Chikungunya virus replication in skeletal muscle cells is required for disease development
Anthony J. Lentscher, … , Thomas E. Morrison, Terence S. Dermody
Anthony J. Lentscher, … , Thomas E. Morrison, Terence S. Dermody
Published December 3, 2019
Citation Information: J Clin Invest. 2020;130(3):1466-1478. https://doi.org/10.1172/JCI129893.
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Research Article Infectious disease Virology

Chikungunya virus replication in skeletal muscle cells is required for disease development

Abstract

Chikungunya virus (CHIKV) is an arbovirus capable of causing a severe and often debilitating rheumatic syndrome in humans. CHIKV replicates in a wide variety of cell types in mammals, which has made attributing pathologic outcomes to replication at specific sites difficult. To assess the contribution of CHIKV replication in skeletal muscle cells to pathogenesis, we engineered a CHIKV strain exhibiting restricted replication in these cells via incorporation of target sequences for skeletal muscle cell–specific miR-206. This virus, which we term SKE, displayed diminished replication in skeletal muscle cells in a mouse model of CHIKV disease. Mice infected with SKE developed less severe disease signs, including diminished swelling in the inoculated foot and less necrosis and inflammation in the interosseous muscles. SKE infection was associated with diminished infiltration of T cells into the interosseous muscle as well as decreased production of Il1b, Il6, Ip10, and Tnfa transcripts. Importantly, blockade of the IL-6 receptor led to diminished swelling of a control CHIKV strain capable of replication in skeletal muscle, reducing swelling to levels observed in mice infected with SKE. These data implicate replication in skeletal muscle cells and release of IL-6 as important mediators of CHIKV disease.

Authors

Anthony J. Lentscher, Mary K. McCarthy, Nicholas A. May, Bennett J. Davenport, Stephanie A. Montgomery, Krishnan Raghunathan, Nicole McAllister, Laurie A. Silva, Thomas E. Morrison, Terence S. Dermody

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Figure 1

CHIKV engineered to contain target sequences for skeletal muscle–specific miR-206 is specifically restricted by its cognate miRNA.

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CHIKV engineered to contain target sequences for skeletal muscle–specifi...
(A) Schematic of the CHIKV genome. Sequences were engineered in-frame within the E3 protein-coding region (shown in grey). (B) Insert cassettes consist of 4 target elements in tandem appended at the 3′ end by sequences of the FMDV 2A protease. (C) Nucleotide sequence of insert cassettes. Four target sequences exhibiting perfect complementarity to skeletal muscle–specific miR-206 were introduced into SKE. Silent mutations were engineered into target sequences to produce SKE mismatch (MM). (D) U-2 OS cells were adsorbed with WT SL15649, SKE MM, or SKE at an MOI of 0.01 PFU/cell. Supernatants were collected at the times shown after adsorption, and viral titer was quantified by plaque assay. (E) U-2 OS cells were transfected with siRNA directed against luciferase (Luc; dotted lines), CHIKV nsP1 (dashed lines), or muscle-specific miR-206-mimic siRNA (solid lines) and adsorbed with WT SL15649 (black), SKE MM (blue), or SKE (red) at an MOI of 0.01 PFU/cell. Supernatants were collected at the times shown after adsorption, and viral titer was determined by plaque assay. (D and E) Results are expressed as the mean viral titer from duplicate wells of 3 independent experiments. Error bars indicate SEM. Dashed lines indicate the limit of detection. P values were determined at 12 and 24 hpi by ANOVA followed by Tukey’s post hoc test. The following comparisons were statistically significant (****P < 0.0001): SKE MM-Luc versus SKE MM-nsP1 and SKE-Luc versus SKE-nsP1 (E, left), WT-206 versus SKE-206, and SKE MM-206 versus SKE-206 (E, right).