Selective cleavage of ncRNA and antiviral activity by human RNase2/EDN in a macrophage infection model

Abstract

RNase2, also named the Eosinophil derived Neurotoxin (EDN), is one of the main proteins secreted by the eosinophil secondary granules. RNase2 is also expressed in other leukocyte cells and is the member of the human ribonuclease A family most abundant in macrophages. The protein is endowed with a high ribonucleolytic activity and participates in the host antiviral activity. Although RNase2 displays a broad antiviral activity, it is mostly associated to the targeting of single stranded RNA viruses. To explore RNase2 mechanism of action in antiviral host defence we knocked out RNase2 expression in the THP1 monocyte cell line and characterized the cell response to human Respiratory Syncytial Virus (RSV). We observed that RSV infection induced the RNase2 expression and protein secretion in THP1 macrophage-derived cells, whereas the knockout (KO) of RNase2 resulted in higher RSV burden and reduced cell viability. Next, by means of the cP-RNAseq methodology, which uniquely amplifies the RNA 2'3'cyclic-phosphate-end products released by an endonuclease cleavage, we compared the ncRNA population in native and RNase2-KO cell lines. Among the ncRNAs accumulated in WT versus KO cells, we found mostly tRNA-derived fragments and secondly miRNAs. Analysis of the differential sequence coverage of tRNAs molecules in native and KO cells identified fragments derived from only few parental tRNAs, revealing a predominant cleavage at anticodon loops and secondarily at D-loops. Inspection of cleavage region identified U/C and A, at 5' and 3' sides of cleavage sites respectively (namely RNase B1 and B2 base binding subsites). Likewise, only few selected miRNAs were significantly more abundant in WT versus RNase2-KO cells, with cleavage sites located at the end of stem regions with predominance for pyrimidines at B1 but following an overall less defined nucleotide specificity. Complementarily, by screening of a tRF/tiRNA PCR array we identified an enriched population of tRNA-derived fragments associated to RNase2 expression and RSV infection. The present results confirm the contribution of the protein in macrophage response against virus infection and provide the first evidence of its cleavage selectivity against ncRNA population. A better understanding of the mechanism of action of RNase2 recognition of cellular RNA during the antiviral host defence should pave the basis for the design of novel antiviral drugs.