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

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.

Identification of a Functional Small Noncoding RNA of African Swine Fever Virus

“…This was in contrast to VACV-infected cells, which had a 30% reduction in miRNA reads at an early time point (6 hpi) and over 50% reduction at late times (24 hpi) ( Fig. 2b ) ( 21 ). To assess the extent of ASFV-induced 3′ modification of miRNAs, all trimmed sequences were analyzed for nontemplated nucleotide 3′ additions after nucleotide position 19.…”

“…VACV, the prototypic poxvirus and NCLDV member, has been shown to induce widespread disruption of cellular miRNAs via a process of 3′ polyadenylation and decay ( 20 , 21 ). To investigate if ASFV shares the ability of VACV to induce miRNA polyadenylation and decay, porcine alveolar macrophages (PAMs) were infected with the pathogenic ASFV Benin 97/1 strain and Vero cells were infected with the Vero cell-adapted ASFV strain, Ba71v.…”

“…In parallel, PAMs and Vero cells were infected with VACV WR, with the samples collected and processed for Northern blotting as described. The miRNA miR-27b-3p was selected as a probe for this experiment, since it is extensively polyadenylated and degraded in VACV-infected cells ( 21 ). As expected, in VACV-infected Vero cells at 0 h postinfection (hpi), mature miR-27b-3p was seen as a lower band ( Fig.…”

“…These accounted for, on average, 73% of total trimmed small RNA reads. Data from a previous study investigating the effect of VACV infection on host miRNAs was used as a comparison ( 21 ). In ASFV-infected cells, there was no variation in the miRNA proportion of the total small RNA reads at both early and late time points compared to uninfected cells ( Fig.…”

“…MCMV induces degradation of cellular miR-27a and miR-27b, which are important for MCMV replication in vivo ( 19 ). A more nonspecific and global effect is wrought by vaccinia virus (VACV), the prototypic poxvirus and NCLDV member, which induces widespread disruption of host miRNAs by a process of 3′ polyadenylation and decay ( 20 , 21 ). As RNAi is the major antiviral pathway in invertebrates, a number of arthropod-borne (arboviruses) are known to manipulate sncRNA during replication to evade this immune response.…”