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We have shown that Macrobrachium rosenbergii nodavirus (MrNV) was able to infect Sf9 cells and that MrNV virus-like particles (MrNV-VLPs) were capable nanocontainers for delivering nucleic acid-based materials. Here, we demonstrated that chymotryptic removal of a C-terminal peptide and its truncated variant (F344-MrNV-VLPs) exhibited a drastically reduced ability to interact and internalize into Sf9 cells. Electron microscopic observations revealed that the loss of C-terminal domain either from enzyme hydrolysis or genetic truncation did not affect the generated MrNV-VLPs' icosahedral conformation, but did drastically affect the VLPs' internalization ability into Sf9 cells. Homology-based modelling of the MrNV capsid with other icosahedral capsid models revealed that this chymotrypsin-sensitive C-terminal domain was not only exposed on the capsid surface, but also constituted the core of the viral capsid protrusion. These results therefore suggest the importance of the C-terminal domain as a structure for targeted cell interaction which is presumably localized at the protruding domain. This work thus provided the functional insights into the role of the MrNV C-terminal domain in viral entry into Sf9 cells and lead to the development of strategies in combatting MrNV infection in susceptible cells., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this study we demonstrated that Macrobrachium rosenbergii nodavirus (MrNV) was able to internalize and replicate in Sf9 insect cells, with levels of infection altered by substances affecting the caveolin-(CAV) mediated endocytosis pathway. The use of Sf9 cells for efficient MrNV replication and propagation was demonstrated by confocal microscopy and PCR amplification, through which early viral binding and internalization were initially detectable at 30min post-infection; whereas at 72h, the distinguishable sign of late-MrNV infection was observable as the gradual accumulation of a cytopathic effect (CPE) in the cells, ultimately resulting in cellular disruption. Moreover, during the early period of infection, the MrNV signals were highly co-localized with CAV1 signals of the CAV-mediated endocytosis pathway. The use of genistein as an inhibitor of the CAV-mediated endocytosis pathway significantly reduced MrNV and CAV1 co-localization, and also reduced the levels of MrNV infection in Sf9 cells as shown by PCR and ELISA. Moreover, the addition of the pathway agonist okadaic acid not only recovered but also augmented both the levels of MrNV co-localization with CAV1 and of Sf9 infection in the presence of genistein inhibition; therefore demonstrating that MrNV infection in Sf9 cells was associated with the CAV-mediated endocytosis pathway machinery., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Virus-like particles (VLPs) are potential candidates in developing biological containers for packaging therapeutic or biologically active agents. Here, we expressed Macrobrachium rosenbergii nodavirus (MrNv) capsid protein (encoding amino acids M1-N371 with 6 histidine residuals) in an Escherichia coli BL21(DE3). These easily purified capsid protein self-assembled into VLPs, and disassembly/reassembly could be controlled in a calcium-dependent manner. Physically, MrNv VLPs resisted to digestive enzymes, a property that should be advantageous for protection of active compounds against harsh conditions. We also proved that MrNv VLPs were capable of encapsulating plasmid DNA in the range of 0.035-0.042 mol ratio (DNA/protein) or 2-3 plasmids/VLP (assuming that MrNV VLPs is T=1, i made up of 60 capsid monomers). These VLPs interacted with cultured insect cells and delivered loaded plasmid DNA into the cells as shown by green fluorescent protein (GFP) reporter. With many advantageous properties including self-encapsulation, MrNv VLPs are good candidates for delivery of therapeutic agents., (Copyright © 2013 Elsevier B.V. All rights reserved.)