Functional Analyses of Bovine Foamy Virus-Encoded miRNAs Reveal the Importance of a Defined miRNA for Virus Replication and Host–Virus Interaction

Abstract

In addition to regulatory or accessory proteins, some complex retroviruses gain a repertoire of micro-RNAs (miRNAs) to regulate and control virus–host interactions for efficient replication and spread. In particular, bovine and simian foamy viruses (BFV and SFV) have recently been shown to express a diverse set of RNA polymerase III-directed miRNAs, some with a unique primary miRNA double-hairpin, dumbbell-shaped structure not known in other viruses or organisms. While the mechanisms of expression and structural requirements have been studied, the functional importance of these miRNAs is still far from understood. Here, we describe the in silico identification of BFV miRNA targets and the subsequent experimental validation of bovine Ankyrin Repeat Domain 17 (ANKRD17) and Bax-interacting factor 1 (Bif1) target genes in vitro and, finally, the suppression of ANKRD17 downstream genes in the affected pathway. Deletion of the entire miRNA cassette in the non-coding part of the U3 region of the long terminal repeats attenuated replication of corresponding BFV mutants in bovine cells. This repression can be almost completely trans-complemented by the most abundant miRNA BF2-5p having the best scores for predicted and validated BFV miRNA target genes. Deletion of the miRNA cassette does not grossly affect particle release and overall particle composition.

The Unique, the Known, and the Unknown of Spumaretrovirus Assembly

“…Elimination of the whole miRNA cassette from non-coding sequences in the U3 region of the long terminal repeats (LTRs) does not grossly affect structural protein expression, processing and particle release but it does impair overall BFV fitness and replication competence. These data indicate that the miRNAs do not play an obvious or prominent role in FV assembly and release [ 66 ].…”

Identification of Feline Foamy Virus-derived MicroRNAs

“…Therefore, although we did not search for the target genes of FFV-derived miRNAs in the present study, it is important to predict target genes when reliable target gene prediction tools become available in cats. Since previous studies reported that FV-derived miRNAs, such as SFVcae, SFVmfu, and BFV, suppress genes related to immune responses and tumor suppression in the host, it is possible that FFV-derived miRNAs also act on these metabolic pathways and help FFV infect hosts ( Kincaid et al , 2014 ; Cao et al , 2020 ; Hashimoto-Gotoh et al , 2020 ). In addition, an evaluation of infectivity using the miRNA deletion mutants of BFV showed that BFV-derived miRNAs contributed to improved infection efficiency ( Cao et al , 2020 ).…”

“…Since previous studies reported that FV-derived miRNAs, such as SFVcae, SFVmfu, and BFV, suppress genes related to immune responses and tumor suppression in the host, it is possible that FFV-derived miRNAs also act on these metabolic pathways and help FFV infect hosts ( Kincaid et al , 2014 ; Cao et al , 2020 ; Hashimoto-Gotoh et al , 2020 ). In addition, an evaluation of infectivity using the miRNA deletion mutants of BFV showed that BFV-derived miRNAs contributed to improved infection efficiency ( Cao et al , 2020 ). Therefore, studies using miRNA deletion mutants will provide novel insights into the biological functions of FFV-derived miRNAs in the future.…”

“…Small RNA deep sequencing analyses revealed that BLV ( Kincaid et al , 2012 ), bovine FV (BFV) ( Whisnant et al , 2014 ), SFVcae derived from African green monkeys ( Chlorocebus aethiops ) ( Kincaid et al , 2014 ), and SFVmfu derived from Japanese macaques ( Macaca fuscata ) ( Hashimoto-Gotoh et al , 2020 ) express miRNAs. In SFVcae, SFVmfu, and BFV, virus-derived miRNAs have been shown to repress the genes involved in the interferon response, solid tumors, and pro-inflammatory signaling, respectively ( Kincaid et al , 2014 ; Cao et al , 2020 ; Hashimoto-Gotoh et al , 2020 ).…”

Foamy Viruses, Bet, and APOBEC3 Restriction

“…In addition, FV encoded micro-RNAs (miRNAs) as shown in BFV [ 127 ] and SFVs [ 128 ] could act as regulators of the innate immune response. BFV miR-BF2–5p can suppress the expression of IFN-β and NF-kB mRNAs by targeting Ankyrin Repeat Domain 17 (ANKRD17), an upstream regulator in the innate immune system and Bax-interacting factor 1 (Bif-1, official name SH3GLB1) [ 129 ].…”