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

Amy H Buck,Amy H Buck,Philippa M Beard,Neil Turnbull,Nicola Craig,Alexandre Houzelle,Katrina Gordon,Katrina Gordon,Alice Roche,Alasdair Ivens,Alasdair Ivens

doi:10.1371/journal.pone.0131787

Amy H Buck, Amy H Buck + Show 9 more

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https://doi.org/10.1371/journal.pone.0131787

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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. miR-29a-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.

Highlights

MicroRNAs have emerged as important regulators of protein expression, influencing many biological pathways including those associated with viral infection [1]
Vaccinia virus (VACV) infection causes widespread reduction of cellular miRNAs at 24 hpi but has varied effects at 6 hpi VACV has previously been shown to degrade both exogenous and endogenous miRNAs in human cells via addition of a 3’ polyadenosine tail catalysed by the VACV polymerase VP55 [11]
To examine further the requirement of a full replication cycle on miR-27b-3p reduction, we examined the levels of this miRNA in Human cervix carcinoma epithelial cells (HeLa) cells infected with Modified Vaccinia virus Ankara (MVA), a replication deficient VACV strain lacking 15% of the parental genome

Summary

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]. 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 guide strand targets mRNAs primarily through complementarity at positions 2–8 of the 50 end of the miRNA (termed the “seed”) [5]

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