The NF-κB/leukemia inhibitory factor/STAT3 signaling pathway in antibody-mediated suppression of Sindbis virus replication in neurons

Abstract

Alphaviruses are positive-sense, enveloped RNA viruses that are important causes of viral encephalomyelitis. Sindbis virus (SINV) is the prototype alphavirus and preferentially infects neurons in rodents to induce an encephalomyelitis similar to the human disease. Using a mouse model of SINV infection of the nervous system, many of the immune processes involved in recovery from viral encephalomyelitis have been identified. Antibody specific to the SINV E2 glycoprotein plays an important role in recovery and is sufficient for noncytolytic suppression of virus replication in vivo and in vitro. To investigate the mechanism of anti-E2 antibody-mediated viral suppression, a reverse-phase protein array was used to broadly survey cellular signaling pathway activation following antibody treatment of SINV-infected differentiated AP-7 neuronal cells. Anti-E2 antibody induced rapid transient NF-κB and later sustained Y705 STAT3 phosphorylation, outlining an intracellular signaling cascade activated by antiviral antibody. Because NF-κB target genes include the STAT3-activating IL-6 family cytokines, expression of these messenger RNAS (mRNAs) was assessed. Expression of leukemia inhibitory factor (LIF) cytokine mRNA, but not other IL-6 family member mRNAs, was up-regulated by anti-E2 antibody. LIF induced STAT3 Y705 phosphorylation in infected differentiated AP-7 cells but did not inhibit virus replication. However, anti-E2 antibody localized the LIF receptor to areas of E2 expression on the infected cell surface, and LIF enhanced the antiviral effects of antibody. These findings identify activation of the NF-κB/LIF/STAT3 signaling cascade as involved in inducing antibody-mediated viral suppression and highlight the importance of nonneutralizing antibody functions in viral clearance from neurons.

Keywords: alphavirus; encephalomyelitis; virus clearance.

Fig. 1.

Anti-E2 antibody suppresses SINV replication and improves cell viability in differentiated AP-7 cells. (A) Schematic diagram of general experimental timeline. Differentiated AP-7 cells were infected with the TE strain of SINV (MOI 10). Four hours postinfection, the cells were treated with 5 μg/mL antibody that was maintained in the culture media until sample collection. (B–D) Cell viability determined by trypan blue exclusion and expressed as percentage of day 0 cells following SINV infection and treatment with media (mock), anti-E2 antibody, or anti-E1 antibody of (B) differentiated AP-7, (C) cycling AP-7, and (D) BHK-21 cells. (E) Intracellular SINV subgenomic and genomic RNA levels quantified by qRT-PCR and normalized to copy numbers of GAPDH RNA. (F) Immunoblot of whole cell lysates probed with antibody against SINV structural proteins and β-actin as a loading control representative of three independent experiments. Data for B–E are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

Fig. 2.

Anti-E2 antibody induces rapid canonical NF-κB activation and late, prolonged STAT3 activation in SINV-infected dAP-7 cells. (A) RPPA results indicating changes in protein expression or posttranslational modification in whole cell lysates from differentiated AP-7 cells infected with SINV and treated with media (mock), anti-E2 antibody, or anti-E1 antibody at 4 h after infection (small arrows). Fold-change values are relative to mock-infected lysates at each time point. (B) Immunoblot confirmation of sustained phosphorylated STAT3 Y705 expression and IκBα degradation in whole cell lysates from differentiated AP-7 cell lysates treated with media or anti-E2 antibody. The blot was stripped and probed with antibody against total STAT3 and actin as loading controls. (C) Densitometric analysis of levels of phosphorylated STAT3 normalized to total STAT3. Data in A and C are presented as mean ± SD from three independent experiments ***P < 0.001, ****P < 0.0001, ^#P < 0.05, ^####P < 0.0001.

Fig. 3.

SINV infection of dAP-7 cells induces late up-regulation of multiple IL-6 family cytokine mRNAs, while anti-E2 antibody treatment induces LIF mRNA alone early. Differentiated AP-7 cells were infected with SINV and treated with media or anti-E2 antibody 4 h after infection (small arrows). At 6, 12, and 24 h after infection, RNA was assessed by qRT-PCR for levels of (A) IL-6 family and related cytokine mRNAs (IL-11, IL-6, OSM, CNTF, Csf3, LIF, Ctf2p, Clcf1, and Ctf1) and (B) gp130 and LIFRβ mRNAs. Fold-changes relative to mock-infected cells were determined by ΔΔCT. Panels with genes up-regulated by SINV infection are outlined in blue. The panel with the single mRNA (LIF) up-regulated with anti-E2 antibody treatment is outlined in red. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

Fig. 4.

Treatment of SINV-infected dAP-7 cells with recombinant LIF and IL-6 does not affect viral replication. Differentiated AP-7 cells were infected with SINV and treated with 10 μg/mL recombinant rat LIF or IL-6 4 h after infection. (A) Immunoblot analysis of phosphorylated STAT3 Y705, total STAT3, SINV structural proteins, and β-actin levels. (B) Densitometric analysis of phosphorylated STAT3 normalized to STAT3 and SINV pE2 normalized to β-actin. (C) Cell viability determined by trypan blue exclusion and expressed as a percentage of day 0 cells. (D) Intracellular SINV RNA levels quantified by qRT-PCR and expressed as log₁₀ copies per GAPDH RNA. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001.

Fig. 5.

Combined anti-E2 antibody and exogenous LIF treatment results in greater suppression of SINV replication. Differentiated AP-7 cells were infected with SINV and treated with media (mock), LIF (10 μg/mL), anti-E2 antibody (5 μg/mL), or LIF and anti-E2 antibody at 4 h after infection. (A) Immunoblot analysis for phosphorylated STAT3 Y705, SINV structural proteins, and total STAT3. (B) Densitometric analysis of phosphorylated STAT3 normalized to total STAT3 and SINV pE2 normalized to β-actin. (C) Intracellular SINV RNA levels quantified by qRT-PCR and normalized to copy numbers of GAPDH RNA. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 6.

LIFR and anti-E2 antibody colocalize on the surface of infected neurons. Differentiated AP-7 cells were grown on coverslips and infected with SINV or media. Four hours after infection, cells were treated with media, anti-E2 antibody (5 μg/mL), or anti-E1 antibody (5 μg/mL). After infection, cells were fixed with 4% paraformaldehyde for immunocytochemistry. Cells were stained with DAPI, anti-mouse IgG (red) to stain anti-E1 and anti-E2 antibodies, and anti-LIFR (green) antibody. The white arrow indicates a region of anti-E2 antibody and LIFR colocalization.

Fig. 7.

Model of antibody-induced intracellular signaling that leads to the host antiviral response. Because SINV buds from the plasma membrane, SINV E2 glycoprotein is present in high concentrations on the surface of infected neurons. Bivalent antibody binding of SINV E2 results in cross-linking of E2 molecules that induces rapid transient canonical NF-κB pathway activation. NF-κB induces expression of the IL-6 family cytokine LIF that, in turn, acts in a paracrine and/or autocrine manner to induce sustained, protective STAT3 pathway activation. Antibody cross-linking of E2 also results in the aggregation of surface LIFR that amplifies the effects of LIF binding in infected neurons.