Kaposi Sarcoma (KS)–Associated Herpesvirus MicroRNA Sequence Analysis and KS Risk in a European AIDS‐KS Case Control Study

Abstract

Background We recently identified polymorphisms in KSHV encoded microRNA (miRNA) sequences from clinical subjects. Here, we examine whether any of these may contribute to KS risk in a European AIDS-KS case control study. Methods KSHV viral load in peripheral blood was determined by real-time quantitative PCR. Samples that had detectable viral loads were used to amplify the 2.8 kbp microRNA encoding region plus a 646 bp fragment of the K12/T0.7 gene. Additionally, we characterized an 840 bp fragment of the K1 gene to determine KSHV subtypes. Results KSHV viral DNA was detected in PBMC of 49.6% cases and 6.8% controls and viral loads tended to be higher in cases. Sequences from the miRNA encoding regions were conserved overall but distinct polymorphisms were detected some of which occurred in pri-miRNAs, pre-miRNAs or mature miRNAs. Conclusions Patients with Kaposi’s sarcoma were more likely to have detectable viral loads than controls without disease. Despite high conservation in KSHV miRNA encoded sequences, polymorphisms were observed including some that have been previously reported. Some polymorphisms could affect mature miRNA processing and appear to be associated with KS risk.

Kaposi's Sarcoma-Associated Herpesvirus Encodes a Mimic of Cellular miR-23

“…Primer extension analysis also confirmed miR-K3ϩ1 expression in BC-1, VG-1, and BCLM cells, as previously reported (12). Interestingly, this SNP has been reported previously to be present in 3 of 19 analyzed KS patients, 13 of 23 KSHV-infected patients diagnosed with multicentric Castleman's disease (MCD), and 4 of 7 patients with KSHV-associated inflammatory cytokine syndrome, but its consequences for KSHV miRNA expression had not been explored (39,42,43).…”

“…As expected, miR-K3ϩ1 and miR-23a expression resulted in comparable target repression, confirming that these miRNAs share their seed targets. miR-K3ϩ1 expression is not conserved among all KSHV isolates, however, implying that miR-K3ϩ1 is dispensable for the normal KSHV life cycle and also for the development of KS, MCD, PEL, and KSHV-associated inflammatory cytokine syndrome (39,42,43). We therefore explored the relationship between miR-23 and miR-K3, which is always expressed by KSHV.…”

Kaposi's Sarcoma-Associated Herpesvirus MicroRNA Single-Nucleotide Polymorphisms Identified in Clinical Samples Can Affect MicroRNA Processing, Level of Expression, and Silencing Activity

“…Previously, we observed single-nucleotide polymorphisms (SNPs) in pre-microRNA sequences of miR-K12-2, -4, -5, -6, -7, -9, and -10 in clinical samples and PEL cell lines (15,16). The SNPs evaluated here are summarized in Table 1, which also indicates which SNPs were observed in clinical samples and which were also present in sequences from PEL cell lines.…”

“…2A). In our previous studies of KSHV microRNAencoded sequences in clinical samples, most subjects were observed to have the miR-K12-5 SNP (15,16). In contrast, SNPs within the terminal loop region of miR-K12-4 and -7 showed reduced expression.…”

“…BCBL-1 was designated the wild type because it is commonly used as a model for KSHV research and it has a KSHV microRNA sequence that was found to cluster in the conserved arm of the phylogenetic tree in our previous study (15). KSHV wt and variant pre-microRNA sequences were amplified from previously characterized clinical samples using Pfu DNA polymerase (Stratagene), with the exception of wt pre-microRNA K12-6, which was synthesized (Operon) (16). The pre-microRNAs included in this study are listed in Table 1.…”