Figure 6

Analyses of ebv-sisRNA-1. (A) Northern blot for ebv-sisRNA-1 using RNA from EBV-negative BJAB cells and from isogenic EBV-positive BJAB-B1 cells. The bottom arrow points to the additional band observed in the EBV-positive BJAB-B1 lane that likely corresponds to the 81 nt ebv-sisRNA-1, while the upper arrow indicates the 106 nt human U6 snRNA (present in both cell types). (B) RT-PCR for EBER1 and ebv-sisRNA-1 using cDNA from BJAB-B1 nuclear RNA and primers complementary to the ends of EBER1 and the 5′ end plus nt 52 to 71 of ebv-sisRNA-1. (C) Fold-enrichment in nuclear versus cytoplasmic RNA as measured by RT-qPCR. Shown are the results for the sisRNA and control 18S and U2 snRNA, which are cytoplasmically and nuclearly enriched, respectively. Error bars indicate the standard error of three biological replicates, each with three technical replicates. (D) MAFFT sequence alignment of ebv-sisRNA-1 and those of seven other lymphocryptoviruses: Pongine herpesvirus 1 (PoHV1, AJ311196.1), Pongine herpesvirus 3 (PoHV3, AJ311194.1), Macacine herpesvirus 4 (MHV4, NC_006146), Cercopithicine herpesvirus 15 (CeHV15, AJ311199), Herpesvirus MF-1 from Macaca fascicularis (HVMF1, X77781), Cercopithecine herpesvirus 12 (CeHV12, AF200364.1), and Callitrichine herpesvirus 3 (CaHV3, NC_004367.1). The question mark at the first position in HVMF1 represents missing data, not a true gap. 100% conserved positions are indicated with stars below the alignment. (E) Predicted structure for ebv-sisRNA-1. Compensatory mutations in the upstream hairpin that convert a GC pair to an AU pair (in CaHV3) are indicated with bold blue nts. This sequence is repeated seven times in EBV: genome (NC_009334.1) coordinates 14603 to 14683, 17673 to 17753, 20743 to 20823, 23813 to 23893, 26883 to 26963, 29953 to 30033, and 33023 to 33103.