Vaccinia Virus Encodes a Novel Inhibitor of Apoptosis That Associates with the Apoptosome

Abstract

Apoptosis is an important antiviral host defense mechanism. Here we report the identification of a novel apoptosis inhibitor encoded by the vaccinia virus (VACV) M1L gene. M1L is absent in the attenuated modified vaccinia virus Ankara (MVA) strain of VACV, a strain that stimulates apoptosis in several types of immune cells. M1 expression increased the viability of MVA-infected THP-1 and Jurkat cells and reduced several biochemical hallmarks of apoptosis, such as PARP-1 and procaspase-3 cleavage. Furthermore, ectopic M1L expression decreased staurosporine-induced (intrinsic) apoptosis in HeLa cells. We then identified the molecular basis for M1 inhibitory function. M1 allowed mitochondrial depolarization but blocked procaspase-9 processing, suggesting that M1 targeted the apoptosome. In support of this model, we found that M1 promoted survival in Saccharomyces cerevisiae overexpressing human Apaf-1 and procaspase-9, critical components of the apoptosome, or overexpressing only conformationally active caspase-9. In mammalian cells, M1 coimmunoprecipitated with Apaf-1-procaspase-9 complexes. The current model is that M1 associates with and allows the formation of the apoptosome but prevents apoptotic functions of the apoptosome. The M1 protein features 14 predicted ankyrin (ANK) repeat domains, and M1 is the first ANK-containing protein reported to use this inhibitory strategy. Since ANK-containing proteins are encoded by many large DNA viruses and found in all domains of life, studies of M1 may lead to a better understanding of the roles of ANK proteins in virus-host interactions.IMPORTANCE Apoptosis selectively eliminates dangerous cells such as virus-infected cells. Poxviruses express apoptosis antagonists to neutralize this antiviral host defense. The vaccinia virus (VACV) M1 ankyrin (ANK) protein, a protein with no previously ascribed function, inhibits apoptosis. M1 interacts with the apoptosome and prevents procaspase-9 processing as well as downstream procaspase-3 cleavage in several cell types and under multiple conditions. M1 is the first poxviral protein reported to associate with and prevent the function of the apoptosome, giving a more detailed picture of the threats VACV encounters during infection. Dysregulation of apoptosis is associated with several human diseases. One potential treatment of apoptosis-related diseases is through the use of designed ANK repeat proteins (DARPins), similar to M1, as caspase inhibitors. Thus, the study of the novel antiapoptosis effects of M1 via apoptosome association will be helpful for understanding how to control apoptosis using either natural or synthetic molecules.

Keywords: Apaf-1; M1L; ankyrin repeat; apoptosis; apoptosome; caspase-9; poxvirus; vaccinia virus.

FIG 1

Creation and characterization of an M1L-expressing MVA virus. (A) Schematic showing insertion of tandem GFP and M1L genes into the del III region of the MVA genome, in which the GFP gene is under the control of the poxvirus p11 promoter (p11) and the M1L gene is under the control of its natural promoter (NP). Primers used for verifying insertion of this GFP-M1L cassette are shown. (B) CEF monolayers were mock infected or infected with MVA, MVA/M1L, or WR (MOI = 10). At 24 h p.i., infected cells were harvested and lysed. DNA was subjected to PCR amplification using either the M1F and M1R primer set to amplify the M1L gene or the F1 and R1 primers to PCR amplify the MVA del III region. A portion of each PCR amplification reaction mixture was separated by agarose gel electrophoresis, and DNA was visualized using ethidium bromide staining. Reactions were analyzed in the same gel, and gel images were spliced for labeling purposes. (C) Detection of M1L gene transcription using semiquantitative RT-PCR. PMA-stimulated THP-1 cells were mock infected or infected with the indicated viruses (MOI = 2). At 6 h p.i., cells were collected and total RNA was extracted from lysed cells. Total RNA was reverse transcribed into cDNA. A portion of cDNA was incubated with primers either nested inside the M1L gene or for the actin gene as a control. A portion of each PCR was analyzed by agarose gel electrophoresis, and PCR amplicons were detected by using ethidium bromide staining of the gel. Reactions were analyzed in the same gel, and gel images were spliced for labeling purposes. (D) CEF or RK13 cellular monolayers were infected with MVA, MVA/M1L, or WR (50 PFU/well of a six-well plate). At 24 h p.i., cells were fixed and incubated in a solution containing anti-vaccinia virus antiserum, followed by a solution containing HRP-conjugated goat anti-rabbit antiserum. The diameters of at least 10 foci per condition were measured, and results are presented as the mean focus size ± SEM for each sample.

FIG 2

M1L increases viability during MVA infection. PMA-stimulated THP-1 cells were either mock infected or infected with MVA or MVA/M1L (MOI = 5) or incubated in medium containing 1 μM staurosporine (STS). (A) At 24 h p.i. or post-STS treatment, all cells were collected and cell death was quantified by using a trypan blue dye exclusion assay. Results are presented as the mean percentage of cells that excluded trypan blue (live) cells divided by the total number of live and dead cells. Cells from each sample were counted in triplicate, and data shown here are the mean ± SD from three independent experiments. (B) At 24 h p.i. or post-STS treatment, PrestoBlue reagent was added to the wells. The fluorescence of each well was quantified using a microplate reader. Results are presented as the mean fluorescence ± SD for each sample. PrestoBlue-based assays were performed in technical triplicate. The graphs shown here represent data obtained from at least three independent experiments. Asterisks indicate conditions in which the viability of MVA/M1L-infected cells was statistically different from that of MVA-infected cells (P < 0.05).

FIG 3

M1L inhibits MVA-induced apoptosis. PMA-treated THP-1 cells (A and B), Jurkat cells (C and D), or caspase-8-deficient (C8^−/−) Jurkat cells (E and F) were either mock infected or infected with MVA or MVA/M1L (MOI = 2). A separate set of uninfected cells was incubated in medium containing 1 μM staurosporine (STS). (A, C, and E) At 16 h p.i. or post-STS treatment, cells were collected and lysed in RIPA buffer. Thirty micrograms of each sample was separated by using SDS–12% PAGE, and proteins were transferred to a PVDF membrane. Membranes were probed with an anti-PARP-1 antibody that detects full-length (116-kDa) and cleaved (24-kDa) PARP-1. An asterisk denotes a nonspecific band. Blots subsequently were incubated with either anti-E3 or anti-actin antisera and redeveloped. Data are representative of at least three independent experiments. In panels A and C, reactions were analyzed in the same gel and gel images were spliced for labeling purposes. (B, D, and F) At 24 h p.i. or post-STS treatment, cells were lysed, and caspase-3 and -7 activities were quantified using the Caspase-Glo 3/7 assay kit. Results are presented as the mean fold induction of caspase activity for each sample ± SD above that for mock-infected cells, whose value was set to 1. Data were obtained from at least three independent experiments. Statistically significant changes in values between MVA and MVA/M1L are indicated by asterisks (P < 0.05).

FIG 4

M1L inhibits MVA-induced procaspase-9 cleavage. PMA-treated THP-1 cells (A), Jurkat cells (B), or caspase-8-deficient (C8^−/−) Jurkat cells (C) were either mock infected or infected with MVA or MVA/M1L (MOI = 2). A separate set of uninfected cells was incubated in medium containing 1 μM staurosporine (STS) for 12 h. At either 12 (A), 20 (B), or 16 (C) h p.i., cells were collected and lysed in RIPA buffer. Thirty micrograms of each sample was analyzed by SDS–10% PAGE, and proteins were transferred to a PVDF membrane. The membrane was probed with anti-caspase-9 antiserum, which detects the 47-, 37-, and 35-kDa forms of caspase-9. Blots were subsequently incubated with either anti-E3 or anti-actin antisera and developed. Data shown are representative of data obtained from at least three independent experiments.

FIG 5

Biochemical hallmarks of staurosporine-induced apoptosis are reduced when M1 is expressed independently of infection. Subconfluent HeLa cellular monolayers were transfected with 1 μg of either empty vector (pCI) or pM1L-V5. At 24 h posttransfection, cells were treated with medium either lacking (−) or containing 0.5 μM staurosporine (STS). At 2, 4, or 6 h post-STS incubation, cells were collected and lysed in RIPA buffer. Lysates were separated by SDS–12% PAGE, and proteins were transferred to a PVDF membrane. Membranes were probed with either anti-PARP-1, anti-caspase-3, or anti-caspase-9 antisera. Membranes were developed and bands were detected using chemiluminescence. Blots were subsequently incubated with either anti-V5, anti-FLAG, or anti-actin antiserum and redeveloped. Data shown are representative of at least three independent experiments.

FIG 6

M1 does not block the loss of mitochondrial membrane potential during staurosporine-induced apoptosis. Subconfluent HeLa cellular monolayers were transfected with 1 μg of either empty vector (pCI), pM1L-V5, or pF1L-FLAG. At 24 h posttransfection, cells were treated with medium lacking (−) or containing (+) 5 μM staurosporine (STS) for 2 h. Cells were incubated in 0.2 μM TMRE for 30 min at 37°C. The percentage of cells with decreased TMRE fluorescence is shown in each histogram. Data shown are representative of at least three independent experiments. A portion of each cellular population was collected and lysed in RIPA buffer, and vaccinia virus protein levels were examined by immunoblotting. This same blot was incubated with anti-actin antiserum and subsequently developed.

FIG 7

M1 inhibits yeast lethality that is triggered by a reconstituted apoptosome or constitutively active caspase-9. Yeast cells were transformed with the indicated expression plasmids, and lethality was induced by overexpressing either Apaf-1, procaspase-9 (Casp-9), and procaspase-3 (Casp-3) or Bax (A) or reverse caspase-9 (rCasp-9) and procaspase-3 (B). Suspensions containing equivalent concentrations of each transformant were serially diluted, and 5 μl of each dilution was spotted onto plates containing galactose (to induce transgene expression) or glucose (to repress transgene expression). Growth on inducing plates indicates survival and proliferation of yeast cells expressing the transgenes.

FIG 8

M1 interacts with and does not disrupt procaspase-9–Apaf-1 interactions. Subconfluent 293T cellular monolayers were cotransfected with 500 ng C9DN-FLAG, 500 ng N-Apaf-1-myc, and 1,000 ng of either pCI, pM1L-V5, or pHA-K1L. At 24 h posttransfection, cells were lysed in NP-40 lysis buffer. Some lysates were set aside to monitor protein expression. For the remaining clarified cellular lysates, immunoprecipitations (IP) were performed using murine IgG or anti-FLAG (A), rabbit IgG or anti-V5 (B), or murine IgG or anti-HA antibodies (C) conjugated to protein G-Sepharose beads. Immunoprecipitated samples or 20 μg of cellular lysates was analyzed by SDS–8% PAGE, and proteins were transferred to a PVDF membrane for immunoblotting. Membranes were probed with the indicated antibodies to detect the epitope-tagged versions of C9DN, N-Apaf-1, M1, K1 or cellular actin. For panel B, all immunoprecipitated and cellular lysates were analyzed in the same gel and gels were spliced for labeling purposes.