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ER intrabody-mediated inhibition of interferon ? secretion by mouse macrophages and dendritic cells.

ABSTRACT: Interferon ? (IFN?) counteracts viral infections by activating various IFN?-stimulated genes (ISGs). These genes encode proteins that block viral transport into the host cell and inhibit viral replication, gene transcription and translation. Due to the existence of 14 different, highly homologous isoforms of mouse IFN?, an IFN? knockout mouse has not yet been established by genetic knockout strategies. An scFv intrabody for holding back IFN? isoforms in the endoplasmic reticulum (ER) and thus counteracting IFN? secretion is reported. The intrabody was constructed from the variable domains of the anti-mouse IFN? rat monoclonal antibody 4EA1 recognizing the 5 isoforms IFN?1, IFN?2, IFN?4, IFN?5, IFN?6. A soluble form of the intrabody had a KD of 39 nM to IFN?4. It could be demonstrated that the anti-IFN? intrabody inhibits clearly recombinant IFN?4 secretion by HEK293T cells. In addition, the secretion of IFN?4 was effectively inhibited in stably transfected intrabody expressing RAW 264.7 macrophages and dendritic D1 cells. Colocalization of the intrabody with IFN?4 and the ER marker calnexin in HEK293T cells indicated complex formation of intrabody and IFN?4 inside the ER. Intracellular binding of intrabody and antigen was confirmed by co-immunoprecipitation. Complexes of endogenous IFN? and intrabody could be visualized in the ER of Poly (I:C) stimulated RAW 264.7 macrophages and D1 dendritic cells. Infection of macrophages and dendritic cells with the vesicular stomatitis virus VSV-AV2 is attenuated by IFN? and IFN?. The intrabody increased virus proliferation in RAW 264.7 macrophages and D1 dendritic cells under IFN?-neutralizing conditions. To analyze if all IFN? isoforms are recognized by the intrabody was not in the focus of this study. Provided that binding of the intrabody to all isoforms was confirmed, the establishment of transgenic intrabody mice would be promising for studying the function of IFN? during viral infection and autoimmune diseases.

SUBMITTER: Bussow K

PROVIDER: S-EPMC6467385 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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ER intrabody-mediated inhibition of interferon α secretion by mouse macrophages and dendritic cells.

Büssow Konrad K Themann Philipp P Luu Sabine S Pentrowski Paul P Harting Claudia C Majewski Mira M Vollmer Veith V Köster Mario M Grashoff Martina M Zawatzky Rainer R Van den Heuvel Joop J Kröger Andrea A Böldicke Thomas T

PloS one 20190416 4

Interferon α (IFNα) counteracts viral infections by activating various IFNα-stimulated genes (ISGs). These genes encode proteins that block viral transport into the host cell and inhibit viral replication, gene transcription and translation. Due to the existence of 14 different, highly homologous isoforms of mouse IFNα, an IFNα knockout mouse has not yet been established by genetic knockout strategies. An scFv intrabody for holding back IFNα isoforms in the endoplasmic reticulum (ER) and thus co ...[more]

PMID: 30990863

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Project description:Type I interferon inhibits viruses through inducing the expression of antiviral proteins, including the myxovirus resistance (Mx) proteins. Compared to the human MxA protein, which inhibits a wide range of viruses, the MxB protein has been reported to specifically inhibit primate lentiviruses, including HIV-1, and herpesviruses. Further, the role of endogenous MxB in alpha interferon-mediated inhibition of HIV-1 infection was questioned by a recent study showing that MxB knockout did not increase the level of infection by HIV-1 which carried the G protein of vesicular stomatitis virus (VSV), allowing infection of CD4-negative HT1080 cells. In order to further examine the anti-HIV-1 activity of endogenous MxB, we have used CRISPR/Cas9 to deplete MxB in different cell lines and observed a substantial restoration of HIV-1 infection in the presence of alpha interferon treatment. However, this rescue effect of MxB knockout became much less pronounced when infection was performed with HIV-1 carrying the VSV G protein. Interestingly, a CRISPR/Cas9 knockout screen of alpha interferon-stimulated genes in U87-MG cells revealed that the genes for interferon-induced transmembrane protein 2 (IFITM2) and IFITM3 inhibited VSV G-pseudotyped HIV-1 much more strongly than the rest of the genes tested, including the gene for MxB. Therefore, our results demonstrate the importance of MxB in alpha interferon-mediated inhibition of HIV-1 infection, which, however, can be underestimated if infection is performed with VSV G protein-pseudotyped HIV-1, due to the high sensitivity of VSV G-mediated infection to inhibition by IFITM proteins.IMPORTANCE The results of this study reconcile the controversial reports regarding the anti-HIV-1 function of alpha interferon-induced MxB protein. In addition to the different cell types that may have contributed to the different observations, our data also suggest that VSV G protein-pseudotyped HIV-1 is much less inhibited by alpha interferon-induced MxB than HIV-1 itself is. Our results clearly demonstrate an important contribution of MxB to alpha interferon-mediated inhibition of HIV-1 in CD4+ T cells, which calls for using HIV-1 target cells and wild-type virus to test the relevance of the anti-HIV-1 activity of endogenous MxB and other restriction factors.

Dataset Information

ER intrabody-mediated inhibition of interferon ? secretion by mouse macrophages and dendritic cells.

Publications

ER intrabody-mediated inhibition of interferon α secretion by mouse macrophages and dendritic cells.

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