Evolutionarily conserved function of a viral microRNA

Abstract

MicroRNAs (miRNAs) are potent RNA regulators of gene expression. Some viruses encode miRNAs, most of unknown function. The majority of viral miRNAs are not conserved, and whether any have conserved functions remains unclear. Here, we report that two human polyomaviruses associated with serious disease in immunocompromised individuals, JC virus and BK virus, encode miRNAs with the same function as that of the monkey polyomavirus simian virus 40 miRNAs. These miRNAs are expressed late during infection to autoregulate early gene expression. We show that the miRNAs generated from both arms of the pre-miRNA hairpin are active at directing the cleavage of the early mRNAs. This finding suggests that despite multiple differences in the miRNA seed regions, the primary target (the early mRNAs) and function (the downregulation of early gene expression) are evolutionarily conserved among the primate polyomavirus-encoded miRNAs. Furthermore, we show that these miRNAs are expressed in individuals diagnosed with polyomavirus-associated disease, suggesting their potential as targets for therapeutic intervention.

FIG. 1.

JCV and BKV encode miRNAs homologous to the SV40 miRNAs. (A to C) Northern blot analyses of the JCV and BKV miRNAs. (A) Diagram of probes used. (B) JCV miRNAs. (C) BKV miRNAs. The arrows indicate the bands corresponding to the miRNAs. Ethidium bromide staining of the low-molecular-weight RNA is shown as a loading control. Mock, uninfected. hpi, hours postinfection. (D) Alignment of the BKV and JCV 5p and 3p miRNAs with those of SV40. The 5p and 3p miRNAs are aligned with asterisks marking the conserved nucleotides. Note that the seed region (boxed; nucleotides 2 to 8), which is essential for target recognition (9, 10), is not conserved. Nucleotides that differ among the SEED sequences are in bold.

FIG. 2.

5′ RACE analysis demonstrates that each miRNA is active at directing the cleavage of the early mRNAs. A modified RACE protocol was utilized to screen for miRNA-mediated cleavage fragments. Portions of the early RNAs corresponding to the coding regions for the large TAgs are shown 5′ to 3′. The positions of the predominant 5′ ends of the cleavage fragments are denoted with black arrowheads, and the numbers of corresponding amplicons are shown. Gray arrowheads indicate the positions of the 5′ ends of less-common amplicons. miRNAs from the complementary strand are shown 3′ to 5′.

FIG. 3.

The JCV miRNAs expressed late during infection downregulate early protein levels. (A) A diagram of a Renilla luciferase reporter containing the region of the early genome complementary to the JCV miRNAs is shown (top). The graph shows luciferase levels from cells transfected with the reporter plus a plasmid expressing the JCV miRNAs (JCmiRNAs) or a negative control plasmid. The Renilla luciferase (Rluc) values were normalized to the values for firefly luciferase (Fluc; cotransfection control). CMV, cytomegalovirus; ds Renilla luciferase, double-stranded Renilla luciferase cDNA; 4X 150 bp JCV TAg, four copies of the JCV TAg construct. (B) A quantitative immunoblot analysis of the large TAg proteins shows that transfection with specific inhibitors (3′/5′ AS) of the JCV miRNAs increased early protein levels during infection. Results from transfection with an irrelevant control oligonucleotide are also shown. The data presented are the averages of results from three independent experiments, plotted as percentages of the protein standards loaded onto each gel.

FIG. 4.

JCV miRNAs are detected in the brain tissue of PML patients. Total RNA was harvested from sections of brain tissue from patients diagnosed with PML. Northern blot analyses with probes specific for the JCV 5p and the loading-control let-7 miRNAs are shown. Northern blotting with a probe specific for the loop region of pre-miRNA showed that the JCV miRNA bands detected in brain tissue were not degradation fragments. (A) Full-length blot showing the detection of the JCV miRNA and pre-miRNA with the 5p probe. (B) Diagram of probes used. (C) Full-length blot from panel A, stripped and reprobed with the terminal loop (TL)-probe. Note that the control loop probe and the 5p probe both detect the pre-miRNA but that only the 5p probe detects bands at ∼22 nucleotides. This pattern rules out the possibility that degradation may account for the miRNA-specific bands detected. The miRNAs detected with the 5p probe are indicated with black arrows. The band corresponding to the pre-miRNA is indicated with a gray arrow. Numbers to the left of the blots indicate sizes in nucleotides. N, normal tissue samples.