Abstract
Sindbis virus (SINV) is an alphavirus that causes age-dependent encephalomyelitis in mice. Within 7–8 days after infection infectious virus is cleared from neurons through the antiviral effects of antibody and interferon-gamma (IFNγ), but RNA persists. To better understand changes in viral RNA associated with immune-mediated clearance we developed recombinant strains of SINV that have genomic and subgenomic viral RNAs tagged with the Broccoli RNA aptamer that binds and activates a conditional fluorophore for live cell imaging of RNA. Treatment of SINV-Broccoli-infected cells with antibody to the SINV E2 glycoprotein had cell type-specific effects. In BHK cells, antibody increased levels of intracellular viral RNA and changed the primary location of genomic RNA from the perinuclear region to the plasma membrane without improving cell viability. In undifferentiated and differentiated AP7 (dAP7) neuronal cells, antibody treatment decreased levels of viral RNA. Occasional dAP7 cells escaped antibody-mediated clearance by not expressing cell surface E2 or binding antibody to the plasma membrane. IFNγ decreased viral RNA levels only in dAP7 cells and synergized with antibody for RNA clearance and improved cell survival. Therefore, analysis of aptamer-tagged SINV RNAs identified cell type- and neuronal maturation-dependent responses to immune mediators of virus clearance.
Highlights
Sindbis virus (SINV) is an alphavirus that causes age-dependent encephalomyelitis in mice
baby hamster kidney (BHK) cells are highly permissive for SINV replication and were used for initial experiments to determine the effects of multiplicity of infection (MOI) and anti-E2 antibody treatment on visualization of Broccoli-tagged viral RNA
To determine the effect of anti-E2 antibody treatment on genomic RNA, BHK cells were infected with TE-nsP3–4Br, a recombinant SINV with 4 Broccoli aptamer copies incorporated into the hypervariable domain of the nsP3 gene in genomic RNA (Fig. 2)
Summary
Sindbis virus (SINV) is an alphavirus that causes age-dependent encephalomyelitis in mice. Infectious SINV is non-cytolytically cleared from neurons in the brain and spinal cord within 7–8 days after infection by a combination of the site-specific effects of antibody to the E2 glycoprotein and interferon (IFN)-γ7–10 These immune mediators are produced within the central nervous system (CNS) by infiltrating lymphocytes[11,12]. In a recent advance over the imaging available with the Spinach[2] aptamer[20], multiple copies of the small bright Broccoli aptamer in a F30 scaffold[21,22] were used to tag both genomic and subgenomic SINV RNAs (Nilaratanakul, Sci Rep, in press) These Broccoli-tagged SINVs replicate well in both neuronal and non-neuronal cells and viral RNA can be visualized in differentiated neurons, as well as more permissive BHK cells and undifferentiated neurons. IFNγ showed synergism with antibody for improved viral RNA clearance and cell survival in differentiated AP7 cells, but had no effect on RNA clearance from BHK or undifferentiated AP7 cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
