RT-PCR detection of HERV-H spliced transcripts in colon cancer cell lines.

<p><b>A.</b> Schematic demonstration of the primer locations. TSS, transcription start site; TTS, transcription termination site. <b>B.</b> RT-PCR was performed to detect HERV-H spliced transcripts in colon cancer cell lines with/without demethylation/histone acetylation treatment using the DNA demethylation agent DAC and the histone deacetylase inhibitor TSA. H2O and genomic DNA mixture (gDNA mix) were used as controls. Genomic DNA mixture produced bands distinct from cDNA samples, which were reverse transcribed from DNase-treated RNA.</p

Gel electrophoresis of RT-PCR products of HERV-H-related transcripts in colon tumor and adjacent normal tissues.

<p>M, DL2000 ladder; T, tumor; N, normal.</p

The loci of active HERV-Hs in colon tumor and adjacent normal tissues.

<p>*Active elements individually contributing to more than 10% of the transcripts in tumor or adjacent normal samples are highlighted in boldface and their transcript abundances are indicated in the ‘Abundance’ column (individual and total, respectively).</p><p>**HERV-HX is the colon cancer-related HERV-H element identified by us previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029950#pone.0029950-Liang2" target="_blank">[14]</a>. Inserts of PCR product clones were all HERV-HX fragments but not spliced sequences, in concordance with our previous finding that no spliced transcripts were produced from HERV-HX in colon tumor samples.</p><p>***The element located at 1q31.3 consists of 5′LTR and 3′LTR, with the entire protein coding region (<i>gag-pro-pol-env</i>) missing.</p><p>****Open reading frames (ORFs) were predicted by the online program ORF Finder and putative peptide sequences were subjected to Blastp search against the Non-redundant protein sequences at <a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>. Only predicted ORFs≥303 nt (peptide sequence ≥100 aa) and with Blastp matches are included. RT, Reverse transcriptase; CREB5, cAMP response element-binding protein 5; Gag, group-specific antigen.</p

The loci of active HERV-H elements in colon cancer cell lines.

<p>*Two adjacent HERV-H elements at 1p32.3 were combinedly active by making use of 5′ LTR of the first HERV-H and 3′ LTR of the second one (representative transcript sequence JK017392).</p><p>**The elements located at 16q24.1 and 19q13.31 are also actively transcribed in both tumor and adjacent normal colon tissues, while the one located at 20p12.1 is active in tumor tissue.</p><p>***Open reading frames (ORFs) were predicted by the online program ORF Finder and putative peptide sequences were subjected to Blastp search against the Non-redundant protein sequences at <a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>. Only predicted ORFs≥303 nt (peptide sequence ≥100 aa) and with Blastp matches are included. Gag, group-specific antigen; RT, Reverse transcriptase; CREB5, cAMP response element-binding protein 5.</p

Characterization of the active HERV-H elements in colon cancer cell lines and their spliced transcripts.

<p><b>A.</b> Schematic of the six commonly deleted regions in the active HERV-H elements in colon cancer cell lines. <b>B.</b> Schematics of the two extraordinarily short HERV-H elements and their transcripts. Pair-wise alignments for each HERV-H element were performed with the HERV-H consensus constructed by Jern P, <i>et al</i>. The shortened alignment results were shown to indicate the missing regions precisely. Color density represents the extent of homology with the HERV-H consensus. Gray areas represent deleted regions in the HERV-H elements as compared with the HERV-H consensus. Spliced transcripts are shown above the alignment results accordingly. Thick bars represent exons, and lines represent introns. Regions of LTRs, pre-gag, <i>gag</i>, <i>pro</i>, <i>pol</i> and <i>env</i> are labeled below. <b>C.</b> Schematics of the two combinedly active HERV-H elements located at 1p32.3 in HT29.</p

Effect of mutation of HIV-1 Vif on its activity against A3DE.

<p>(A) Vif DR14/15 and W79 are required for A3DE degradation. HEK293T cells were cotransfected with A3DE plus a control vector, HIV-1 Vif, or one of the indicated Vif mutant expression vectors. A3DE stability was assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003963#pone-0003963-g002" target="_blank">Fig. 2A</a>. (B) Mutation of Vif DR14/15 and W79 inhibits Vif function, resulting in the packaging of A3DE into HIV-1 virions. HEK293T cells were co-transfected with NL4-3ΔVif and A3DE plus a control vector, WT Vif, or one of the indicated mutant expression vectors. Virus was purified and evaluated for A3DE packaging as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003963#pone-0003963-g002" target="_blank">Fig. 2D</a>. (C) Effect of WT or mutant Vif on the infectivity of NL4-3△Vif in the presence of A3DE. HIV viruses were produced in HEK293T cells coexpressing A3DE in the presence of WT or mutant Vif as indicated. Virus infectivity was assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003963#pone-0003963-g001" target="_blank">Fig. 1B</a>.</p

The C-CCD of A3F behaves like the full-length A3F.

<p>(A) Alignment of A3C and the C-CCD of AF. (B) Alignment of A3C and the N-CCD of AF. (C) Interaction of A3F and A3F-C with HIV-1 Vif. HEK293T cells were cotransfected with HIV-1 Vif-myc plus control vector, full-length A3F-HA, or A3F-C-HA. The cells were treated with 10 µM MG132 12 h prior to harvesting., and A3F-HA proteins were immunoprecipitated from cell lysates with an anti-HA antibody conjugated to agarose beads. The interaction of A3F with HIV-1 Vif molecules was detected by immunoblotting with antibodies against A3F-HA and Vif antibody. (D) HIV-1 Vif induces the degradation of the C-CCD of A3F. HEK293T cells were transfected with an expression vector encoding the C-CCD of A3F plus a control vector, WT Vif, or one of the indicated Vif mutant expression vectors. A3F-C stability was assessed by immunoblotting with antibodies against V5, Vif-myc, and β-tubulin as a loading control.</p

Models of HIV-1 Vif mediated interaction and polyubiquitination of A3G (A) and A3F (B).

<p>(A) Two distinct domains of HIV-1 Vif (G-box and FG-box) mediate interaction with the amino-terminal domain of A3G. However, the carboxyl-terminal domain of A3G is also required for Vif-mediated polyubiquitination and degradation. (B) Three distinct domains of HIV-1 Vif (F1-box, F2-box, and FG-box) mediate interaction with the carboxyl-terminal domain of A3F. The carboxyl-terminal domain of A3F is sufficient for Vif-mediated A3F degradation.</p

The D¹⁴RMR¹⁷ and Trp79 domains mediate the interaction between Vif and A3DE.

<p>HIV-1 Vif DR14/15 and W79 all showed reduced interaction with A3DE when compared to WT Vif. HEK293T cells were cotransfected with A3DE and a control vector, WT Vif, or one of the indicated Vif mutants. At 48 h post-transfection, cell lysates were prepared and immunoprecipitated with anti-myc antibody and agarose-conjugated protein A/G. Cell lysates (A) and the interaction of A3C with WT or mutant Vif molecules(B) were detected by immunoblotting with antibodies against A3DE-HA and Vif-myc.</p

Effect of mutations in HIV-1 Vif on Vif function.

<p>(A) Diagram of the functional domains of HIV-1 Vif. The BC-box structure mediates the interaction with ElonginB/C. A zinc-binding domain, Hx2YFxCFx4Φx2AΦx7-8Cx5H, is important for Cul5 selection. The N-terminal of Vif has been proposed to bind to APOBEC3 cytidine deaminases. (B) Effect of HIV-1 WT or mutant Vif proteins on the infectivity of SIVagmTan△Vif in the presence of A3C. SIV viruses were produced in HEK293T cells co-expressing A3C in the presence of HIV-1 WT or mutant Vif as indicated. Virus infectivity was assessed by Magi assay, with virus infectivity in the presence of WT Vif set to 100%. Error bars represent the standard deviations from triplicate wells. (C) Effect of HIV-1 WT or mutant Vif proteins on the infectivity of NL4-3△Vif in the presence of A3G. HIV viruses were produced in HEK293T cells co-expressing A3G in the presence of HIV-1 WT or mutant Vif as indicated. Virus infectivity was assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003963#pone-0003963-g001" target="_blank">Fig. 1B</a>.</p