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

Buck AH, Ivens A, Gordon K, Craig N, Houzelle A, Roche A, Turnbull N, Beard PM - PLoS ONE (2015)

Bottom Line: 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.We also demonstrate that intermediate and late VACV gene expression are required for optimal repression of some miRNAs including miR-27-3p.

View Article: PubMed Central - PubMed

Affiliation: Centre for Immunity, Infection and Evolution, University of Edinburgh, King's Buildings, Edinburgh, United Kingdom.

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.

No MeSH data available.


Related in: MedlinePlus

VACV generates long 3’ polyA extensions to endogenous miRNAs.(A) The 3’ modifications of miRNAs (defined by a non-templated nucleotide after position 19 in the mature sequence) were classified by the nucleotide identity (C, G or U) or A, which was then further classified by the number of sequential adenosines: 1, 2, 3–4, 5–9 or 10+. The number of reads plotted on the Y axis are the average of 3 replicates and error bars indicate standard deviation. The actual number of reads in each condition is noted above the column. (B) The number of distinct miRNAs with at least 2 modified reads detected is indicated in green; of these, the miRNAs showing modifications with at least 5 sequential adenosines are depicted in blue or modified by at least 10 sequential adenosines in red.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4498801&req=5

pone.0131787.g003: VACV generates long 3’ polyA extensions to endogenous miRNAs.(A) The 3’ modifications of miRNAs (defined by a non-templated nucleotide after position 19 in the mature sequence) were classified by the nucleotide identity (C, G or U) or A, which was then further classified by the number of sequential adenosines: 1, 2, 3–4, 5–9 or 10+. The number of reads plotted on the Y axis are the average of 3 replicates and error bars indicate standard deviation. The actual number of reads in each condition is noted above the column. (B) The number of distinct miRNAs with at least 2 modified reads detected is indicated in green; of these, the miRNAs showing modifications with at least 5 sequential adenosines are depicted in blue or modified by at least 10 sequential adenosines in red.

Mentions: In parallel with the reduction of unmodified miRNA reads we detected a marked increase in modified miRNA reads in VACV infected cells (Fig 1). Modified reads were defined as containing a non-templated nt at position 19 or higher in the mature sequence (Materials and Methods); the reads were then categorised according to the number of adenosine residues present (Fig 3a). In mock-infected cells at 6 hpi 12% of the reads mapping to miRNAs contained modifications at the 3’ end, dominated by a single adenosine. Only 0.007% of the modified reads contained 5 or more sequential adenosine nts. In comparison, 46% of the reads mapping to miRNAs in cells infected with VACV were modified at the 3’ end, with 19% of the modifications consisting of polyA “tails” 5 nts or longer in length (Fig 3a). Treatment of cells with AraC did not decrease the extent of miRNA polyadenylation (Fig 3a), consistent with the fact that the enzyme responsible for polyadenylation is the RNA polymerase protein VP55 [11] encoded by the VACV early viral gene E1L [36].


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

Buck AH, Ivens A, Gordon K, Craig N, Houzelle A, Roche A, Turnbull N, Beard PM - PLoS ONE (2015)

VACV generates long 3’ polyA extensions to endogenous miRNAs.(A) The 3’ modifications of miRNAs (defined by a non-templated nucleotide after position 19 in the mature sequence) were classified by the nucleotide identity (C, G or U) or A, which was then further classified by the number of sequential adenosines: 1, 2, 3–4, 5–9 or 10+. The number of reads plotted on the Y axis are the average of 3 replicates and error bars indicate standard deviation. The actual number of reads in each condition is noted above the column. (B) The number of distinct miRNAs with at least 2 modified reads detected is indicated in green; of these, the miRNAs showing modifications with at least 5 sequential adenosines are depicted in blue or modified by at least 10 sequential adenosines in red.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4498801&req=5

pone.0131787.g003: VACV generates long 3’ polyA extensions to endogenous miRNAs.(A) The 3’ modifications of miRNAs (defined by a non-templated nucleotide after position 19 in the mature sequence) were classified by the nucleotide identity (C, G or U) or A, which was then further classified by the number of sequential adenosines: 1, 2, 3–4, 5–9 or 10+. The number of reads plotted on the Y axis are the average of 3 replicates and error bars indicate standard deviation. The actual number of reads in each condition is noted above the column. (B) The number of distinct miRNAs with at least 2 modified reads detected is indicated in green; of these, the miRNAs showing modifications with at least 5 sequential adenosines are depicted in blue or modified by at least 10 sequential adenosines in red.
Mentions: In parallel with the reduction of unmodified miRNA reads we detected a marked increase in modified miRNA reads in VACV infected cells (Fig 1). Modified reads were defined as containing a non-templated nt at position 19 or higher in the mature sequence (Materials and Methods); the reads were then categorised according to the number of adenosine residues present (Fig 3a). In mock-infected cells at 6 hpi 12% of the reads mapping to miRNAs contained modifications at the 3’ end, dominated by a single adenosine. Only 0.007% of the modified reads contained 5 or more sequential adenosine nts. In comparison, 46% of the reads mapping to miRNAs in cells infected with VACV were modified at the 3’ end, with 19% of the modifications consisting of polyA “tails” 5 nts or longer in length (Fig 3a). Treatment of cells with AraC did not decrease the extent of miRNA polyadenylation (Fig 3a), consistent with the fact that the enzyme responsible for polyadenylation is the RNA polymerase protein VP55 [11] encoded by the VACV early viral gene E1L [36].

Bottom Line: 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.We also demonstrate that intermediate and late VACV gene expression are required for optimal repression of some miRNAs including miR-27-3p.

View Article: PubMed Central - PubMed

Affiliation: Centre for Immunity, Infection and Evolution, University of Edinburgh, King's Buildings, Edinburgh, United Kingdom.

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.

No MeSH data available.


Related in: MedlinePlus