Differentiation of Neurons Restricts Arbovirus Replication and Increases Expression of the Alpha Isoform of IRF-7

Abstract

Susceptibility to alphavirus infection is age dependent, and host maturation is associated with decreased virus replication and less severe encephalitis. To identify factors associated with maturation-dependent restriction of virus replication, we studied AP-7 rat olfactory bulb neuronal cells, which can differentiate in vitro. Differentiation was associated with a 150-to 1,000-fold decrease in replication of the alphaviruses Sindbis virus and Venezuelan equine encephalitis virus, as well as La Crosse bunyavirus. Differentiation delayed synthesis of SINV RNA and protein but did not alter the susceptibility of neurons to infection or virion maturation. Additionally, differentiation slowed virus-induced translation arrest and death of infected cells. Differentiation of uninfected AP-7 neurons was associated with changes in expression of antiviral genes. Expression of key transcription factors was increased, including interferon regulatory factor 3 and 7 (IRF-3 and IRF-7) and STAT-1, suggesting that neuronal maturation may enhance the capacity for antiviral signaling upon infection. IRF-7 produced by undifferentiated AP-7 neurons was exclusively the short dominant negative ␥-isoform, while that produced by differentiated neurons was the full-length ␣-isoform. A similar switch in IRF-7 isoforms also occurred in the brains of maturing C57BL/6J mice. Silencing of IRF expression did not improve virus multiplication in differentiated neurons. Therefore, neuronal differentiation is associated with upregulation of transcription factors that activate antiviral signaling, but this alone does not account for maturation-dependent restriction of virus replication. IMPORTANCEViral encephalomyelitis is an important cause of age-dependent morbidity and mortality. Because mature neurons are not readily regenerated, recovery from encephalitis suggests that mature neurons utilize unique antiviral mechanisms to block infection and/or clear virus. To identify maturational changes in neurons that may improve outcome, we compared immature and mature cultured neurons for susceptibility to three encephalitic arboviruses and found that replication of Old World and New World alphaviruses and a bunyavirus was reduced in mature compared to immature neurons. Neuronal maturation was associated with increased baseline expression of interferon regulatory factor 3 and 7 mRNAs and production of distinct isoforms of interferon regulatory factor 7 protein. Overall, our studies identified maturational changes in neurons that likely contribute to assembly of immunoregulatory factors prior to infection, a more rapid antiviral response, increased resistance to virus infection, and improved survival. Development of age-dependent resistance to fatal disease is a characteristic of many virus infections of the central nervous system (CNS) (1-9). We have used Sindbis virus (SINV), the prototype alphavirus in the family Togaviridae, as a model system to understand maturation-mediated restriction of virus multiplication in neurons. SINV preferentially i...

Alphavirus Encephalomyelitis: Mechanisms and Approaches to Prevention of Neuronal Damage

“…1a) [13][14][15]. Maturation-dependent restriction of virus replication is also observed in cultured primary neurons (e.g., dorsal root ganglia cells) and neuronal cell lines differentiated in vitro (e.g., CSM14.1, AP-7) (Fig.…”

“…Neuronal maturation in the absence of infection is associated with increased expression of interferon (IFN)-β, transcription factors IFN regulatory factor (IRF)-3 and IRF-7, and several IFN-stimulated gene (ISG) mRNAs (e.g., 2,5OAS, RNaseL, β2 m, IFIT1, IFIT3, ISG20) [15]. IRF-7 is a key transcription factor, with multiple splice variants, that regulates and amplifies the IFN response through induction of the IFN-α genes, as well as ISGs [19].…”

“…IRF-7 is a key transcription factor, with multiple splice variants, that regulates and amplifies the IFN response through induction of the IFN-α genes, as well as ISGs [19]. The IRF-7 protein produced by neurons evolves with maturation from the short dominant negative γ isoform to the full-length active α isoform necessary for transcribing antiviral protein genes [15]. With maturation, uninfected neurons produce small amounts of IFN-β that results in priming the cell for an antiviral response.…”

“…In response to infection, differentiated, but not undifferentiated, neurons rapidly produce IFN and upregulate ISGs to restrict virus replication. Therefore, neuronal maturation is associated with antiviral priming characterized by increased basal levels of important transcription factors that rapidly activate antiviral signaling in response to infection, and thus reduce virus replication in mature neurons [15].…”

“…a Virus replication in the brains of 1-day and 4-week-old mice after intracerebral inoculation [8]. b Virus replication in immature undifferentiated and mature differentiated AP-7 rat neuronal cells [15] Role of the Immune Response…”