NF-κB Activation Promotes Alphavirus Replication in Mature Neurons

Abstract

Alphaviruses are enveloped, positive-sense RNA viruses that are important causes of viral encephalomyelitis. Sindbis virus (SINV) infects the neurons of rodents and is a model for studying factors that regulate infection of neuronal cells. The outcome of alphavirus infection of the central nervous system is dependent on neuronal maturation status. Differentiated mature neurons survive and control viral replication better than undifferentiated immature neurons. The cellular factors involved in age-dependent susceptibility include higher levels of antiapoptotic and innate immune factors in mature neurons. Because NF-κB pathway activation is required for the initiation of both apoptosis and the host antiviral response, we analyzed the role of NF-κB during SINV infection of differentiated and undifferentiated rat neuronal cells. SINV infection induced canonical NF-κB activation, as evidenced by the degradation of IκBα and the phosphorylation and nuclear translocation of p65. Inhibition or deletion of the upstream IκB kinase substantially reduced SINV replication in differentiated but not in undifferentiated neuronal cells or mouse embryo fibroblasts. NF-κB inhibition did not affect the establishment of infection, replication complex formation, the synthesis of nonstructural proteins, or viral RNA synthesis in differentiated neurons. However, the translation of structural proteins was impaired, phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) was decreased, and host protein synthesis was maintained, suggesting that NF-κB activation was involved in the regulation of translation during infection of mature neurons. Inhibition or deletion of double-stranded RNA-activated protein kinase (PKR) also decreased eIF2α phosphorylation, the translation of viral structural proteins, and virus production. Therefore, canonical NF-κB activation synergizes with PKR to promote SINV replication in differentiated neurons by facilitating viral structural protein translation. IMPORTANCE Mosquito-borne alphaviruses are a significant and growing cause of viral encephalomyelitis worldwide. The outcome of alphaviral neuronal infections is host age dependent and greatly affected by neuronal maturation status, with differentiated, mature neurons being more resistant to infection than undifferentiated, immature neurons. The biological factors that change during neuronal maturation and that influence the outcome of viral infection are currently only partially defined. These studies investigated the role of NF-κB in determining the outcome of alphaviral infection in mature and immature neurons. Inhibition of canonical NF-κB activation decreased alphavirus replication in mature neurons by regulating protein synthesis and limiting the production of the viral structural proteins but had little effect on viral replication in immature neurons or fibroblasts. Therefore, NF-κB is a signaling pathway that influences the maturation-dependent outcome of alphaviral infection in neurons and that highlights the importance of cellular context in determining the effects of signal pathway activation.

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

“…Previous studies indicated that bivalent antibody against the SINV E2 glycoprotein suppressed SINV replication in infected primary rat DRG neurons and immortalized AT3-bcl-2 rat prostatic carcinoma cells and improved cell survival (18)(19)(20)(21). To model infection of mature neurons, the current in vitro experiments used an immortalized AP-7 rat olfactory sensory neuron cell line that can be differentiated into nondividing mature neuronal cells (dAP-7) (23)(24)(25). The dAP-7 cells were infected with the TE strain of SINV at a multiplicity of infection (MOI) of 10 (Fig.…”

“…1D), suggesting that antibody-induced viral suppression is more effective in mature neurons (22,24,27). Because SINV replicates faster and to higher titers in cAP-7 and BHK-21 cells than in dAP-7 cells (24,25), it is possible that antibody added earlier or at a higher concentration might also have an effect on viability of these cell types.…”

“…Finally, the current study reinforces previous findings that antiviral antibody is more protective in mature neurons than in immature neurons or nonneuronal cell types. One possible explanation for this is increased expression of antiapoptotic factors and host innate immune factors in mature neurons that restrict viral replication and allow for prolonged survival following infection (24,25,58), increasing the window of time that antiviral mechanisms can function. Another possibility is that baseline levels of transcription factor expression or epigenetic history differ and lead to divergent outcomes.…”

“…Cell Culture. AP-7 immortalized rat olfactory sensory neuron-derived cells (gift from Dale Hunter, Tufts University, Boston, MA) (23) were grown and differentiated for 7 d as previously described (25). Baby hamster kidney (BHK-21) cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS).…”

Neuronal hyperexcitability is a DLK-dependent trigger of HSV-1 reactivation that can be induced by IL-1

“…The downstream signaling elicited by IL-1 in neurons has not been clearly defined and likely varies between different subtypes of neurons. NF-κB has been reported to be absent in certain subtypes of neurons but constitutively active in others 65,66 , and a recent study suggests that NF-κB levels increase with neuronal maturation 67 , which may be why we only observed IL-1-mediated reactivation in mature neurons. Additional studies have found a role for p38MAPK signaling and AKT/mTOR signaling in neuronal IL-1-mediated responses 68,69 .…”