Peer Review #2 of "Linear Lepidopteran ambidensovirus 1 sequences drive random integration of a reporter gene in transfected Spodoptera frugiperda cells (v0.1)" [peer_review]

2018 unpublished
Background. The Lepidopteran ambidensovirus 1 isolated from Junonia coenia (hereafter JcDV) is an invertebrate parvovirus considered as a viral transduction vector as well as a potential tool for the biological control of insect pests. Previous works showed that JcDV-based circular plasmids experimentally integrate into insect cells genomic DNA. Methods. In order to approach the natural conditions of infection and possible integration, we generated linear JcDV-gfp based molecules which were
more » ... ules which were transfected into non permissive Spodoptera frugiperda (Sf9) cultured cells. Cells were monitored for the expression of GFP and DNA was analysed for integration of transduced viral sequences. Non-structural protein modulation of the VP-gene cassette promoter activity was additionally assayed. Results. We show that linear JcDV-derived molecules are capable of long term genomic integration and sustained transgene expression in Sf9 cells. As expected, only the deletion of both inverted terminal repeats (ITR) or the polyadenylation signals of NS and VP genes dramatically impairs the global transduction/expression efficiency. However, all the integrated viral sequences we characterized appear "scrambled" whatever the viral content of the transfected vector. Despite a strong GFP expression, we were unable to recover any full sequence of the original constructs and found rearranged viral and nonviral sequences as well. Cellular flanking sequences were identified as non-coding ones. On the other hand, the kinetics of GFP expression over time led us to investigate the apparent down-regulation by non-structural proteins of the VP-gene cassette promoter. Conclusions. Altogether, our results show that JcDV-derived sequences included in linear DNA molecules are able to drive efficiently the integration and expression of a foreign gene into the genome of insect cells, whatever their composition, provided that at least one ITR is present. However, the transfected sequences were extensively rearranged with cellular DNA during or after random integration in the host cell genome. Last, the non-structural proteins seem to participate in the regulation of p9 promoter activity rather than to the integration of viral sequences. Manuscript to be reviewed 1 Linear lepidopteran ambidensovirus 1 sequences drive random integration of a reporter 2 gene in transfected Spodoptera frugiperda cells Abstract 40 Background. The Lepidopteran ambidensovirus 1 isolated from Junonia coenia (hereafter 41 JcDV) is an invertebrate parvovirus considered as a viral transduction vector as well as a 42 potential tool for the biological control of insect pests. Previous works showed that JcDV-based 43 circular plasmids experimentally integrate into insect cells genomic DNA. 44 Methods. In order to approach the natural conditions of infection and possible integration, we 45 generated linear JcDV-gfp based molecules which were transfected into non permissive 46 Spodoptera frugiperda (Sf9) cultured cells. Cells were monitored for the expression of GFP and 47 DNA was analysed for integration of transduced viral sequences. Non-structural protein 48 modulation of the VP-gene cassette promoter activity was additionally assayed. 49 Results. We show that linear JcDV-derived molecules are capable of long term genomic 50 integration and sustained transgene expression in Sf9 cells. As expected, only the deletion of both 51 inverted terminal repeats (ITR) or the polyadenylation signals of NS and VP genes dramatically 52 impairs the global transduction/expression efficiency. However, all the integrated viral sequences 53 we characterized appear "scrambled" whatever the viral content of the transfected vector. 54 Despite a strong GFP expression, we were unable to recover any full sequence of the original 55 constructs and found rearranged viral and non-viral sequences as well. Cellular flanking 56 sequences were identified as non-coding ones. On the other hand, the kinetics of GFP expression 57 over time led us to investigate the apparent down-regulation by non-structural proteins of the 58 VP-gene cassette promoter. 59 Conclusions. Altogether, our results show that JcDV-derived sequences included in linear DNA 60 molecules are able to drive efficiently the integration and expression of a foreign gene into the 61 genome of insect cells, whatever their composition, provided that at least one ITR is present. PeerJ reviewing PDF |
doi:10.7287/peerj.4860v0.1/reviews/2 fatcat:zqxq4gckofbjvbi2rlzrnks3ky