Persistent Spodoptera frugiperda rhabdovirus infection in Sf9 cells is not restricted by Wolbachia wMelPop-CLA and wAlbB strains and is targeted by the RNAi machinery

Parry, Rhys; Malmanche, Henry de; Asgari, Sassan

doi:10.1016/j.virol.2021.08.013

Cited by 2 publications

(2 citation statements)

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“…If the siRNA pathway is indeed involved in such a process, then Sf9 cells would be expected to contain siRNAs that map onto the genome of SfRV, indicative of the processing of viral dsRNA intermediates by Dicer-2. Indeed, an analysis of an existing dataset of small RNAs extracted from Sf9 cells revealed that there are large numbers of 20 nt small RNA reads mapping onto both polarities of the SfRV genome [36]. Furthermore, an analysis of a dataset of deep-sequenced small RNAs bound to Ago2 in BmN cells [35] revealed abundant 20 nt reads mapping to the genome of SfRV in both polarities [58].…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, such a function for Dicer-2 has been demonstrated during the infection of D. melanogaster cells by vesicular stomatitis virus, another member of Rhabdoviridae [34]. By analysing deep sequencing datasets of small RNAs, it was recently shown that SfRV is targeted by the RNAi machinery in two lepidopteran cell lines, including the Sf9 cell line [35,36], further suggesting the possibility that Dicer-2 and the siRNA response may restrict SfRV replication to a level that does not compromise cell viability.…”

Section: Introductionmentioning

confidence: 99%

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Knockout of Dicer-2 in the Sf9 cell line enhances the replication of Spodoptera frugiperda rhabdovirus and conditionally increases baculovirus replication

Malmanche

Hussain

Marcellin

et al. 2022

Journal of General Virology

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The Sf9 cell line, originally isolated from the ovarian tissue of Spodoptera frugiperda larvae, is widely used in academia and industry for the baculovirus-mediated production of recombinant proteins and virus-like particles. RNA interference (RNAi) is a conserved antiviral pathway present in eukaryotic organisms and is the primary antiviral defence mechanism in insects. Recent evidence has implicated RNAi as an antiviral response to baculovirus infection in Sf9 cells. To test this hypothesis, CRISPR/Cas9 technology was used to disable the RNAi pathway in Sf9 cells by knocking out Dicer-2, the protein responsible for cleaving viral double-stranded RNA precursors into short interfering RNAs. Infection of Dicer-2 knockout Sf9 cells with either the wild-type baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV), recombinant AcMNPV (rAcMNPV) expressing β-galactosidase (β-gal), or rAcMNPV expressing a wasp venom protein (Vn50) at a multiplicity of infection (m.o.i.) of 1 resulted in a modest increase in virus replication compared to control Sf9 cells under adherent culture conditions. In contrast, Dicer-2 knockout Sf9 monolayer or suspension cultures infected by the rAcMNPV expressing β-gal at higher m.o.i.s (3.5 and 20) did not exhibit increases in either viral DNA replication or β-gal production. Intriguingly, during long-term passaging in suspension, Dicer-2 knockout Sf9 cultures underwent transient crashes in cell proliferation and viability. It was discovered that these periods of low growth and viability coincided with a dramatic increase in the RNA levels of S. frugiperda rhabdovirus, a recently identified adventitious virus that persistently infects the Sf9 cell line, suggesting a role for Dicer-2 in managing chronic viral infections in this industrially relevant insect cell line.

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Section: Discussionmentioning

confidence: 99%