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. 2020 Nov 24;17(10):2800–2817. doi: 10.1080/15548627.2020.1846301

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© 2020 Chinese Academy of Agricultural Sciences Harbin Veterinary Research Institute. Published by Informa UK Limited, trading as Taylor & Francis Group.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

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Figure 2. — EIAV Rev decreases equine SAMHD1 expression. (A) EIAV infection decreases levels of EfSAMHD1 expression at the protein level. eMDM cells were infected with EIAV_DLV36 (RT = 200 ng). EfSAMHD1 protein levels were monitored at 12 and 24 hpi. Western blotting was performed with indicated antibodies. The densities of EfSAMHD1 bands were analyzed to calculate the values relative to that of ACTB. Results were normalized to uninfected cells as control group. (B) EIAV infection does not decrease levels of EfSAMHD1 mRNA. eMDM cells were infected with EIAV_DLV36 (RT = 200 ng). EfSAMHD1 mRNA levels were monitored at 12 and 24 hpi. mRNA levels were analyzed using real-time PCR. Data represent means and SD of three independent experiments. P > 0.05 was considered NS, P < 0.05 was considered statistically significant. (C) EIAV infection decreases levels of EfSAMHD1 protein in a dose-dependent manner. eMDMs were mock-infected or infected with EIAV_DLV36 for 24 h (RT = 200 ng; RT = 400 ng). (D) EIAV Rev decreases protein levels of EfSAMHD1. HEK293T cells were transfected with the indicated plasmids. Protein expression was analyzed using western blotting with indicated antibodies at 48 hpt. The densities of EfSAMHD1 bands were analyzed to calculate the values relative to that of ACTB. Results were normalized to control cells. (E) Immunoblot analysis of SAMHD1 expression from the EIAV Rev and SAMHD1mutant co-expression experiment. HEK293T cells were transfected with indicated plasmids. Protein expression was analyzed using western blotting with indicated antibodies at 48 hpt. The densities of SAMHD1 mutant bands were analyzed to calculate the values relative to that of ACTB. Results were normalized to respective control cells. (F) The effect of EIAV infection on different SAMHD1 mutants expression. EIAV with VSV-G (RT = 400 ng) was used to infect stable U937 cell lines expressing different SAMHD1 mutants. Protein expression was analyzed using western blotting with indicated antibodies at 48 hpi. The densities of SAMHD1 mutant bands were analyzed to calculate the values relative to that of ACTB. Results were normalized to respective control cells. (G) HIV-1 Rev does not decrease expression levels of EfSAMHD1 or huSAMHD1 proteins