Characterization of an In Vitro Model of Alphavirus Infection of Immature and Mature Neurons

Abstract

Terminally differentiated, mature neurons are essential cells that are not easily regenerated. Neurotropic viruses, such as Sindbis virus (SV), cause encephalomyelitis through their ability to replicate in neurons. SV causes the death of immature neurons, while mature neurons can often survive infection. The lack of a reproducible and convenient neuronal cell culture system has hindered a detailed study of the differences in levels of virus replication between immature and mature neurons and the molecular events involved in virus clearance from mature neurons. We have characterized SV replication in immortalized CSM14.1 rat neuronal cells that can be differentiated into neurons. During differentiation, CSM14.1 cells ceased dividing, developed neuronal morphology, and expressed neuron-specific cell markers. SV infection of undifferentiated CSM14.1 cells was efficient and resulted in high levels of virus replication and cell death. SV infection of differentiated CSM14.1 cells was less efficient and resulted in the production of 10-to 100-fold less virus and cell survival. In undifferentiated cells, SV induced a rapid shutdown of cellular protein synthesis and pE2 was efficiently processed to E2 (ratio of E2 to pE2, 2.14). In differentiated cells, the SV-induced shutdown of cellular protein synthesis was transient and pE2 was the primary form of E2 in cells (ratio of E2 to pE2, 0.0426). We conclude that age-dependent restriction of virus replication is an intrinsic property of maturing neurons and that the CSM14.1 cell line is a convenient model system for investigating the interactions of alphaviruses with neurons at various stages of differentiation.Viral encephalitis due to infection with arthropod-borne viruses is an important cause of mortality and often results in significant long-term neurological deficits in those that survive (8,31,46). The enveloped, message-sense RNA virus Sindbis virus (SV) is the prototype alphavirus in the family Togaviridae and is related to viruses known to cause encephalitis and arthritis in humans (47). Neurons are the primary target cell for SV in mice (28), and the outcome of infection is determined by both host and viral factors (20,21,37,50,52). An important host determinant is the maturity of the neuronal population infected. Immature animals replicate virus in the central nervous system (CNS) to higher titers than mature animals and are highly susceptible to fatal disease, dying within 3 to 4 days after infection (20,29,37). Adult animals infected with the same strain of SV survive and, in the absence of a virus-specific immune response, develop persistent infection (20, 34). Immunocompetent mature animals can recover from infection and clear virus from the CNS through noncytolytic mechanisms involving anti-viral glycoprotein antibody (34) and gamma interferon (3). However, an understanding of the mechanisms underlying these virus-host interactions has been hampered by the lack of a convenient in vitro system for study.The availability of a reproducible and convenient in vit...

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

“…CSM14.1 neurons were grown in DMEM-10% FBS with penicillin, streptomycin, and glutamine at 31°C in 5% CO 2 . For differentiation, CSM14.1 cells were shifted to the nonpermissive culture conditions of DMEM-1% FBS with penicillin, streptomycin, and glutamine at 39°C in 5% CO 2 for at least 3 weeks as described previously (15). BHK-21 cells were grown at 37°C in 5% CO 2 in DMEM-10% FBS with penicillin, streptomycin, and glutamine.…”

“…The molecular basis for this maturation-dependent restriction of virus replication is unclear, but it has previously been attributed to decreased expression of viral receptors, proapoptotic molecules, and inflammatory response genes and to increased expression of fractalkine and interferon (IFN)-inducible genes (12,13). Although changes in neuronal receptor expression may contribute to age-dependent susceptibility to SINV (14), the reduced production of virus by mature neurons compared to immature neurons suggests a postentry restriction of replication (15). Because neurons are terminally differentiated essential cells, for host recovery neural function must be preserved during viral clearance.…”

The contribution of rodent models to the pathological assessment of flaviviral infections of the central nervous system

“…Several host factors have been implicated in age-related susceptibility to different viral pathogens. A number of viruses, including JEV [120] and Sindbis virus [183] exhibit a marked tropism for immature neurons. This predisposes young animals with developing central nervous systems to neurological disease.…”

Alphavirus Encephalomyelitis: Mechanisms and Approaches to Prevention of Neuronal Damage

“…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. 1b), facilitating mechanistic studies [13,[16][17][18].…”

“…With maturation, uninfected neurons produce small amounts of IFN-β that results in priming the cell for an antiviral response. Neutralization of IFN increases replication of SINV, suggesting that the low levels of IFN constitutively produced by mature neurons are important for resistance [16]. In response to infection, differentiated, but not undifferentiated, neurons rapidly produce IFN and upregulate ISGs to restrict virus replication.…”