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. 2013 Jan 20;435(2):269-80.
doi: 10.1016/j.virol.2012.09.008. Epub 2012 Oct 17.

An attenuating mutation in a neurovirulent Sindbis virus strain interacts with the IPS-1 signaling pathway in vivo

Amy C Wollish  1 , Martin T Ferris, Lance K Blevins, Yueh-Ming Loo, Michael Gale Jr, Mark T Heise
Affiliations

An attenuating mutation in a neurovirulent Sindbis virus strain interacts with the IPS-1 signaling pathway in vivo

Amy C Wollish et al. Virology. .

Abstract

The AR86 strain of Sindbis virus causes lethal neurologic disease in adult mice. Previous studies have identified a virulence determinant at nonstructural protein (nsP) 1 position 538 that regulates neurovirulence, modulates clearance from the CNS, and interferes with the type I interferon pathway. The studies herein demonstrate that in the absence of type I interferon signaling, the attenuated mutant exhibited equivalent virulence to S300 virus. Furthermore, both S300 and nsP1 T538I viruses displayed similar neurovirulence and replication kinetics in IPS-1-/- mice. TRIF dependent signaling played a modest role in protecting against disease by both S300 and nsP1 T538I, but did not contribute to control of nsP1 T538I replication within the CNS, while MyD88 played no role in the disease process. These results indicate that the control of the nsP1 T538I mutant virus is largely mediated by IPS-1-dependent RLR signaling, with TRIF-dependent TLR signaling also contributing to protection from virus-induced neurologic disease.

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Conflict of interest statement

Conflict of Interest Statement. The authors report no conflict of interest.

Figures

Figure 1
Figure 1. The mutant virus nsP1 T538I is attenuated in C57BL/6J mice as compared to S300 wild-type virus
Adult 9–12 week old female C57BL/6J mice were infected i.c. with 103 PFU of S300 or nsP1 T538I virus and monitored daily for (A) survival, (B) weight loss, and (C) brain and (D) spinal cords were harvested at 1, 3, 5, and 6 days post infection for viral titer by plaque assay. Survival data are presented as the pooled results of at least 3 experiments. Weight loss data are presented as the means + the standard deviation of 16 experiments. Viral titer data are shown as the mean of a single experiment and are representative of at least three experiments. The number of mice infected per virus is indicated in parentheses. Asterisks denote p< 0.01(**), p< 0.001(***), or p< 0.0001(****). The horizontal dashed line indicates the lower limit of assay sensitivity.
Figure 2
Figure 2. Both S300 and nsP1 T538I show equivalent and enhanced virulence in the absence of the IFN-α/β response
Adult 9–12 week old female C57BL/6J and IFN-α/βR deficient mice were infected i.c. with 103 PFU of S300 or nsP1 T538I virus. Equivalent and rapid (A) lethality and (B) weight loss with both S300 and nsP1 T538I infection of IFN-α/βR−/− mice. The number of mice infected per virus is indicated in parentheses, and data are compiled from a minimum of two experiments. Infectious virus data are shown as the mean of 2 pooled experiments, and was assayed at day 3 pi within the (C) brain and (D) spinal cord of C57BL/6J and IFN-α/βR−/− mice. Asterisks denote p< 0.0001(****). The dashed line indicates the lower limit of assay sensitivity.
Figure 3
Figure 3. TLR signaling through MyD88 is not required for protection or control of nsP1 T538I infection
Adult 9–12-week-old female C57BL/6J and MyD88−/− mice were infected i.c. with 103 PFU of S300 or nsP1 T538I virus. Differential (A) survival and (B) weight loss between S300 and nsP1 T538I infected MyD88−/− mice. The number of mice infected per group is indicated in parentheses. Infectious virus was assayed at 5 days pi in the (C) brain and (D) spinal cord. Asterisks denote p< 0.01(**) or p< 0.001(***). Titer data are representative of two or more experiments. The dashed line indicates the lower limit of assay sensitivity.
Figure 4
Figure 4. TRIF signaling is not required for viral replication control of nsP1 T538I, but TRIF may play a minor role in protection from SINV-induced disease
Adult 9–12-week-old female C57BL/6J and TRIF−/− mice were infected i.c. with 103 PFU of S300 or nsP1 T538I virus. (A) Survival and (B) weight loss of S300 and nsP1 T538I infected C57BL/6J or TRIF−/− mice are shown. The number of mice infected per group is indicated in parentheses. Infectious virus was assayed at 5 days pi within the (C) brain and (D) spinal cord. Titer data are representative of two or more experiments. Asterisks denote p< 0.05(*) or p< 0.01(**). The dashed line indicates the lower limit of assay sensitivity. Mice were perfused with 15 mL of 4% PFA for in situ hybridization and H&E, and brains were paraffin embedded and cut sagitally. Sections were subjected to in situ hybridization with a riboprobe specific for SINV AR86 and shown are (E) hippocampus and (F) spinal cord regions. Nonspecific binding controls included sections from mock-infected mice probed with the AR86 riboprobe, and sections from infected mice probed with a riboprobe specific for Epstein Barr virus (not shown). (G) H&E stained brain sections depicting the hippocampus.
Figure 5
Figure 5. IPS-1 is required for protection from nsP1 T538I and contributes to control of nsP1 T538I
Adult 9–12-week-old female C57BL/6J and IPS-1−/− mice were infected i.c. with 103 PFU of S300 or nsP1 T538I virus. Similar (A) survival and (B) weight loss curves between S300 and nsP1 T538I infected IPS-1−/− mice. The number of mice infected per group is indicated in parentheses. Infectious virus was assayed at 1, 3, and 5 days pi in the (C) brain and (D) spinal cord. Titer data are representative of two or more experiments. Asterisks denote p< 0.05(*), p< 0.01(**), p< 0.01(***), and p< 0.0001(****). The dashed line indicates the lower limit of assay sensitivity.
Figure 6
Figure 6. Increased quantity and distribution of viral RNA and neuronal damage in IPS-1−/− mice infected with S300 and nsP1 T538I as compared with C57BL/6 mice infected with nsP1 T538I
C57BL/6J and age-matched IPS-1−/− mice were infected i.c. with 103 PFU of S300 or nsP1 T538I virus. Brains were harvested at 5 days pi. (A, B) Mice were perfused with 15 mL of PBS, and RNA was harvested prior to qRT-PCR using primer/probe sets specific for SINV (A) 49S genomic RNA or (B) 26S subgenomic RNA. Asterisks denote p< 0.05(*), p< 0.01(**), p< 0.001(***), orp< 0.0001(****). Mice were perfused with 15 mL of 4% PFA for in situ hybridization and H&E, and brains were paraffin embedded and cut sagitally. Sections were subjected to (C, D) in situ hybridization with a riboprobe specific for SINV AR86 or (E) H&E. Nonspecific binding controls included sections from mock-infected mice probed with the AR86 riboprobe, and sections from infected mice probed with a riboprobe specific for Epstein-Barr virus (not shown). Data shown are representative of three experiments with at least three mice per group.
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