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Novel Role of vBcl2 in the Virion Assembly of Kaposi's Sarcoma-Associated Herpesvirus

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Novel Role of vBcl2 in the Virion Assembly of Kaposi's Sarcoma-Associated Herpesvirus

Qiming Liang et al. J Virol.

Abstract

The viral Bcl-2 homolog (vBcl2) of Kaposi's sarcoma-associated herpesvirus (KSHV) displays efficient antiapoptotic and antiautophagic activity through its central BH3 domain, which functions to prolong the life span of virus-infected cells and ultimately enhances virus replication and latency. Independent of its antiapoptotic and antiautophagic activity, vBcl2 also plays an essential role in KSHV lytic replication through its amino-terminal amino acids (aa) 11 to 20. Here, we report a novel molecular mechanism of vBcl2-mediated regulation of KSHV lytic replication. vBcl2 specifically bound the tegument protein open reading frame 55 (ORF55) through its amino-terminal aa 11 to 20, allowing their association with virions. Consequently, the vBcl2 peptide derived from vBcl2 aa 11 to 20 effectively disrupted the interaction between vBcl2 and ORF55, inhibiting the incorporation of the ORF55 tegument protein into virions. This study provides new insight into vBcl2's function in KSHV virion assembly that is separable from its inhibitory role in host apoptosis and autophagy.IMPORTANCE KSHV, an important human pathogen accounting for a large percentage of virally caused cancers worldwide, has evolved a variety of stratagems for evading host immune responses to establish lifelong persistent infection. Upon viral infection, infected cells can go through programmed cell death, including apoptosis and autophagy, which plays an effective role in antiviral responses. To counter the host response, KSHV vBcl2 efficiently blocks apoptosis and autophagy to persist for the life span of virus-infected cells. Besides its anti-programmed-cell-death activity, vBcl2 also interacts with the ORF55 tegument protein for virion assembly in infected cells. Interestingly, the vBcl2 peptide disrupts the vBcl2-ORF55 interaction and effectively inhibits KSHV virion assembly. This study indicates that KSHV vBcl2 harbors at least three genetically separable functions to modulate both host cell death signaling and virion production and that the vBcl2 peptide can be developed as an anti-KSHV therapeutic application.

Keywords: KSHV; Kaposi's sarcoma-associated herpesvirus; ORF55 tegument; autophagy; lytic replication; vBcl2; virion assembly.

Figures

FIG 1
FIG 1
vBcl2-derived peptide (vBcl2p) blocks KSHV progeny production. (A) Identification of the functional regions of vBcl2 for KSHV lytic replication. iSLK-BAC16-ΔvBcl2 cells were complemented with lentivirus carrying vBcl2 and its mutants, as indicated, and treated with doxycycline (1 μg/ml) and sodium butyrate (1 mM) to induce lytic replication. After a 3-day induction, the supernatants containing progeny viruses were used to infect SLK cells, and the infectious units were quantified by fluorescence-activated cell sorter (FACS) analysis with GFP-positive cells. (B and C) iSLK-BAC16 cells were pretreated with increasing amounts of the indicated peptides, and lytic replication was subsequently induced. The progeny viruses were quantified by FACS analysis as described above for panel A.
FIG 2
FIG 2
vBcl2 peptide blocks KSHV assembly. (A) Genome-wide analysis of KSHV transcripts using NanoString technology with treatment with the Tat control peptide or vBcl2p. iSLK-BAC16 cells were pretreated with the Tat or vBcl2 peptide and then lytically induced for 48 h. RNA was isolated, and viral mRNA expression was analyzed with an nCounter analysis system using nSolver analysis software 2.0 (NanoString Technologies). The graph shows relative levels of KSHV transcripts upon treatment with the Tat or vBcl2 peptide. (B) vBcl2 peptide does not affect KSHV protein expression. iSLK-BAC16 cells were pretreated with the Tat or vBcl2 peptide and then lytically induced for 24, 48, and 72 h as described above. Whole-cell lysates were analyzed by immunoblotting (IB) using the indicated antibodies. (C) Relative amounts of viral DNA were quantified by using ORF11-specific primers as described previously (34). (D) iSLK-BAC16 cells were pretreated with the Tat or vBcl2 peptide and then lytically induced for 48 h. Cells were harvested and processed for transmission electron microscopy analysis. The numbers of viral particles in each cell were quantified (P < 0.05).
FIG 3
FIG 3
vBcl2 peptide does not affect autophagy and apoptosis. (A) SLK-vector and SLK-vBcl2 cells were pretreated with the indicated peptides for 0.5 h, followed by rapamycin (Rapa) (2 μM) stimulation for 4 h. Cells were harvested, and whole-cell lysates were subjected to immunoblotting with anti-LC3 and antiactin antibodies. DMSO, dimethyl sulfoxide. (B) SLK-GFP-LC3-vector and SLK-GFP-LC3-vBcl2 stable cells were pretreated with the indicated peptides and then stimulated with rapamycin (2 μM) for 4 h. Cells were fixed, stained with DAPI (4′,6-diamidino-2-phenylindole), and subjected to confocal microscopy. The numbers of GFP-LC3 punctae per cell and the percentages of GFP-LC3 puncta-positive cells were quantified. (C) SLK-vector and SLK-vBcl2 stable cells were pretreated with the indicated peptides and then treated with TNF-α (5 ng/ml) and CHX (5 μg/ml) for 12 h. After treatment, cells were collected and subjected to TUNEL staining, and the numbers of apoptotic cells were quantified. Whole-cell lysates were subjected to an ELISA to measure caspase-3 cleavage. (D and E) vBcl2 peptide is not toxic to cells. SLK cells were treated with H2O, the Tat peptide, or the vBcl2 peptide. Cell viability and growth were monitored as indicated (P < 0.05).
FIG 4
FIG 4
vBcl2 binds to ORF55 through its aa 11 to 20. (A) vBcl2 binds to ORF55 during KSHV lytic replication. iSLK-BAC16-vBcl2-HA cells were lytically induced for 72 h as described in the legend of Fig. 1A. Whole-cell lysates (WCL) were subjected with immunoprecipitation (IP) and immunoblotting (IB) or fractionated and analyzed with the indicated antibodies (C). (B) vBcl2 specifically binds to ORF55. HEK293T cells were transfected with the indicated plasmids, and whole-cell lysates were harvested at 48 h posttransfection and subjected to immunoprecipitation and immunoblotting with the indicated antibodies. (C) iSLK-BAC16-vBcl2-HA cells were lytically induced for 72 h, and whole-cell lysates were fractionated and analyzed with the indicated antibodies. (D) vBcl2 binds to ORF55 through its A3-A4 region (aa 11 to 20). Forty-eight hours after transfection with the indicated plasmids, HEK293T cells were harvested, and whole-cell lysates were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. FL, full length. (E) The vBcl2 E14A mutant cannot bind to ORF55. Forty-eight hours after transfection with the indicated plasmids, HEK293T cells were harvested, and whole-cell lysates were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. (F) vBcl2 binds to ORF55 through its A3-A4 region (aa 11 to 20) in yeast-two hybrid assays. The bait vector containing the indicated vBcl2 WT or mutant constructs and the prey vector harboring ORF55 were cotransformed into yeast and screened in 2-dropout (2DO) plates to check transformation and in 4-dropout (4DO) plates with X-αgal to check the interaction. (G) The vBcl2 peptide impairs the interaction between vBcl2 and ORF55 during KSHV lytic replication. iSLK-BAC16-vBcl2-HA cells were pretreated with the Tat or vBcl2 peptide and then lytically induced for 72 h. Whole-cell lysates were subjected to immunoprecipitation and immunoblotting with the indicated antibodies.
FIG 5
FIG 5
vBcl2 is required for tegument integrity during KSHV lytic replication. (A) vBcl2 is detected in mature KSHV virions. iSLK-BAC16-vBcl2-HA cells were lytically induced for 96 h. Virions were harvested from supernatants, concentrated by ultracentrifugation, and then subjected to immunoblotting (IB) with the indicated antibodies. (B) KSHV virions were treated with Triton X-100 as described previously (35) and subjected to immunoblotting with the indicated antibodies. (C) KSHV virions were concentrated from iSLK-BAC16 cells (2 10-cm dishes), iSLK-BAC16-ΔvBcl2 cells (50 10-cm dishes), iSLK-BAC16-E14A cells (50 10-cm dishes), iSLK-BAC16 cells with vBcl2 peptide treatment (50 10-cm dishes), or iSLK-BAC16-ΔvBcl2 cells with lentivirus-mediated V5-vBcl2 expression (2 10-cm dishes). Purified virions were normalized by the viral DNA copy number, and equal amounts of virions were subjected to immunoblotting with the indicated antibodies. (D) iSLK-BAC16-vBcl2-HA cells were pretreated with the Tat or vBcl2 peptide and then lytically induced. Nuclear and cytosolic fractions were separated and subjected to immunoblotting with the indicated antibodies.

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