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. 2021 Jan 28;17(1):e1009255. doi: 10.1371/journal.ppat.1009255

Fig 1. MCMV must use infected hematopoietic cells for dissemination in C57BL/6 mice after i.n. inoculation.

Fig 1

A. Schematic of miR-142-3p targeted viruses (MCMV-IE3-142 and MCMV-GFP-142). Four target sites for miR-142-3p were inserted into the 3’ untranslated region of the essential viral gene IE3 or into a GFP-SIINFEKL fusion construct inserted into the IE2 locus. Control viruses contain shuttle vector sequences without miR-142-3p target sites in the same location. B. Targeting IE3 with miR-142-3p binding sites prevents viral replication in miR-142-3p-expressing macrophages. Multi-step growth curves of the MCMV-IE3-015 control virus and the MCMV-IE3-142 virus in 3T3 fibroblasts and IC-21 macrophages. C. The miR-142-3p targets IE3 gene and inhibits its gene expression. IE1 and IE3 gene expression were evaluated by RT-PCR after infection of M2-10B4 cells and bone marrow-derived macrophages and dendritic cells with either MCMV-IE3-015 or MCMV-IE3-142 viruses. Gene expression was normalized to β-actin in each sample and compared between MCMV-IE3-142 and MCMV-IE3-015 infected samples using the ΔΔCT method. The bar graph represents the mean value. Each symbol represents an individual cell sample, and error bars represent the standard deviation. Data are from one representative experiment of three independent experiments. D-E. Targeting IE3 with miR-142-3p binding sites inhibits gene expression. Regulation of gene expression by miR-142-3p is visualized by GFP expression after infection of 3T3 fibroblasts and IC-21 macrophages (D) or M2-10B4 cells and bone marrow-derived macrophages and dendritic cells (E) with either MCMV-GFP-015 or MCMV-GFP-142 viruses. F-G. Productive infection of hematopoietic cells is necessary for viral dissemination after i.n. inoculation, but not after i.p. inoculation. Virus titers in the nasal mucosa, lungs and salivary glands at 14 days after i.n. inoculation (F) or i.p. inoculation (G), with 106 PFU MCMV-IE3-142, or control virus MCMV-IE3-015. Each symbol represents an individual animal. The solid line shows the mean titer, and error bars represent the SEM. Dashed lines show the detection limit (50 PFU/g). Data are combined from two independent experiments.