miRNA regulation of BK polyomavirus replication during early infection

Abstract

Viral microRNAs (miRNAs) play an important role during infection by posttranscriptionally regulating both host and viral gene expression. However, the function of many viral miRNAs remains poorly understood. In this study, we investigated the role of the BK polyomavirus (BKPyV) miRNA in regulating virus replication. The function of the polyomavirus miRNA was investigated in archetype BKPyV, which is the transmissible form of the virus and thought to establish a persistent infection in the host urinary tract. In agreement with previous studies, we show that the BKPyV miRNA targets early mRNAs. Importantly, we show that the miRNA plays a significant role in limiting archetype BKPyV replication in a natural host cell model of infection. This regulation is accomplished through the balance of regulatory elements located within the noncoding control region that control early gene expression and miRNA expression before genome replication. We therefore provide evidence for a unique function of the polyomavirus miRNA that may have important implications for the mechanism of viral persistence.

Fig. 1.

Schematic of the BKPyV genome. The viral genome is a double-stranded circular DNA molecule. The early primary transcript (counter clockwise bold arrow) and the late primary transcript (clockwise bold arrow) are divergently transcribed from the NCCR by the early and late promoters, respectively. The miRNA is complementary to the early coding region and transcribed from the late strand.

Fig. 2.

The BKPyV miRNA mutant is unable to target early mRNAs. (A) Predicted fold of the WT and the mutant (mut) pre-miRNAs (29). Point mutations are indicated by circles and bases recognized by the stem-loop RT primer are in bold. (B) The BKPyV miRNA mutant virus does not produce mature miRNA. 293TT cells were infected with purified WT or miRNA mutant virus at an MOI of 0.01. Total cell RNA was harvested 5 dpi. Mature 5P and 3P miRNAs were detected by Northern blotting. The blot was hybridized using the 5P probe (which recognizes the 5P arm), stripped, and then rehybridized using the 3P probe. Ethidium-stained rRNA bands are shown as a loading control. (C) BKPyV 5P miRNA expression was quantified by stem-loop RT qPCR and normalized to the cellular control hsa-let-7a. The A WT sample was arbitrarily set to 1. No miRNA expression was detected in mock, mock infected; A, archetype; R, rearranged; WT, WT miRNA; mut, mutant miRNA; ND, not detected. (D) Relative luciferase levels from 293 cells cotransfected with the luciferase reporter plasmid and a plasmid expressing the WT BKPyV miRNA, mutant BKPyV miRNA, or empty vector (EV). Renilla luciferase (Rluc) values were normalized to firefly luciferase (Ffluc) values as a transfection control. Each bar is the average from three (C) or four (D) independent experiments and the error bars are SD. ND, not detected; **P < 0.01. (The two-tailed, unpaired Student t test was performed for statistical analyses).

Fig. 3.

The BKPyV miRNA limits viral replication in RPTE cells. RPTE cells were infected with purified WT or miRNA mutant virus at an MOI of 0.01. Total cell RNA was harvested 3 dpi. (A) BKPyV 5P miRNA expression was quantified by stem-loop RT qPCR and normalized to the cellular control hsa-let-7a. (B) Early transcript was measured by qRT-PCR normalized to GAPDH transcript. The A WT sample was arbitrarily set to 1. (C) Total protein was harvested at 3 dpi and analyzed by Western blotting for the expression of TAg, VP1, and GAPDH. Light and dark exposures of TAg are shown. Blots shown are representative of three independent experiments. (D) Low molecular weight DNA was harvested at 1 dpi and 3 dpi and BKPyV DNA was quantified by qPCR. Replicated DNA (3 dpi) was normalized to input DNA (1 dpi). (E) Viral lysates were harvested from cells 3 dpi and progeny were quantified by an IU assay. Each bar is the average from three independent experiments and the error bars are SD. No mature miRNA, early transcript, viral genome, or progeny were detected in mock-infected samples. ND, not detected; A, archetype; R, rearranged; WT, WT miRNA; mut, mutant miRNA; ╪, below the limit of detection; *P < 0.05.

Fig. 4.

Balance of NCCR regulatory elements in archetype virus vs. rearranged variant. (A) Schematic of the WT and flip NCCR constructs. RPTE cells were infected with 1 × 10⁹ genomes of crude stock virus in the presence of AraC. Total cell RNA was harvested 2 dpi. (B) BKPyV 5P miRNA expression was quantified by stem-loop qPCR and normalized to the cellular control hsa-let-7a. (C) Late transcript was measured by qRT-PCR and normalized to GAPDH transcript. The A WT sample was arbitrarily set to 1. Each bar represents the average from three independent experiments and the error bars are SEM. No mature miRNA or late transcript was detected in mock-infected samples. A, archetype; R, rearranged.

Fig. 5.

Model of miRNA control of archetype virus replication. (A) In rearranged variants, high levels of early mRNA are expressed from high early promoter activity, whereas the miRNA is only weakly expressed. TAg, translated from early mRNA, binds to the origin of replication and drives DNA replication. (B) Archetype virus early mRNA is weakly expressed from the early promoter, whereas miRNA is robustly expressed and targets early mRNA for degradation. Therefore, DNA replication is blocked in archetype virus in RPTE cells.