Figure 1. Restriction of MMTV replication by BST-2 present in host target cells and modulation of BST-2 by MMTV.

Homozygote WT and BST-2−/− mice were crossed to generate the heterozygote BST-2+/− mice. The genotypes were confirmed by PCR with primers that detect endogenous BST-2 and neomycin following previously described protocol (Swiecki et al., 2012) (A). WT, BST-2+/− (+/−), and BST-2−/− (−/−) mice (n = 5/genotype) were phenotyped for BST-2 expression by RT-qPCR using BST-2 specific primers (B) or by FACS - fluorescence activated cell sorting (C). Lymphocytes and splenocytes obtained from WT, BST-2+/− (+/−), and BST-2−/− (−/−) mice were infected ex vivo with MMTV. DNA isolated from cells was examined for level of infection by qPCR 24 hours later (D). Naïve age-matched WT, BST+/−, and BST-2−/− mice (n = 5/genotype/time point) were infected with MMTV by subcutaneous injection on the hind footpad. On days 1 and 7, groups of 5 mice/genotype were sacrificed. Single cells from the lymph nodes and spleens were used for qPCR (E and F), FACS (G and H), and RT-qPCR (I and J) to determine the level of virus replication, surface level of BST-2, and BST-2 transcripts respectively. (K) RT-based plasma viral loads as determined by Molecular Probes EnzCheck Reverse Transcriptase Assay was performed with cell-free plasma from WT, BST-2+/−, and BST-2−/− mice (n = 5) infected with MMTV for 1 and 7 days. RT activity of the samples was extrapolated from the RT standard curve and presented as fold change of RT units in 1 µl of WT plasma. PCR data are normalized to GAPDH and presented as fold change relative to proviral DNA (VDNA) or BST-2 mRNA in WT mice. Error bars are standard deviation; ** is significance with p value less than 0.01 and * is significance with p value less than 0.05. The numbers next to histogram legend denote mean florescence intensity (MFI) of surface BST-2 levels. Experiments were performed with 5 mice per genotype and repeated at least 3 times with similar results.