Figure 1.
Structure of the HERV-K genome and spliced mRNAs showing the primers used for reverse transcription and PCR.
A genetic map of a HERV-K provirus (gray) inserted into flanking host genome sequences is shown with a 5 or 6 bp target duplicated sequence (TDS) indicated as black boxes. The unspliced primary viral transcript, singly-spliced env mRNA and doubly-spliced rec and np9 mRNAs are shown below the viral genome, along with the singly spliced RNA [56] that is not known to encode any protein. 3′ poly(A) tails are indicated (AAAA). The 292 nucleotide deletion of type 1 HERV-K proviruses spanning the pol-env junction is indicated (Δ292). Type 2 HERV-K proviruses and their transcripts contain these nucleotides. Positions of the PCR primer pairs are shown at the bottom as black arrows with the names by which they are identified throughout the paper. The gray arrows identify primers used for nested PCR. The dashed, angled line shows the excised intronic sequences that the 1X-env and 2X-rec primer pairs were designed to cross. The primer pair used for quantitative RT-PCR is also shown (q-env).
Figure 2.
Detection of HERV-K transcripts in 13 cancer cell lines.
RT-PCR was performed to detect viral transcripts at five different positions in HERV-K genome, two of which across splicing junctions to detect RNAs spliced at the conventional env mRNA splice junction (1x-env) and the rec mRNA splice junctions. GAPDH RT-PCR was performed simultaneously and served as a positive control for RNA integrity and as loading comparison. The products were resolved by agarose gel electrophoresis. Genomic positions of the primers used are shown in Fig. 1. Parallel controls were performed without reverse transcriptase (−RT) as controls to exclude DNA contamination. DNA size markers are shown on the left.
Figure 3.
Detection of HERV-K transcripts in 13 cancer cell lines.
Nested PCR was performed on RT-PCR products shown in Fig. 2 to detect viral transcripts at three different positions in HERV-K genome. Two of RT-PCRs cross splicing junctions to detect RNAs spliced at the conventional env mRNA splice junction (1x-env) and rec and np9 mRNA (2X-rec, 2X-np9) splice junctions. The products were resolved by electrophoresis. Genomic positions of the primers used are shown in Fig. 1. Parallel controls were performed without reverse transcriptase (-RT) as controls to exclude DNA contamination. DNA size markers are shown on the left.
Table 1.
Polymorphisms in cDNA sequences from pro amplicons in 13 cancer cell lines and reference to 22 full-length or near full-length proviruses.
Figure 4.
DNA sequencing chromatographs of RT-PCR products generated from RNAs isolated from four arbitrarily chosen cell lines are shown in the upper panel, one for each cancer type used in the study.
In the lower panel, the HERV-K full-length proviruses used as references to identify the loci of origin of mRNA transcripts are shown. On the left, the major branches of extant hominoids are shown, along with the approximate times of their branching from the lineage leading to modern humans in millions of years ago (Ma). Proviruses that are inserted at precisely orthologous positions in humans and other hominoids, and thus are identical by descent, are indicated.
Table 2.
Individual HERV-K loci identified by sequencing of nested RT-PCR, 1X-env RNA, spliced products.
Figure 5.
HERV-K RNA splice sites determined by sequencing of RT-PCR products.
The 5′ and 3′ portions of a HERV-K provirus are shown at the top separated by/and/. 5′SS and 3′SS indicate the conventional splicing sites of HERV-K, and their positions are marked with black circles. The positions of the outer PCR primers used for the nested PCR are shown as arrows. Structures of the env spliced RT-PCR products from the cell lines and proviruses indicated are diagrammed below the viral genome. Dashed angled lines show the excised introns. Red circles show the positions of the sequences where unconventional splicing occurred. For each spliced product, the top sequence shows the inferred primary transcript sequence determined from that of the cognate genomic locus, and the bottom sequence shows the sequence of the RT-PCR product. Red nucleotides were joined in the spliced product. Gray nucleotides show the ends of the excised intronic sequences. The position of the 292 nucleotide deletion definitive of type 1 proviruses is shown.
Figure 6.
Quantitation of HERV-K expression by qRT-PCR in 12 cancer cell lines after irradiation.
Genomic positions of the primers used are shown in Fig. 1. Five different ionizing radiation doses were used (0, 2.5, 5, 10, and 20 Gy), each applied in a single dose, are shown in different colors at five different times following the γ-irradiation (1, 3, 8, 24, and 72 hours). Fold change relative to 0 Gy for each time point was obtained by normalization to 3 housekeeping genes. Bars represent the means of three independent RT-PCR replicates performed for each of three experiments. Error bars show standard deviations.
Table 3.
P-values for effects of ionizing radiation at 24 hours.