G mutant CT5K/R, in which cytoplasmic lysine residues at positions 491, 493, 496, 497, and 511 were mutated to arginine residues, were created by inserting overlapping PCR fragments into MfeI/XhoI-digested pC-NLenv and KpnI/XhoI-digested pVSVg-T7E, respectively. The hemagglutinin (HA)-tagged version of the WT and its lysine mutant NL Env expression plasmid were created by inserting the HA-tag linker into the BamHI site located at the gp41 cytoplasmic domain of pC-NLenv and its CT2K/R mutant. The MARCH8 mutant 222AxxL225 or 232AxxV235, in which a tyrosine residue at position 222 or 232 was mutated to an alanine residue, was generated by inserting overlapping PCR fragments into the KpnI/XhoI-digested pCAGGS or XhoI-NotI-digested pCAGGS-NHA to create an untagged or N-terminally HA-tagged expression plasmid. All constructs were verified by a DNA sequencing service (FASMAC).
Cell maintenance and generation of stable cell lines
293T, MT4, HeLa, MAGIC5 (HeLa derivative13), and HOS cells were maintained under standard conditions. Cells were originally obtained from ATCC (except MAGIC5 cells) and routinely tested negative for mycoplasma contamination (PCR Mycoplasma Detection kit, Takara).
Virion production and infectivity assays
To prepare VSV-G-pseudotyped or HIV-1-Env-pseudotyped luciferase reporter viruses, 2.5 × 105 293T cells were cotransfected with 120 ng of the MARCH expression plasmid (WT or RING-CH mutants), 20 ng of pC-VSVg, pC-VSVg-CT5K/R, pC-NLenv, or pC-NLenv-CT2K/R, 500 ng of pNL-Luc2-E(-), and an empty vector up to 1 μg of total DNA, using FuGENE 6. Sixteen hours later, the cells were washed with phosphate-buffered saline, and 1 ml of fresh complete medium was added. After 24 h, supernatants were treated as described above and then harvested. The p24 antigen levels in viral supernatants were measured by an HIV-1 p24 antigen capture ELISA (XpressBio). Transfection efficiencies were normalized to the luciferase activity. To determine viral infectivity, 1 × 104 MAGIC5 cells were incubated with 1 ng of p24 antigen from the HIV-1 supernatants. After 48 h, cells were lysed in 100 μl of One-Glo Luciferase Assay Reagent (Promega). The firefly luciferase activity was determined with a Centro LB960 (Berthold) luminometer.
Immunoblotting assays
Protein expression of constructs was confirmed by Western blot analyses as described elsewhere10,12. Briefly, cells transfected as described above were lysed in 500 μl of lysis buffer containing 1.25% n-octyl-β-D-glucoside, and Complete protease inhibitor mixture (Roche Applied Science). Cell extracts were then subjected to gel electrophoresis and transferred to a nitrocellulose membrane, followed by probing with an anti-HA mouse monoclonal antibody (Sigma-Aldrich, H9658) or an anti-β-actin mouse monoclonal antibody (Sigma-Aldrich, A5316). Proteins were then visualized by chemiluminescence using an ECL Western blotting detection system (GE Healthcare) and monitored by using a LAS-3000 imaging system (FujiFilm).
Viral entry assays
A β-lactamase (BlaM)-fused viral protein R (Vpr)-based entry assay was performed as described elsewhere 10,12. Briefly, HIV-1 particles containing a fusion protein of Vpr and BlaM-Vpr were produced by cotransfection of 293T cells with pNL4-3, pMM31014 encoding BlaM-Vpr, and either pC-MARCH8, pC-MARCH8-222AxxL225, pC-MARCH8-232AxxV235, or the control vector. Similarly, VSV-G-pseudotyped HIV-1 particles containing BlaM-Vpr were prepared by cotransfection with pNL-Luc-E(-), pC-VSVg, pMM310, and either pC-MARCH8, pC-MARCH8-222AxxL225, pC-MARCH8-232AxxV235, or the control vector. The produced viruses were normalized to the p24 antigen level (100 ng) and used for infection of the CD4+ T cell line MT4 (5 × 105 cells) at 37 °C for 4 h to allow viral entry. After extensive washing with Hank’s balanced salt solution (HBSS; Invitrogen), cells were incubated with 1 μM CCF2-AM dye (Invitrogen), a fluorescent substrate of BlaM, in HBSS containing 1 mg ml−1 Pluronic F-127 surfactant (Invitrogen) and 0.001% acetic acid for 1 h at room temperature and then washed with HBSS. Cells were further incubated for 14 h at room temperature in HBSS supplemented with 10% FBS, washed three times with PBS and fixed in a 1.2% paraformaldehyde solution. Fluorescence was monitored at 520 and 447 nm by flow cytometry using BD FACS Canto II (BD Bioscience), and the data were collected and analyzed with BD FACS Diva Software (BD Bioscience).
Env incorporation assays
HIV-1 gp120 ELISA-based Env incorporation assays were described elsewhere12.
Ubiquitination assays
293T cells (5 × 105) were cotransfected with 0.8 μg of pC-VSVg-T7E, 0.2 μg of the empty vector, and 0.8 μg of pC-HA-MARCH8. After 48 h, cells were lysed in TBS-T buffer (50 mM Tris-HCl buffer (pH 7.5), 0.15 M NaCl, 1% Triton X-100, and 0.5% deoxycholic acid) containing a protease inhibitor cocktail and 10 mM N-ethylmaleimide, as an inhibitor of deubiquitination enzymes. The mixture was centrifuged at 21,500 × g for 15 min, and the supernatant was used as total cell lysate for immunoblotting or immunoprecipitation. Fifty microliters of Protein A-coupled Sepharose 4B (GE Healthcare, 17-0780-01) was preincubated for 2 h at 4 °C with 4 μg of the appropriate antibody (anti-T7 epitope rabbit polyclonal antibody; MBL, PM022, H6908, anti-ubiquitin mouse monoclonal antibody; Clone FK2, Cayman, 14220). Total cell lysate was incubated with antibody-coupled Sepharose for 20 h at 4 °C. The Sepharose was washed three times with TBS-T buffer and one time with PBS before the immunoprecipitated proteins were eluted with SDS sample buffer. To evaluate the ubiquitination states of the immunoprecipitated proteins, proteins immunoprecipitated with an anti-T7 epitope rabbit antibody were subjected to Western blotting with an anti-ubiquitin mouse antibody, whereas proteins immunoprecipitated with the anti-ubiquitin mouse antibody were subjected to Western blotting with the anti-T7 epitope rabbit antibody. Immunoreactive bands were detected using an ECL detection kit (ATTO, EzWestLumi plus, WSE-7120) with a ChemiDoc imaging system (Bio-Rad).
Immunofluorescence microscopy
HOS cells were plated on 13-mm coverslips, cotransfected with the indicated plasmids, 0.5 μg of either pC-NLenv or pC-VSVg-T7E, 0.1 μg of pC-Gag-Pol, 0.05 μg of pCa-Rev, and 0.3 μg of the pC-HA-MARCH8 expression plasmids (WT or CS-mutant) using FuGENE6 and cultured for 24 h. For the total staining of both VSV-G and MARCH8, cells were fixed with 4% paraformaldehyde for 30 min on ice and permeabilized with 0.05% saponin. The fixed cells were incubated with both primary antibodies anti-T7 epitope mouse monoclonal antibody (Novagen, 69522-4) and anti-HA goat polyclonal antibody (GenScript, A00168-40). The secondary antibodies, Alexa 488 donkey anti-mouse IgG (Molecular Probes, A-21202) and Alexa 647 donkey anti-goat IgG (Molecular Probes, A-21447) were used for the double staining assay. For the cell surface staining of gp120 protein, cells were incubated with an anti-gp120 goat polyclonal antibody (Abcam, Ab21179) at 4 °C for 5 min and washed with PBS at 4 °C before fixation. Fixation was performed with 4% paraformaldehyde for 30 min on ice, and fixed cells were permeabilized with 0.05% saponin (Sigma-Aldrich) to detect the intracellular expression and localization of MARCH8 proteins. The coverslips were incubated with the anti-HA mouse monoclonal antibody (Sigma Aldrich, H9658) for 1 h, washed with PBS and incubated for 30 min with the secondary antibodies Alexa 488 donkey anti-goat IgG (Molecular Probes, A-11055) and Alexa 647 donkey anti-mouse IgG (Molecular Probes, A-31571) for the double staining assay. Confocal images were obtained with a FluoView FV10i automated confocal laser-scanning microscope (Olympus; Tokyo).
AUTHOR CONTRIBUTIONS
Y.Z., T.T., S.O., H.F. and K.T. performed the experiments and analyzed the data. Y.Z., H.F. and K.T. discussed the data. S.K. provided reagents. K.T. conceived the study, supervised the work and wrote the paper. All authors read and approved the final manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
This work was supported by grants from the Japan Society for the Promotion of Science (KAKENHI, 18K07156) to K.T.