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. 2016 Jul;97(7):1693-1698.
doi: 10.1099/jgv.0.000482. Epub 2016 Apr 13.

MCPIP1/regnase-I inhibits simian immunodeficiency virus and is not counteracted by Vpx

Hongmei Li  1 , Tony T Wang  2
Affiliations

MCPIP1/regnase-I inhibits simian immunodeficiency virus and is not counteracted by Vpx

Hongmei Li et al. J Gen Virol. 2016 Jul.

Abstract

We have previously shown that the cellular RNase MCPIP1/regnase-1 potently blocks HIV-1 infection in resting CD4+ T-cells. As simian immunodeficiency virus (SIV) encodes an accessory protein named Vpx, which enhances viral replication in resting CD4+ T-cells by degrading the cellular restriction factor SAMHD1, we investigated whether MCPIP1 restricts SIV infection and whether Vpx protein antagonizes MCPIP1-mediated restriction. In co-transfection studies, human MCPIP1 markedly reduced the production of infectious SIV, whereas MCPIP2 and MCPIP3 had little effect. MCPIP1 derived from cynomolgus monkey also inhibited human immunodeficiency virus (HIV-1) and SIV production, albeit to a lesser degree. Lastly, expression of SIV Vpx protein did not reduce MCPIP1 at the protein level, nor did it ablate the MCPIP1-mediated restriction. In conclusion, both human and cynomolgus monkey MCPIP1 restrict SIV replication. Unlike SAMHD1, MCPIP1-mediated HIV-1 restriction cannot be overcome by SIV Vpx.

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Figures

Fig. 1.
Fig. 1.
MCPIP1 restricts SIV infection. To package SIV-GFP virus carrying VSV glycoprotein (VSV-G), 2 µg pSIvec1.GFP, 0.2 µg VSV-G and 0.1 µg Rev plasmid were transfected into 293T cells along with 0.5 µg hMCPIP1 or the plasmids indicated. For HIV-GFP virus, 0.5 µg pTrip-GFP, 0.5 µg pCMVR8.2 and 0.2 µg VSV-G were co-transfected with 0.5 µg of the MCPIP1 plasmids indicated; for MLV-GFP virus, 0.25 µg pFB-GFP, 0.2 µg MLV-Gag-pol,and 0.1 µg VSV-G were co-transfected with 0.25 µg MCPIP1 plasmid. Forty-eight hours after transfection, cells were imaged by Nikon TS-100 fluorescence microscope. 525 or 125 µl supernatants were added to natïve 293T cells for virus titration. Infected cells were both imaged and quantified by flow cytometry for GFP+ cells in order to calculate infectious titre [infectious units (i.u.) ml−1]; n=3.
Fig. 2.
Fig. 2.
SIV Vpx does not antagonize MCPIP1. (a) 293T cells were transfected with 0.2 µg HA-SAMHD1 or 0.1 µg Flag-hMCPIP1 or 0.1 µg Flag-MonMCPIP1 along with 0.1 or 0.2 µg Myc-Vpx plasmid for 48 h. A representative Western blot image of three independent experiments was presented here. Anti-HA, anti-Flag and anti-Myc antibodies were used in order to detect SAMHD1, MCPIP1 and Vpx. β-Actin was measured as loading control. (b) HIV-GFP virus was packaged as described in Fig. 1 except that an increasing amount of Vpx plasmid was added into the transfection reaction. The infectious titres of the newly released virus were plotted. n=4.
Fig. 3.
Fig. 3.
MCPIP1 localizes to cytoplasm. (a) Two distribution patterns of GFP-hMCPIP1 were observed in HEK293-Dox-GFP-MCPIP1 cells after doxycycline induction. Bars, 10 µm (cytoplasmic), 5 µm (punctate). (b) Flag-hMCPIP1 was immunostained using the Millipore mab-MCPIP1 (2H8.2, 1 : 100) in HeLa-Dox-Flag-hMCPIP1 cells after induction. A representative image is presented of cells expressing low amounts of Flag-MCPIP1. Bars, 5 µm (control), 10 µm (Flag-MCPIP1). (c–e) 293T cells were transfected with the plasmids indicated and fixed in methanol prior to immunostaining. Images were captured using Carl Zeiss LSM 700 confocal microscopy. The doxycycline-inducible clones were previously established in the lab (Liu et al., 2013a). Bars, 5 µm.
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