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. 2013 Dec;447(1-2):140-5.
doi: 10.1016/j.virol.2013.08.024. Epub 2013 Sep 26.

Persistently adenovirus-infected lymphoid cells express microRNAs derived from the viral VAI and especially VAII RNA

Yuki Furuse  1 , David A Ornelles, Bryan R Cullen
Affiliations

Persistently adenovirus-infected lymphoid cells express microRNAs derived from the viral VAI and especially VAII RNA

Yuki Furuse et al. Virology. 2013 Dec.

Abstract

Human adenovirus can establish latent infections in lymphoid tissues in vivo and persistent, infections in cultured lymphoid cell lines. During lytic infection, adenovirus expresses microRNAs (miRNAs) derived from the viral non-coding RNAs VAI and, especially, VAII. Here, we demonstrate that persistently adenovirus-infected human BJAB cells also produce adenovirus-derived miRNAs primarily derived from the viral VAII RNA, which contributes ~2.7% of all RNA-induced silencing complex (RISC)-associated RNAs. However, our data indicate that the 5' end of the predominant VAII-derived viral RNA, and hence its seed sequence, differs from what has been previously reported. Our data demonstrate that adenovirus expresses viral miRNAs in chronically infected lymphoid cells and raise the possibility that these may contribute to the maintenance of the latently adenovirus-infected lymphoid cells previously observed in mucosal-associated lymphoid tissues in vivo.

Keywords: Adenovirus; MicroRNAs; Persistent infection; RNA interference.

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Figures

Figure 1
Figure 1. Determination of AdV DNA levels in persistently infected lymphoid cells
A master lysate plate was prepared by diluting persistently AdV-infected BJAB or KE37 cells at 1000, 100, 10, 1, and 0.1 cells per well and twelve wells for each dilution were tested for AdV DNA y a nested PCR assay. The percentage of positive wells was plotted with a best-fit three-parameter logistic fit constrained between 0% and 100%.
Figure 2
Figure 2. Detection of VA RNAs and other viral transcription units
Relative expression levels of various AdV RNAs in the persistently AdV-infected BJAB cells were determined by qRT-PCR and then normalized to VAI. RT indicates samples with reverse transcription and no-RT indicates negative controls prepared without reverse transcriptase. Error bars indicate standard deviation of 3 independent experiments. Black bars indicate VA RNAs. Light gray and dark gray bars represent transcription units for AdV early (E) and late (L) genes, respectively.
Figure 3
Figure 3. Small RNA deep sequencing
A) Length distribution of reads mapped to the AdV or human genome, and unmapped small RNA reads. B) Breakdown of reads mapped to human genome according to the class of RNA that they originate from. C) Breakdown of reads mapped to the AdV genome according to origin.
Figure 4
Figure 4. Site-by-site coverage of VAI by small RNAs
A) A schematic secondary structure for VAI is shown. Red, yellow and green indicate the terminal stem, the central domain and the apical region, respectively. B) The percentage of site-by-site coverage relative to total coverage (both human and AdV genome) is plotted. The X-axis shows positions from the 5’ end to 3’ end of VAI. Open dots reflect the result of deep-sequencing the total small RNA population while filled gray dots represent reads obtained using RNA isolated by RIP against Ago2 (only for BJAB cells). Regions of origin in VAI are specified by colors that correspond to panel A.
Figure 5
Figure 5. Site-by-site coverage of VAII by small RNAs
The figure is for VAII and was generated exactly as described for VAI in Fig. 4.
Figure 6
Figure 6. Small RNAs derived from VAI
Each bar represents both the expression level and origin of each VAI-derived small RNA in either the total small RNA library or the RISC-associated small RNA library. The percentage contribution of each small RNA population to the total number of reads obtained is shown to the left of each bar. Dashed lines indicate the 5’ nucleotide of each RNA.
Figure 7
Figure 7. Small RNAs derived from VAII
The figure for VAII-derived small RNAs was generated as described for VAI in Fig. 6.
See this image and copyright information in PMC

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