Quantitative Analysis of MicroRNAs in Vaccinia virus Infection Reveals Diversity in Their Susceptibility to Modification and Suppression

RESEARCH ARTICLE Quantitative Analysis of MicroRNAs in Vaccinia virus Infection Reveals Diversity in Their Susceptibility to Modification and Suppression Amy H. Buck1*, Alasdair Ivens1, Katrina Gordon1, Nicola Craig2¤a, Alexandre Houzelle2, Alice Roche2¤b, Neil Turnbull2, Philippa M. Beard2* a11111 OPEN ACCESS Citation: Buck AH, Ivens A, Gordon K, Craig N, Houzelle A, Roche A, et al. (2015) Quantitative Analysis of MicroRNAs in Vaccinia virus Infection Reveals Diversity in Their Susceptibility to Modification and Suppression. PLoS ONE 10(7): e0131787. doi:10.1371/journal.pone.0131787 Editor: James P. Stewart, University of Liverpool, UNITED KINGDOM Received: April 26, 2015 Accepted: June 8, 2015 Published: July 10, 2015 Copyright: © 2015 Buck et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Sequencing data are available in NCBI GEO database (GSE54235). Funding: The Roslin Institute receives Institute Strategic Grant funding from the BBSRC. This work is supported by a Wellcome Trust RCDF (WT097394AIA) and BBSRC project (BB/J001279/1) awarded to A. Buck as well as a Wellcome Trust strategic award (095831MA) to the Centre for Immunity, Infection and Evolution supporting A. Ivens. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 Centre for Immunity, Infection and Evolution, University of Edinburgh, King’s Buildings, Edinburgh, United Kingdom, 2 Infection and Immunity, The Roslin Institute / Royal (Dick) School of Veterinary Studies, Easter Bush, Midlothian, United Kingdom ¤a Current address: Institute of Biodiversity, Animal Health and Comparative Medicine, University of Glasgow, Garscube Estate, Glasgow, United Kingdom ¤b Current address: Biological Engineering Department, Polytech Clermont-Ferrand/ Blaise Pascal University, Clermont-Ferrand, Auvergne, France * (AHB); (PMB) Abstract Vaccinia virus (VACV) is a large cytoplasmic DNA virus that causes dramatic alterations to many cellular pathways including microRNA biogenesis. The virus encodes a poly(A) polymerase which was previously shown to add poly(A) tails to the 3’ end of cellular miRNAs, resulting in their degradation by 24 hours post infection (hpi). Here we used small RNA sequencing to quantify the impact of VACV infection on cellular miRNAs in human cells at both early (6 h) and late (24 h) times post infection. A detailed quantitative analysis of individual miRNAs revealed marked diversity in the extent of their modification and relative change in abundance during infection. Some miRNAs became highly modified (e.g. miR29a-3p, miR-27b-3p) whereas others appeared resistant (e.g. miR-16-5p). Furthermore, miRNAs that were highly tailed at 6 hpi were not necessarily among the most reduced at 24 hpi. These results suggest that intrinsic features of human cellular miRNAs cause them to be differentially polyadenylated and altered in abundance during VACV infection. We also demonstrate that intermediate and late VACV gene expression are required for optimal repression of some miRNAs including miR-27-3p. Overall this work reveals complex and varied consequences of VACV infection on host miRNAs and identifies miRNAs which are largely resistant to VACV-induced polyadenylation and are therefore present at functional levels during the initial stages of infection and replication. PLOS ONE | DOI:10.1371/journal.pone.0131787 July 10, 2015 1 / 19 Quantification of the Impact of Vaccinia virus on Host MicroRNAs Competing Interests: The authors have declared that no competing interests exist. Introduction MicroRNAs have emerged as important regulators of protein expression, influencing many biological pathways including those associated with viral infection [1]. These small RNAs function by guiding the RNA-induced silencing complex (RISC) to messenger RNAs (mRNAs) with base pair complementarity, resulting in inhibition of translation and/or mRNA destabilization [2]. The biogenesis of a miRNA (reviewed in [3, 4]) begins in the nucleus with synthesis of a primary miRNA transcript (pri-miRNA), generally by RNA polymerase II. In mammals the Drosha nuclear RNase III endonuclease and DGCR8 process the pri-miRNA to a shorter (approximately 70 nt) hairpin structure known as the precursor miRNA (pre-miRNA). This structure is transported to the cytoplasm via exportin 5, where it is processed to a ~ 22 nt duplex RNA by the RNase Dicer and its cofactor TRBP. The RNA duplex is loaded into the Argonaute (Ago) proteins, where one strand (the passenger or star strand) is released and degraded, and the other strand (the guide) retained. The guide strand then targets mRNAs primarily through complementarity at positions 2–8 of the 50 end of the miRNA (termed the “seed”) [5]. Several non-canonical miRNA biogenesis pathways have been described that include the ability to bypass the need for processing in the nucleus [6]. Some viruses have evolved to express their own miRNAs by these canonical or non-canonical pathways [7] and viruses can also be engineered to produce miRNAs [8, 9]. Poxviruses are large cytoplasmic DNA viruses with a complex life cycle that includes viral DNA replication and transcription occurring in specialised cytoplasmic “viral factories” by virally-encoded polymerase enzymes [10]. VACV is the prototypic orthopoxvirus which does not encode any viral miRNAs [1] but induces polyadenylation of mature cellular miRNAs with a concurrent widespread reduction in abundance by 24 h pi [11, 12]. Dysregulation of miRNA expression has been linked with numerous diseases [13] and there is extensive interest in understanding how the levels of these molecules are naturally regulated. While the steps leading to miRNA production and maturation are relatively well understood [14] the mechanisms involved in the decay of miRNAs remain somewhat elusive [15, 16]. In many cellular contexts mature miRNAs appear to be extremely stable with half-lives in the range of days [17–19]. However some miRNAs exhibit rapid downregulation under certain physiological conditions including cell cycle, neuronal activation, viral infection or in response to growth factors [15]. This implies regulated mechanisms for miRNA decay. Several studies in animals have suggested some exonucleases associated with degradation of the miRNAs, reviewed in [15, 20]. Loss of Ago2 in mammals also results in a reduction in miRNA levels, suggesting Ago2 provides a level of protection and stabilisation of the mature miRNAs [21, 22]. One mechanism for selectively degrading a specific miRNA involves recognition of the mature sequence by another non-coding RNA; this has been termed “target mediated miRNA degradation” (TMMD) and requires extensive complementarity between the two RNAs[23]. (...truncated)