Control of Archetype BK Polyomavirus MicroRNA Expression

Abstract

BK polyomavirus (BKPyV) is a ubiquitous human pathogen, with over 80% of adults worldwide persistently infected. BKPyV infection is usually asymptomatic in healthy people; however, it causes polyomavirus-associated nephropathy in renal transplant patients and hemorrhagic cystitis in bone marrow transplant patients. BKPyV has a circular, double-stranded DNA genome that is divided genetically into three parts: an early region, a late region, and a non-coding control region (NCCR). The NCCR contains the viral DNA replication origin and cis-acting elements regulating viral early and late gene expression. It was previously shown that a BKPyV miRNA expressed from the late strand regulates viral large T antigen expression and limits the replication capacity of archetype BKPyV. A major unanswered question in the field is how expression of the viral miRNA is regulated. Typically, miRNA is expressed from introns in cellular genes but there is no intron readily apparent in the BKPyV from which the miRNA could derive. Here we provide evidence for primary RNA transcripts that circle the genome more than once and include the NCCR. We identified splice junctions resulting from splicing of primary transcripts circling the genome more than once, and Sanger sequencing of RT-PCR products indicates that there are viral transcripts that circle the genome up to four times. Our data suggest that the miRNA is expressed from the intron of these greater-than-genome size primary transcripts. IMPORTANCE The BK polyomavirus (BKPyV) miRNA plays an important role in regulating viral large T antigen expression and limiting the replication of archetype BKPyV, suggesting that the miRNA regulates BKPyV persistence. However, how miRNA expression is regulated is poorly understood. Here, we present evidence that the miRNA is expressed from an intron that is generated by RNA polymerase II transcribing the circular viral genome more than once. We identified splice junctions that could only be generated from primary transcripts that contain tandemly repeated copies of the viral genome. The results indicate another way in which viruses optimize expression of their genes using limited coding capacity.

Biology of Polyomavirus miRNA

“…This question was addressed by a recent study that suggested that during BKPyV late gene transcription, RNA polymerase II circles the genome more than once producing transcripts containing tandem repeats of viral genome sequences. These transcripts could then place the miRNA coding sequence in a genome-sized intron (Zou et al, 2020). Splice junctions that could only be derived from this kind of transcript were identified, and consistent with this observation, the study also showed that a circular DNA template expresses higher levels of viral miRNA than a linear template, which cannot produce tandemly-repeated transcripts (Zou et al, 2020).…”

“…These transcripts could then place the miRNA coding sequence in a genome-sized intron (Zou et al, 2020). Splice junctions that could only be derived from this kind of transcript were identified, and consistent with this observation, the study also showed that a circular DNA template expresses higher levels of viral miRNA than a linear template, which cannot produce tandemly-repeated transcripts (Zou et al, 2020). Polyomavirus viral transcripts circling the genome more than once were also observed during MuPyV and MCPyV infection, due to a weak late strand polyadenylation signal (Adami et al, 1989;Batt and Carmichael, 1995;Theiss et al, 2015).…”

“…The authors speculated that the remaining expressed viral miRNA generated from transcripts originated from the NCCR. A recent study on the BKPyV miRNA also suggested that there are sequences both within and outside the NCCR that can regulate miRNA expression (Zou et al, 2020).…”

Long-read sequencing reveals complex patterns of wraparound transcription in polyomaviruses

“…In contrast to SV40, the architecture of BKPyV late transcripts is poorly characterized-prior to this work, the two major classes of late transcripts ("16S" and "19S", reflecting transcript size based on gradient sedimentation properties) were the primary late transcript classifications [15]. Only recently did a study provide some evidence for leader-leader splicing in BKPyV [36]. While the late transcripts of most PyV are thought to encode the canonical late viral proteins, a recent study in JCPyV identified two splice events that lead to the generation of novel proteins containing the N-terminal region of VP1-one of which was validated through western blot [23].…”

BK Polyomavirus bkv-miR-B1-5p: A Stable Micro-RNA to Monitor Active Viral Replication after Kidney Transplantation

“…Transcription in the direction of late genes is also responsible for the generation of a pre-miRNA whose sequence is perfectly complementary to the TAg sequence [22]. Recently, it was shown that the RNA polymerase II could circle the genome multiple times, producing transcripts with tandem repeats of BKPyV sequences [23,24]. This phenomenon places the pre-miRNA sequence in a genomesized intron and promotes a higher miRNA expression level.…”