Ectromelia virus suppresses expression of cathepsins and cystatins in conventional dendritic cells to efficiently execute the replication process.

BACKGROUND: Cathepsins are a group of endosomal proteases present in many cells including dendritic cells (DCs). The activity of cathepsins is regulated by their endogenous inhibitors - cystatins. Cathepsins are crucial to antigen processing during viral and bacterial infections, and as such are a prerequisite to antigen presentation in the context of major histocompatibility complex class I and II molecules. Due to the involvement of DCs in both innate and adaptive immune responses, and the quest to understand the impact of poxvirus infection on host cells, we investigated the influence of ectromelia virus (ECTV) infection on cathepsin and cystatin levels in murine conventional DCs (cDCs). ECTV is a poxvirus that has evolved many mechanisms to avoid host immune response and is able to replicate productively in DCs. RESULTS: Our results showed that ECTV-infection of JAWS II DCs and primary murine GM-CSF-derived bone marrow cells down-regulated both mRNA and protein of cathepsin B, L and S, and cystatin B and C, particularly during the later stages of infection. Moreover, the activity of cathepsin B, L and S was confirmed to be diminished especially at later stages of infection in JAWS II cells. Consequently, ECTV-infected DCs had diminished ability to endocytose and process a soluble antigen. Close examination of cellular protein distribution showed that beginning from early stages of infection, the remnants of cathepsin L and cystatin B co-localized and partially co-localized with viral replication centers (viral factories), respectively. Moreover, viral yield increased in cDCs treated with siRNA against cathepsin B, L or S and subsequently infected with ECTV. CONCLUSIONS: Taken together, our results indicate that infection of cDCs with ECTV suppresses cathepsins and cystatins, and alters their cellular distribution which impairs the cDC function. We propose this as an additional viral strategy to escape immune responses, enabling the virus to replicate effectively in infected cells.

Functional paralysis of GM-CSF-derived bone marrow cells productively infected with ectromelia virus.

Ectromelia virus (ECTV) is an orthopoxvirus responsible for mousepox, a lethal disease of certain strains of mice that is similar to smallpox in humans, caused by variola virus (VARV). ECTV, similar to VARV, exhibits a narrow host range and has co-evolved with its natural host. Consequently, ECTV employs sophisticated and host-specific strategies to control the immune cells that are important for induction of antiviral immune response. In the present study we investigated the influence of ECTV infection on immune functions of murine GM-CSF-derived bone marrow cells (GM-BM), comprised of conventional dendritic cells (cDCs) and macrophages. Our results showed for the first time that ECTV is able to replicate productively in GM-BM and severely impaired their innate and adaptive immune functions. Infected GM-BM exhibited dramatic changes in morphology and increased apoptosis during the late stages of infection. Moreover, GM-BM cells were unable to uptake and process antigen, reach full maturity and mount a proinflammatory response. Inhibition of cytokine/chemokine response may result from the alteration of nuclear translocation of NF-κB, IRF3 and IRF7 transcription factors and down-regulation of many genes involved in TLR, RLR, NLR and type I IFN signaling pathways. Consequently, GM-BM show inability to stimulate proliferation of purified allogeneic CD4+ T cells in a primary mixed leukocyte reaction (MLR). Taken together, our data clearly indicate that ECTV induces immunosuppressive mechanisms in GM-BM leading to their functional paralysis, thus compromising their ability to initiate downstream T-cell activation events.