Cloning and analysis of microRNAs encoded by the primate gamma-herpesvirus rhesus monkey rhadinovirus - PubMed (original) (raw)

Comparative Study

Cloning and analysis of microRNAs encoded by the primate gamma-herpesvirus rhesus monkey rhadinovirus

Alexandra Schäfer et al. Virology. 2007.

Abstract

Several pathogenic human herpesviruses have recently been shown to express virally encoded microRNAs in infected cells. Although the function of these microRNAs is largely unknown, they are hypothesized to play a role in mediating viral replication by downregulating cellular mRNAs encoding antiviral factors. Here, we report the cloning and analysis of microRNAs encoded by Rhesus Monkey Rhadinovirus (RRV), an animal virus model for the pathogenic human gamma-herpesvirus Kaposi's Sarcoma-Associated Herpesvirus (KSHV). RRV expresses several microRNAs that are encoded in the same genomic location as the previously reported KSHV microRNAs, yet these microRNAs are unrelated in primary sequence. These data set the stage for the mutational ablation and phenotypic analysis of RRV mutants lacking one or more viral microRNAs.

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Figures

Fig. 1

Fig. 1

Predicted RNA folding of the primary miRNA stem-loop structures that give rise to ten of the eleven RRV miRNAs listed in Table 1. Cloned RRV miRNAs are indicated in red.

Fig. 2

Fig. 2

Detection of RRV miRNAs using primer extension analysis. The RNA samples analyzed were derived from control 293 cells or from 293 cells that were infected with RRV at high multiplicity and that were beginning to enter lytic replication. The primers used were all designed to be 5 or 6 nt shorter, at the 3’ end, than the mature RRV miRNAs listed in Table 1. In the case of miR-rR1-7-5p, we detected two primer extension products, differing by 1 nt in length, as predicted in Table 1. All other miRNAs gave rise to a single extension product, with the possible exception of miR-rR1-4.

Fig. 3

Fig. 3

RRV miRNA expression is enhanced upon entry into the lytic phase. A) RT-PCR analysis of the expression of a latent RRV mRNA (ORF71), a lytic RRV mRNA (ORF50/RTA) and a control cellular mRNA (GAPDH) using total RNA derived from uninfected 293 cells, 293 cells maintained in the latent phase of the RRV replication cycle by treatment with ganciclovir, or lytically RRV-infected 293 cells. Reactions were controlled for contaminating cellular DNA by performing the PCR amplification without first performing a reverse transcription (RT) step. B) This primer extension analysis was performed as described in Fig. 2 using the RNA preparations described in panel A. The cellular miR-16 miRNA served as a loading control.

Fig. 4

Fig. 4

Schematic representation of the genomic localization of the miRNAs encoded by RRV and KSHV. This schematic shows the viral genes conserved between RRV and KSHV in the latency associated region, as well as the K12/Kaposin gene unique to KSHV. ORF73, ORF72, ORF71 and K12 are expressed during latent infection while ORF68 and ORF69 are lytic genes (indicated in grey). The transcriptional orientation of the viral protein coding genes and of the viral miRNAs is indicated.

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