Understanding piRNA Biogenesis and Function in the Lepidopteran Ovarian Germ Cell Line Hi5

In animals, reproductive fitness depends on PIWI-interacting RNAs (piRNAs), small RNAs that silence transposable elements, thereby maintaining genomic stability. Several Lepidopteran cell lines derive from germ cells and produce piRNAs. The genome of Hi5 cells, an immortalized germ cell line from Trichoplusia ni (cabbage looper), has been fully sequenced, assembled, and annotated, making it a suitable cultured insect cell model to study piRNA biogenesis and function. Hi5 cells express the complete set of piRNA genes, and individual piRNA-processing proteins can be depleted using chemically synthesized siRNAs, allowing us to examine how they function in piRNA biogenesis. Hi5 cells are chronically infected with Tn5 Cell Line Virus (TNCLV), a positive-strand, segmented RNA virus in the Alphanodavirus genus. TNCLV replicates via double-stranded RNA intermediates, and Hi5 cells control TNCLV infection using virus-derived siRNAs. Depleting mRNAs encoding siRNA pathway proteins Ago2 and Dicer-2 reduces viral siRNAs and derepresses viral RNA. Unexpectedly, we also observed that, in the absence of piRNA factors, TNCLV viral RNA load increased by as much as 100-fold. Transcriptome profiling detected no change in the levels of mRNAs encoding known siRNA pathway proteins. Similarly, small RNA sequencing found no change in the steady-state abundance of endogenous siRNAs derived from T. ni genomic loci. Consistent with the ≤100-fold increase in viral transcripts, virus-derived siRNAs also increased by as much as 100-fold. Although highly abundant, these anti-viral siRNAs appear to be double-stranded, suggesting they are not loaded into Ago2, the insect-specific Argonaute protein dedicated to the RNAi pathway. Alphanodaviruses encode an RNAi suppressor, the B2 protein, which binds both long double-stranded RNA and siRNA duplexes. The accumulation of unloaded siRNA duplexes is consistent with the expression of TNCLV-encoded B2 when the piRNA pathway is impaired. Yet we can detect neither piRNAs derived from the TNCLV genome nor the B2 protein sequence integrated into the Hi5 genome. B2 depletion rescues TNCLV silencing caused by loss of Piwi, suggesting a role for the piRNA pathway in silencing B2. We observed that Hi5 cells generate piRNAs with enough partial complementarity to bind and cleave TNCLV transcripts, particularly concentrated in the B2 coding region. This explains how the piRNA pathway contributes to viral silencing. Notably, some of these piRNAs are also expressed in the T. ni ovary and thorax, so they are not exclusively Hi5-specific. To the best of our knowledge, this is the first example of piRNA-based anti-viral immunity in the absence of viral sequences integrated into the genome.