PMC

Production of pseudotype LacZ-MuLV virions. Presence of the pseudotype LacZ-MuLV virus particles in culture supernatants of the GP2-293 packaging cells was confirmed by detection of the env polypeptides using antibody against Rauscher MuLV gp69/71. Arrow indicates the env polypeptides. Supernatants collected from cells transfected with a blank plasmid serves as a negative control (mock).

Coding potential and membrane localization of ENV_OV1 and ENV_OV2 polypeptides. (a) The coding potential of the ENV_OV1 and ENV_OV2 polypeptides was confirmed by overexpression followed by Western blot analysis using antibody against Rauscher MuLV gp69/71 env polypeptide. (b) The cellular distribution of the overexpressed ENV_OV1 and ENV_OV2 polypeptides was examined by immunocytochemistry using antibody against Rauscher MuLV gp69/71 polypeptide, and their membrane staining pattern was evident. The cells transfected with a blank plasmid serves as a negative control (mock). DAPI (4′,6-diamidino-2-phenylindole).

Cytopathic effects of the ENV_OV1 and ENV_OV2 polypeptides. (a) and (b) Cytotoxic property of the ENV_OV2 polypeptide, but not ENV_OV1 polypeptide, was observed during cytotoxicity assay by measurement of lactate dehydrogenase release and morphologic examination of cells (200x magnification). The degree of cytotoxicity was normalized to the no DNA (Panel (a)). (c) ENV_OV2 polypeptide's inhibitory effect on cell proliferation was demonstrated by MTT assay, which measures cell viability, following overexpression. All experiments were performed in triplicate. ∗ and ∗∗ indicate statistical significance (*P < .05; **P < .01).

Effects of ENV_OV1 and ENV_OV2 polypeptides on expression of inflammatory mediators. (a) and (b) The effects of the ENV_OV1 or ENV_OV2 polypeptide in RAW264.7 on the expression of various inflammatory mediators are presented. Differential modulation potentials for inflammatory mediators were observed between ENV_OV1 and ENV_OV2 polypeptides. The densitometric value of each inflammatory mediator was normalized to β-actin, and a graph was formulated. Three different forms of negative control were included in this experiment: no DNA, pcDNA4 (blank pcDNA4/HisMax plasmid), and GR11 (similar insert size as ENV_OV1 and ENV_OV2: mouse glucocorticoid receptor in pcDNA4/HisMax). The assay was performed in triplicate. ∗ and ∗∗ indicate statistical significance (*P < .05; **P < .01).

Identification of full-length env transcripts from the ovary of C57BL/6J mice. (a) A number of MuLV-ERV subgenomic transcripts were expressed in normal tissues (liver, lung, salivary gland, adrenal gland, brain, and skin) of C57BL/6J mice. A schematic diagram indicates the locations of primers used for amplification of the subgenomic transcripts. (b) The amino acid sequences of two intact env genes, named ENV_OV1 and ENV_OV2, which were isolated from the ovary (indicated with an arrow in panel (a)), were compared to reference env polypeptides with known tropism traits (GeneBank accession number: AAG39911 (Eco), ACY30460 (Xeno), AAO37283 (Poly), and AAA88318 (m.Poly)). (c) The putative MuLV-ERV proviruses harboring the ENV_OV1 and ENV_OV2 genes were mapped to chromosomes 8 and 18 of C57BL/6J genome, respectively. SU (surface domain), TM (transmembrane domain), VRA (variable region A), VRB (variable region B), PRR (proline rich region), LTR (long terminal repeat), R (repeat), and U (unique region).