Supplementary data for
Dual roles for the telomeric repeats in chromosomally integrated human herpesvirus-6
Tamae Ohye, Hidehito Inagaki, Masaru Ihira, Yuki Higashimoto, Koji Kato, Junko Oikawa, Hiroshi Yagasaki, Takahiro Niizuma, Yoshiyuki Takahashi, Seiji Kojima, Tetsushi Yoshikawa, and Hiroki Kurahashi*
* Corresponding author
Supplementary Figure S1. Restriction map of the integrated HHV-6B genomes. Restriction enzymes used for the analyses are as follows: MseI (M) (a), PstI and BglII (P and B) (b), and SacI (S) (c). Probes are denoted by small horizontal bars. The expected sizes of the detected restriction fragments are indicated above the schematic diagrams. Diagrams are shown for standard cases (upper panels) as well as cases 28 that include three DR copies (lower panels).
Supplementary Figure S2. PCR analyses of the TRS-1 site within the DR-R region (a) and TRS-2 of the DR-L region (b) of HHV-6. The bracket and arrowhead denote authentic products. Lane M, size markers; lane 1, case 18; lane 2, case 19; lane 3, case 20; lane 4, case 28; lane 5, case 31; lane 6, case 63; lane 7, non-CIHHV-6 case; lane 8, water control. Telomere repeat numbers are indicated below the panel.
Supplementary Figure S3. Southern blot analyses of genomic DNA from the CIHHV-6B study subjects. The restriction enzymes used for these analyses are indicated above the panels. In each panel, the data with a DR probe is shown on the left side, while the data with a probe located at the distal end of the UL region (flanking to the DR) is shown on the right. All of the gels have been run under the same experimental conditions. Triangles indicate bands derived from the DR-R, while arrows indicate the smears derived from the DR-L. The restriction fragments for DR-R are large enough to include two copies of DRs. On the other hand, the restriction fragments for DR-L manifest as smears and the sizes correspond to one copy of the DR. Size markers are indicated on the left. Lane 1, case 18; lane 2, case 28; lane 3, non-CIHHV-6 case.
Supplementary Figure S4. Comparison of the restriction map of the integrated HHV-6B genomes in cases 18 and 28. Restriction enzymes used for the analyses are as follows: EcoRI (E) (a), NdeI (Nd) (b), and NcoI (N) (c). Probes are denoted by small horizontal bars. DR probes are indicated by black bars, while probes located at the distal end of the UL region are indicated by white bars. The expected sizes of the detected restriction fragments are indicated above the schematic diagrams. Diagrams are shown for standard case, case 18 (upper panels). For case 28 that include three DR copies, two possibilities exist: two of the three copies might be located at the proximal region (middle panels) or at the distal region (lower panels).
Supplementary Figure S5a. PCR analysis of the junction of the two DRs in HHV-6B. The arrowhead denotes authentic products. The sizes of the PCR products from cases 28 and 63 are lower than that from the replicating viruses from a patient with exanthema subitum. Lane M, size markers; lane S, sample from a patient with exanthema subitum; lane 1, case 18; lane 2, case 19; lane 3, case 20; lane 4, case 28; lane 5, case 31; lane 6, case 63; lane 7, non-CIHHV-6 case; lane 8, water control.
Supplementary Figure S5b. Southern blot analysis for the long range PCR using telomeric primer. To show the specificity of the PCR products appearing as a smear in Figure 4, we performed Southern hybridization using telomere repeat probe. The results indicated that the PCR products really contained telomeric repeats. Size markers are indicated on the left. Lane 1, non-CIHHV-6 case; lane 2, case 28; lane 3, case 18.
Supplementary Figure S6. Whole image of the gel shown in the Figure 3b.