Sequence analysis of <i>env</i> gene from XMRV proviral DNA-positive offspring samples.

<p>(A) Nested-PCR, cloning, and sequence analysis were performed as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029682#pone.0029682-Sakuma2" target="_blank">[33]</a>. The number of mutations in the <i>env</i> gene from lymph node and heart of the mouse B1 are summarized. A total of 2751 bp and 4366 bp were sequenced from lymph node and heart, respectively. (B) Schematic representation of XMRV Env with partial amino acid sequence of receptor binding domain (RBD) is shown. One Env isolate from heart (B1: GenBank accession no. JN987227) has one amino acid deletion of serine at position 148 in SU as boxed in blue. Amino acids from 1 to 33, from 34 to 444, and from 445 to 645 indicate signal peptide (SP), surface protein (SU), and transmembrane protein (TM), respectively. Variable regions (VRA, VRB, and VRC) are highlighted in gray. (C) Protein expression of wild-type XMRV Env and XMRV Env with S148 mutation was analyzed by Western blotting. Env protein was visualized with goat anti-MLV antibody (1∶2000, ATCC). Actin (1∶2000, sigma) was also detected as a loading control. Statistical analysis showed that protein expression was not statistically significant (<i>p</i> = 0.31). (D) Retroviral vectors pseudotyped with XMRV Env or XMRV Env with S148 mutation were produced by transient transfection in 293T cells. The transduction titer of retrovirus pseudotyped with XMRV Env or XMRV Env with a serine mutation is statistically significant (<i>p</i> = 0.009). Error bars indicate 95% confidence intervals.