Fig 1.
Signaling pathways of programmed cell death: brief introductory overview.
(Casp3;8) caspase 3;8, (DAI/ZBP1) DNA-dependent activator of interferon-regulatory factors/Z-DNA-binding protein 1, (FADD) Fas-associated death domain protein, (MLKL) Mixed lineage kinase domain-like protein, (RIPK 1;3) receptor interacting protein kinase 1;3, (TLR3) Toll-like receptor 3, (TRIF) TIR [Toll/interleukin-1 receptor] domain-containing adaptor protein inducing interferon beta. Only the extrinsic signaling pathway is shown for apoptosis. The sketch was created with BioRender.com.
Fig 2.
Release from apoptosis blockade by deletion of M36 enhances the antiviral CD8 T-cell response.
(A) Sketch illustrating the block in the apoptotic signaling pathway by mCMV protein M36. (ΔM36) mCMV-ΔM36; virus symbol colored green to indicate the approval of apoptosis by deletion of M36. (WT) mCMV-WT; virus symbol colored red to indicate the inhibition of apoptosis by M36. The sketch was created with BioRender.com. (B) Viral epitope-specific CD8 T-cell response determined for the RLN, specifically the PLN, on day 7 after intra-plantar infection of BALB/c or C57BL/6 mice with viruses mCMV-WT (WT) expressing M36, mCMV-ΔM36 (ΔM36) lacking M36, and the revertant virus mCMV-ΔM36-Rev (ΔM36-Rev) expressing M36. (Green color) apoptosis can take place. (Red colors) apoptosis is blocked. Bars represent frequencies of responding CD8 T cells measured in ELISpot assays performed for cohorts of 5 mice per infecting virus. Error bars indicate 95% confidence intervals (CI) determined by intercept-free linear regression analysis based on graded numbers of responder cells. Differences are considered significant when the 95% CI do not overlap.
Fig 3.
Release from apoptosis blockade by deletion of M36 leads to virus growth attenuation in the PLN but not locally at the viral entry site.
(A) Level of infection determined by quantitation of IE1 transcripts in plantar (footpad) tissue (left panel) and in the draining RNL, specifically the PLN (right panel), at 48 hours after intra-plantar infection. (B) Level of infection determined by quantitation of IE1 transcripts in the PLN at 72 hours after intra-plantar infection. At 24 hours before infection, mice were depleted of NK cells (panel α-NK), of CD8 T cells (panel α-CD8), of both (panel α-CD8 + α-NK), or were left undepleted (panel ∅). Different times of analyses in (A) and (B) take into account that virus replication precedes the immune response. Infecting viruses are indicated in the internal legend and color-coded as in Fig 2B. Filled circles represent data from 5 mice per experimental group (n = 5) tested individually. Median values are marked. Levels of significance are indicated for group comparisons of interest: P-values (*) <0.05, (**) <0.01, and (***) <0.001.
Fig 4.
Interruption of apoptotic signaling by virally-encoded dominant-negative FADD functionally reverts virus growth attenuation and the enhancing effect of M36 deletion on the antiviral CD8 T-cell response.
(A) Sketch illustrating the block in the apoptotic signaling pathway by replacement of cellular FADD with virally-encoded FADDDN. (ΔM36) mCMV-ΔM36; virus symbol colored green to indicate the approval of apoptosis by deletion of M36. (ΔM36.FADDDN) mCMV-ΔM36.FADDDN; virus symbol colored red to indicate the interruption of signaling by FADDDN. The sketch was created with BioRender.com. (B) Interruption of apoptotic signaling by virally-encoded dominant-negative FADD functionally reverts virus growth attenuation caused by M36 deletion. Levels of infection were determined by quantitation of IE1 transcripts in plantar (footpad) tissue (left panel) and in the draining RLN, the PLN (right panel), at 48 hours (day 2) after intra-plantar infection (day 0) with the viruses indicated in the internal legend. For further information and statistical evaluation, see the legend of Fig 3. (C) Interruption of apoptotic signaling by virally-encoded dominant-negative FADD functionally reverts the enhancing effect of M36 deletion on the antiviral CD8 T-cell response. The viral epitope-specific CD8 T-cell response was determined for the PLN on day 7 after intra-plantar infection of BALB/c mice (cohorts of 5 mice) with the viruses indicated in the internal legend. (Green color) apoptosis can take place. (Red colors) apoptosis is blocked. For further information, see the legend of Fig 2B.
Fig 5.
Viral genome-wide screening of the viral antigen-specific CD8 T-cell response in dependence on apoptosis.
A viral genome-spanning ORF library of expression plasmids was used to test the overall CD8 T-cell response determined for the spleen on day 7 after intra-plantar infection of BALB/c mice (cohorts of 7 mice) with the viruses indicated. Signals represent frequencies of CD8 T cells activated to express intracellular IFNγ after stimulation with antigenic transfectants. For more prominent signals, the corresponding ORF is named.
Fig 6.
Release from necroptosis blockade by functional deletion of M45 leads to virus growth attenuation and enhances the antiviral CD8 T-cell response.
(A) Sketch illustrating the block in the necroptotic signaling pathway by mCMV protein M45. (M45fs) mCMV-M45fs; virus symbol colored green to indicate the approval of necroptosis by functional deletion of M45. (WT) mCMV-WT; virus symbol colored red to indicate the inhibition of necroptosis by functional M45. The sketch was created with BioRender.com. (B) Release from necroptosis blockade by functional deletion of M45 leads to virus growth attenuation in the PLN but not locally at the viral entry site. Levels of infection were determined by quantitation of IE1 transcripts in plantar (footpad) tissue (left panel) and in the PLN (right panel), at 48 hours (day 2) after intra-plantar infection (day 0) with the viruses indicated in the internal legend. For further information and statistical evaluation, see the legend of Fig 3. (C) Release from necroptosis blockade by functional deletion of M45 enhances the antiviral CD8 T-cell response. The viral epitope-specific CD8 T-cell response was determined for the PLN on day 7 after intra-plantar infection of BALB/c mice (cohorts of 5 mice) with the viruses mCMV-WT (WT) expressing functional M45, mCMV-M45fs (M45fs) lacking functional M45, and the revertant virus mCMV-M45fs-Rev (M45fs-Rev) expressing functional M45. (Green color) necroptosis can take place. (Red colors) necroptosis is blocked. For further information, see the legend of Fig 2B. (D) ORF library screening of the overall CD8 T-cell response determined for the spleen on day 7 after intra-plantar infection of BALB/c mice (cohorts of 7 mice) with the viruses indicated. For further information see the legend of Fig 5.
Fig 7.
Missing contribution of apoptosis to the CD8 T-cell response in C57BL/6-Unc93b13d/3d mice genetically unable to cross-present.
The viral epitope-specific CD8 T-cell response was determined for the PLN on day 7 after intra-plantar infection of cohorts of 4 mice of WT strain C57BL/6 (left panel) and mutant strain Unc93b13d/3d (center and right panel) with the viruses indicated. For further information, see the legend of Fig 2B.