MicroRNA and Nonsense Transcripts as Putative Viral Evasion Mechanisms

Abstract

Viral proteins encode numerous antiviral activities to modify the host immunity. In this article, we hypothesize that viral genomes and gene transcripts interfere with host gene expression using passive mechanisms to deregulate host microRNA (miRNA) activity. We postulate that various RNA viruses mimic or block binding between a host miRNA and its target transcript, a phenomenon mediated by the miRNA seed site at the 5' end of miRNA. Virus-encoded miRNA seed sponges (vSSs) can potentially bind to host miRNA seed sites and prevent interaction with their native targets thereby relieving native miRNA suppression. In contrast, virus-encoded miRNA seed mimics (vSMs) may mediate considerable downregulation of host miRNA activity. We analyzed genomes from diverse RNA viruses for vSS and vSM signatures and found an abundance of these motifs indicating that this may be a mechanism of deceiving host immunity. Employing respiratory syncytial virus and measles virus as models, we reveal that regions surrounding vSS or vSM motifs have features characteristics of pre-miRNA templates and show that RSV viral transcripts are processed into small RNAs that may behave as vSS or vSM effectors. These data suggest that complex molecular interactions likely occur at the host-virus interface. Identifying the mechanisms in the network of interactions between the host and viral transcripts can help uncover ways to improve vaccine efficacy, therapeutics, and potentially mitigate the adverse events that may be associated with some vaccines.

Keywords: RNA viruses; miR; miRNA; microRNA; mimics; seed sequence; sponges.

Figure 1

Distribution of vSMs and vSSs across genera of the Paramyxoviridae family. (A) Viral sequences identical to host miRNA seed site were designated vSMs (open bars) while those complementary to miRNA seed sites were designated as vSSs (filled bars). (B) Ratio of vSS/vSM across genera is shown. Dotted line represents ratio equal to 1.0. HV, hendravirus; HMPV, human metapneumovirus; MV, measles virus; MuV, mumps virus; NDV, New Castle's disease virus; NiV, nipah virus; RSV, respiratory syncytial virus.

Figure 2

Sequence alignments of miRNA vSMs encoded by different paramyxoviruses. Alignments show identity between host miRNA seed sites and the corresponding vSM across Paramyxoviruses. Nucleotide numbers indicated viral genome coordinates while labels on the right indicate encoding viral gene. miRNA seed sequence is indicated in bold. Straight lines indicate perfect identity.

Figure 3

Sequence alignments of miRNA vSSs encoded by different paramyxoviruses. Alignments show complementarity between host miRNA seed site and the corresponding vSS across Paramyxoviruses. Nucleotide numbers indicated viral genome coordinates while labels on the right indicate encoding viral gene. miRNA seed sequence is indicated in bold. Straight lines indicate Watson-Crick base pairing (AU/GC) while colon indicates a non-Watson-Crick base pair (GU).

Figure 4

Viral genomic regions form characteristic stem- loop folds in vSM and vSS encoding regions and are processed into smaller transcripts. Secondary structures of a 100 nt sequence flanking vSM-4719 (A) and−556-3p (B) were predicted using RNAfold and drawn using VARNA. Highlighted region corresponds to the vSM. Internal and terminal loops are prefixed with L and T, respectively. Helices are prefixed with H and the 5′ and 3′ ends of the molecule are as indicated. (C) Size-fractionated small RNA from mock or RSV A2 infected (24 h or 48 h pi) Vero cells was reverse transcribed using RSV G- and L-specific reverse primers. Amplicons obtained using a G/L specific primer pair were electrophoresed on a 12% PAGE gel alongside a molecular ladder and stained with SYBR gold.

Figure 5

Small RNA from RSV-infected respiratory epithelial (A549) (A,B) or neuronal (SHSY5Y) (C,D) cells were sequenced on an Illumina MiSeq. Alignments show RSV vSMs or vSSs in bold.

Figure 6

Model of vSM / vSS modulation of host responses during infection. Host mRNA are depicted as solid lines, microRNAs are depicted as dashed lines with end caps while viral transcripts are depicted as dashed lines. (A) Normal post-transcriptional miRNA regulation of gene expression. Degree of miRNA complementarity determines if gene knockdown proceeds via mRNA decay/translation following miRNA binding to target transcript in the RISC complex. (B) Viral RNAs encoding vSSs/processed vSSs bind to host miRNAs and prevent native suppression of pro-viral factors. (C) Viral RNAs encoding vSMs can supplement host miRNA activity and suppress anti-viral responses.