Anti-IFITM2 monoclonal antibody, clone 2G3

Immune cells play a central role in early pregnancy establishment in cattle. We aimed to: (1) discover novel early-pregnancy-induced genes in peripheral blood mononuclear cells (PBMC); and (2) characterize the temporal pattern of early-pregnancy-induced transcription of select genes in PBMC and peripheral blood polymorphonuclear cells (PMN). Beef heifers were artificially inseminated on D0 and pregnancies were diagnosed on D28. On D10, 14, 16, 18, and 20, blood was collected for isolation of PBMC and PMN from heifers that were retrospectively classified as pregnant (P) or non-pregnant (NP). PBMC samples from D18 were submitted to RNAseq and 220 genes were differentially expressed between pregnant (P) and non-pregnant (NP) heifers. The temporal abundance of 20 transcripts was compared between P and NP, both in PBMC and PMN. In PBMC, pregnancy stimulated transcription of IFI6, RSAD2, IFI44, IFITM2, CLEC3B, OAS2, TNFSF13B, DMKN and LGALS3BP as early as D18. Expression of IFI44, RSAD2, OAS2, LGALS3BP, IFI6 and C1R in PMN was stimulated in the P group from D18. The novel early-pregnancy induced genes discovered in beef heifers will allow both the understanding of the role of immune cells during the pre-attachment period and the development of technologies to detect early pregnancies in beef cattle.

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.