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. 2009 Oct;83(19):9672-81.
doi: 10.1128/JVI.00597-09. Epub 2009 Jul 15.

Molecular mechanism of BST2/tetherin downregulation by K5/MIR2 of Kaposi's sarcoma-associated herpesvirus

Mandana Mansouri  1 , Kasinath Viswanathan, Janet L Douglas, Jennie Hines, Jean Gustin, Ashlee V Moses, Klaus Früh
Affiliations

Molecular mechanism of BST2/tetherin downregulation by K5/MIR2 of Kaposi's sarcoma-associated herpesvirus

Mandana Mansouri et al. J Virol. 2009 Oct.

Abstract

K3/MIR1 and K5/MIR2 of Kaposi's sarcoma-associated herpesvirus (KSHV) are viral members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family and contribute to viral immune evasion by directing the conjugation of ubiquitin to immunostimulatory transmembrane proteins. In a quantitative proteomic screen for novel host cell proteins downregulated by viral immunomodulators, we previously observed that K5, as well as the human immunodeficiency virus type 1 (HIV-1) immunomodulator VPU, reduced steady-state levels of bone marrow stromal cell antigen 2 (BST2; also called CD317 or tetherin), suggesting that BST2 might be a novel substrate of K5 and VPU. Recent work revealed that in the absence of VPU, HIV-1 virions are tethered to the plasma membrane in BST2-expressing HeLa cells. By targeting BST2, K5 might thus similarly overcome an innate antiviral host defense mechanism. Here we establish that despite its type II transmembrane topology and carboxy-terminal glycosylphosphatidylinositol (GPI) anchor, BST2 represents a bona fide target of K5 that is downregulated during primary infection by and reactivation of KSHV. Upon exit of the protein from the endoplasmic reticulum, lysines in the short amino-terminal domain of BST2 are ubiquitinated by K5, resulting in rapid degradation of BST2. Ubiquitination of BST2 is required for degradation, since BST2 lacking cytosolic lysines was K5 resistant and ubiquitin depletion by proteasome inhibitors restored BST2 surface expression. Thus, BST2 represents the first type II transmembrane protein targeted by K5 and the first example of a protein that is both ubiquitinated and GPI linked. We further demonstrate that KSHV release is decreased in the absence of K5 in a BST2-dependent manner, suggesting that K5 contributes to the evasion of intracellular antiviral defense programs.

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Figures

FIG. 1.
FIG. 1.
K5 downregulates IFN-induced BST2 in DMVECs infected with KSHV. (A) BST2 is induced by IFN-β and TNF-α in DMVECs. E-DMVECs were treated with 500 U/ml of human IFN-β or 10 ng/ml TNF-α for 24 h or were left untreated (red) prior to flow cytometry with anti-BST2 (HM1.24). (B) K5 downregulates IFN-β-induced BST2 during de novo infection with KSHV. (Top) E-DMVECs were transduced with Ad-K5 (black), Ad-K3 (red), or Ad-TET (gray) for 24 h prior to treatment with IFN-β and flow cytometry for BST2. (Middle) E-DMVECs were infected with KSHV (white) or mock infected (gray) prior to treatment with IFN-β and staining for BST2. (Bottom) E-DMVECs were treated with K5-specific (white) or scrambled (gray) siRNA and infected with KSHV prior to treatment with IFN-β and staining for BST2. (C) K5-specific siRNA inhibits K5 protein expression during primary infection. Duplicate samples from the middle and lower parts of panel B were used to confirm K5 knockdown by K5 siRNA. Cells were lysed and immunoblotted with anti-K5 or anti-GAPDH antibody.
FIG. 2.
FIG. 2.
KSHV inhibits induction of BST2 protein, but not mRNA, upon reactivation. (A) BST2 downregulation upon viral reactivation. (Left) Uninfected E-DMVECs were treated with IFN-β for 24 h (+IFN; gray) or were left untreated (UT; black) prior to being stained with anti-BST2 (HM1.24). (Middle) E-DMVECs latently infected with KSHV were treated with IFN-β (+IFN; gray) or left untreated (UT; black). (Right) Latently infected E-DMVECs were transduced with Ad-RTA (black) or Ad-TET (gray) prior to treatment with IFN-β for 24 h. (B) KSHV does not inhibit induction of BST2 mRNA by IFN-β. BST2 (top) and K5 (bottom) mRNA levels were quantified by qPCR for cells treated as described for panel A. Results were normalized to a β-actin control, and changes compared to untreated, uninfected E-DMVECs were calculated using the comparative threshold cycle method as described previously (46).
FIG. 3.
FIG. 3.
Degradation of BST2 by K5. (A) BST2 is absent from the cell surface in the presence of K5. E-DMVECs were transduced with Ad-K5 or Ad-TET alone for 24 h, followed by treatment with 500 U/ml IFN-β. After 24 h, cell surface-expressed proteins were biotinylated. Upon cell lysis, biotinylated proteins were captured with avidin and separated by SDS-PAGE. BST2 was visualized by immunoblotting with anti-BST2 (top), anti-K5 (middle), or anti-BAP31 (bottom) as a loading control. (B) K5 reduces steady-state levels of BST2. E-DMVECs were transduced with Ad and treated with IFN-β as described for panel A. Cells were lysed in SDS sample buffer, and the lysate was separated by SDS-PAGE followed by immunoblotting with HM1.24 or anti-GAPDH. (C) Increased degradation of endo H-resistant BST2 by K5. Human fibroblasts stably transfected with the lentivector pCDH-BST2-HA were pulse labeled for 15 min, followed by a chase, as indicated. BST was immunoprecipitated with anti-HA after treatment, followed by treatment with endo H or PNGase F when indicated. SDS-PAGE and autoradiography revealed a 24-kDa glycosylated endo H-sensitive precursor that was converted to an ∼34-kDa endo H-resistant glycoprotein. Deglycosylated BST2 migrated at about 17 kDa, consistent with its predicted molecular mass of 19 kDa. A nonspecific 24-kDa band comigrated with the 24-kDa form of BST2 (*).
FIG. 4.
FIG. 4.
Proteasomal but not lysosomal inhibitors prevent K5-mediated BST2 degradation. (A) Proteasome inhibitors restore cell surface expression of BST2 in the presence of K5. Stable transfectants expressing BST2-HA were mock treated (dashed line) or transduced with Ad-K5 (black line) or Ad-TET (gray area) for 24 h, followed by treatment with MG132 (20 μM) or ConA (50 nM) for 12 h. BST2 expression was monitored by flow cytometry using anti-HA. (B) Proteasome inhibitors prevent K5-mediated BST2 degradation. Human fibroblasts stably expressing BST2-HA were metabolically labeled for 15 min, and the label was chased for the indicated times prior to immunoprecipitation and PNGase F treatment. Prior to being labeled, human fibroblasts were pretreated for 12 h with MG132 (20 μM) or left untreated. Immunoprecipitation was done with anti-HA antibody. (C) Overexpression of dominant-negative VPS4-EQ partially restores BST2 surface expression compared to wild-type VPS4 in K5-transfected cells. HeLa cells were cotransfected with control plasmid, K5, or K3 and with wild-type VPS4 or VPS4-EQ. Twenty-four hours later, transfected cells were analyzed by flow cytometry. Surface expression of BST2 on VPS4-expressing cells (as detected by GFP positivity) was measured by flow cytometry using anti-BST2.
FIG. 5.
FIG. 5.
Ubiquitination of cytoplasmic lysines of BST2 is required for K5-mediated degradation. (A) Cytoplasmic lysines are required for BST2 downregulation from the cell surface. Flow cytometry was performed with human fibroblasts stably transfected with BST2 or lysine-deleted BST2-KR and transduced with Ad-K5 (black) or Ad-TET (gray) for 24 h. BST2 was detected with the anti-BST2 antibody. (B) Lysine-deleted BST2 is resistant to K5-mediated degradation. BST2-KR-HA-expressing fibroblasts were transduced with Ad-K5 or Ad-TET for 24 h, followed by pulse-chase metabolic labeling and immunoprecipitation with antibodies to MHC-I (W6/32) or anti-HA. Note that the half-life of MHC-I molecules was decreased in the presence of K5, whereas that of BST2-KR-HA was unaffected. (C) BST2 is ubiquitinated in the presence of K5. BST2-HA-expressing fibroblast cells were transduced with Ad-TET alone or with Ad-TET and Ad-K5 in the presence of MG132, ConA, or no drug for 10 h. The cells were harvested, immunoprecipitated using anti-HA antibody, and immunoblotted with anti-ubiquitin antibody (P4D1). Likewise, BST2-KR-HA-expressing fibroblasts were transduced with Ad-TET alone or with Ad-TET and Ad-K5 for 10 h and then immunoprecipitated and immunoblotted. Nonspecific and unidentified protein bands are indicated. Ab, antibody.
FIG. 6.
FIG. 6.
KSHV K5 facilitates viral release by removing BST2 from HeLa cell surface. (A) Decreased viral release in the presence of K5 siRNA. HeLa cells infected with rKSHV.219 were transfected in triplicate with two K5 siRNAs or control siRNA. The transfections were repeated in 8 h, and 24 h thereafter, Ad-RTA was added to reactivate the virus. After an additional 48 h, cell supernatants were harvested, filtered, and used to infect 293 cells. Forty-eight hours later, GFP fluorescence was monitored in 293 cells by flow cytometry. (B) BST2 siRNA restores KSHV release in the absence of K5. HeLa cells infected with KSHV were transfected with the indicated siRNAs as described for panel A. (C) BST2 and K5 transcript levels upon siRNA treatment. qPCR analysis was performed to determine the relative amounts of BST2 and K5 transcripts in siRNA-transfected HeLa cells infected with KSHV. The K5 transcript level in Ad-RTA-induced KSHV-infected HeLa cells was set to 100%, whereas the BST2 level was set to 100% in the nonactivated KSHV-infected HeLa cells.
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