Rice dwarf virus is engulfed into and released via vesicular compartments in cultured insect vector cells

Abstract

Vector insect cells infected with Rice dwarf virus had vesicular compartments containing viral particles located adjacent to the viroplasm when examined by transmission electron and confocal microscopy. Such compartments were often at the periphery of infected cells. Inhibitors of vesicular transport, brefeldin A and monensin, and an inhibitor of myosin motor activity, butanedione monoxime, abolished the formation of such vesicles and prevented the release of viral particles from infected cells without significant effects on virus multiplication. Furthermore, the actin-depolymerizing drug, cytochalasin D, inhibited the formation of actin filaments without significantly interfering with formation of vesicular compartments and the release of viruses from treated cells. These results together revealed intracellular vesicular compartments as a mode for viral transport in and release from insect vector cells infected with a plant-infecting reovirus.In vector cells grown in monolayers (VCMs), Rice dwarf virus (RDV), a phytoreovirus, multiplies and spreads from primarily infected cells to neighbouring cells (Wei et al., 2006a) in addition to spreading via mature, free viral particles. Infection via free viral particles was protected by the addition of neutralizing antibodies to the cell culture medium. As part of integrated studies on RDV proliferation in vector insects, we have focused our study on the accumulation of the virus in cells of the insect vector and on the subsequent release of the virus.In electron micrographs of thin sections of insect tissues infected with plant-pathogenic reoviruses, viral particles were sequestered in spherical vesicular compartments (Fukushi et al., 1962;Shikata & Maramorosch, 1965;Shikata, 1969;Vidano, 1970;Omura et al., 1985). However, the biological significance of these inclusions in RDV infection has not been clarified because, for the most part, the tissues examined were in the late stage of infection which made it difficult to gather details on the formation of these compartments. Similar vesicular compartments appear to play a role in the release of viral particles from cultured cells infected with animal viruses, such as severe acute respiratory syndrome coronavirus (Ng et al., 2003) and human immunodeficiency virus (HIV) (Nydegger et al., 2003).A method for the continuous culture of cells from the leafhopper vector Nephotettix cincticeps, consisting of VCMs, has allowed the study of infection with RDV because such infection results in asymptomatic but persistent infection (Peterson & Nuss, 1985;Kimura, 1986). VCMs have been used to reveal fundamental aspects of viral activity at the cellular level, which suggests details of the transmission, multiplication and cytopathology of RDV in vector insects (Wei et al., 2006a(Wei et al., , b, c, 2007(Wei et al., , 2008. In the present study with this system, we investigated the role of vesicular compartments in the transport of RDV and its release from infected VCMs by confocal and electron microscopy, and the use of drugs that i...

Outer-capsid P8 proteins of phytoreoviruses mediate secretion of assembled virus-like particles from insect cells

“…In the case of RDV, the outer capsid shell is composed of the major capsid-protein P8, which plays an important role in the infection of insectvector cells (Omura & Yan, 1999), and the minor outercapsid proteins P9 and P2, which are required for adsorption of the virus to host insect cells (Tomaru et al, 1997;Omura et al, 1998). In our earlier studies of core-like particles composed solely of the P3 core-capsid protein of RDV and expressed using a baculovirus expression system in cells of the insect Spodoptera frugiperda (Sf9 cells; Hagiwara et al, 2004), we detected and purified core-like particles from infected cells but we failed to find them in the culture medium.Infectious and intact RDV and RGDV particles that contained both the P3 core-and the P8 outer-capsid proteins were, however, detected in the culture medium of vector cells grown in monolayers (VCM;Wei et al, 2008). These results and the cell association of P3 proteins noted above suggested that the outer capsid proteins of phytoreoviruses might play a role in the transmembrane transport of viral particles in insect-vector cells.…”

“…Infectious and intact RDV and RGDV particles that contained both the P3 core-and the P8 outer-capsid proteins were, however, detected in the culture medium of vector cells grown in monolayers (VCM; Wei et al, 2008). These results and the cell association of P3 proteins noted above suggested that the outer capsid proteins of phytoreoviruses might play a role in the transmembrane transport of viral particles in insect-vector cells.…”

“…that are exploited by authentic RDV particles for their transport and release from infected insect-host cells (Wei et al, 2008(Wei et al, , 2009a. Our results suggest that assembled VLPs, generated in non-host Sf9 cells, are released from these cells via vesicular compartment-like structures in a manner similar to that of intact viral particles (Wei et al, 2008).…”

“…These results indicated that the capsid proteins RDV P3 and RGDV P8 had selfassembled into VLPs in the baculovirus-infected Sf9 cells and that the VLPs had been secreted from the cells. We reported previously (Wei et al, 2008) that authentic RDV is released via vesicular compartments from cultured VCM (Kimura & Omura, 1988). The results from our present study suggest that core-like particles composed of RDV P3 proteins, to which RGDV outer-capsid P8 proteins had attached, were secreted into the medium in the absence of any other viral structural proteins and nucleic acids.…”

Exosome isolation from hemolymph of white-spotted flower chafer, Protaetia brevitarsis (Kolbe) (Coleoptera: Scarabaeidae).

“…Exosomes are generated by inward budding into multi-vesicular bodies (MVB) and then released from cultured insect cell, and the signaling molecules inside are involved in intracellular communication (Koles and Budnik, 2012;Koppen et al,2011;Korkut et al,2009). Plant-infecting Rice dwarf virus was detected in insect exosome isolated from cultured leafhopper cell (Wei et al, 2009;Wei et al, 2008). The virus engulfed in insect exosome was transported and released from the cell of insect vector.…”