Ectromelia-encoded virulence factor C15 specifically inhibits antigen presentation to CD4+ T cells post peptide loading

Abstract

Smallpox and monkeypox pose severe threats to human health. Other orthopoxviruses are comparably virulent in their natural hosts, including ectromelia, the cause of mousepox. Disease severity is linked to an array of immunomodulatory proteins including the B22 family, which has homologs in all pathogenic orthopoxviruses but not attenuated vaccine strains. We demonstrate that the ectromelia B22 member, C15, is necessary and sufficient for selective inhibition of CD4 + but not CD8 + T cell activation by immunogenic peptide and superantigen. Inhibition is achieved not by down-regulation of surface MHC- II or co-stimulatory protein surface expression but rather by interference with antigen presentation. The appreciable outcome is interference with CD4 + T cell synapse formation as determined by imaging studies and lipid raft disruption. Consequently, CD4 + T cell activating stimulus shifts to uninfected antigen-presenting cells that have received antigen from infected cells. This work provides insight into the immunomodulatory strategies of orthopoxviruses by elucidating a mechanism for specific targeting of CD4 + T cell activation, reflecting the importance of this cell type in control of the virus.

The ectromelia virus virulence factor C15 facilitates early viral spread by inhibiting NK cell-infected cell contacts

“…With the addition of this novel NK-inhibitory function, we have now revealed that in murine cells C15 is able to inhibit NK and CD4 T cell but not CD8 T cell contact and function. While the specific molecular mechanism of C15 antagonism of CD4 T cells remains to be determined, it appears to impact the formation of immunological synapses (Forsyth et al, 2020). Although there are significant distinctions between T cell and NK cell synapses, these contact-dependent interaction sites are also implicated by results shown here.…”

“…B22 family members are well conserved across the virulent OPXVs (including VARV, MPXV and ECTV), but the C15 ORF is truncated in VACV. The size and conservation of B22 family members suggest a large contribution to virulence, and this has been demonstrated for the homologs in MPXV and ECTV (Alzhanova et al, 2014; Reynolds et al, 2017; Forsyth et al, 2020). Functionally, B22 family members have been shown to modulate T cell responses via complex mechanisms.…”

“…All viruses used in this work have been previously described: ECTV expressing eGFP (Moscow strain background) was a kind gift of Dr. Luis Sigal (Fang et al, 2008). eGFP expressing ΔC15 ECTV (Moscow strain background) was generated as previously described (Forsyth et al, 2020); the C15 revertant ECTV that was constructed was demonstrated to behave similarly to WT ECTV and was not used in this work. All viruses were grown in house in TK-143b osteosarcoma cells at high MOI for 3 days and virus was harvested from cells by cycles of freeze-thawing and sonication.…”

“…Functionally, B22 family members have been shown to modulate T cell responses via complex mechanisms. The VARV, MPXV and cowpox virus (CPXV) B22 homologs inhibit primate CD4 and CD8 T cells, but B22 family proteins belonging to OPXV that infect murine cells (CPXV and ECTV) selectively inhibit CD4 but not CD8 T cell function (Alzhanova et al, 2014; Forsyth et al, 2020). These data suggest that primate and murine CD8 T cells differ in ways that dictate susceptibility to B22-mediated inhibition.…”

“…These data suggest that primate and murine CD8 T cells differ in ways that dictate susceptibility to B22-mediated inhibition. While the specific mechanism of this inhibition has not been revealed, our previous work with ECTV demonstrated that C15 interferes with the formation of CD4 T cell synapses (Forsyth et al, 2020).…”