Evidence that Hsc70 Is Associated with Cucumber Necrosis Virus Particles and Plays a Role in Particle Disassembly.
Uncoating of a virus particle to expose its nucleic acid is a critical aspect of the viral multiplication cycle, as it is essential for the establishment of infection. In the present study, we investigated the role of plant HSP70 homologs in the uncoating process of Cucumber necrosis virus (CNV), a nonenveloped positive-sense single-stranded RNA [(+)ssRNA] virus having a T=3 icosahedral capsid. We have found through Western blot analysis and mass spectrometry that the HSP70 homolog Hsc70-2 copurifies with CNV particles. Virus overlay and immunogold labeling assays suggest that Hsc70-2 is physically bound to virions. Furthermore, trypsin digestion profiles suggest that the bound Hsc70-2 is partially protected by the virus, indicating an intimate association with particles. In investigating a possible role of Hsc70-2 in particle disassembly, we showed that particles incubated with Hsp70/Hsc70 antibody produce fewer local lesions than those incubated with prebleed control antibody on Chenopodium quinoa In conjunction, CNV virions purified using CsCl and having undetectable amounts of Hsc70-2 produce fewer local lesions. We also have found that plants with elevated levels of HSP70/Hsc70 produce higher numbers of local lesions following CNV inoculation. Finally, incubation of recombinant Nicotiana benthamiana Hsc70-2 with virus particles in vitro leads to conformational changes or partial disassembly of capsids as determined by transmission electron microscopy, and particles are more sensitive to chymotrypsin digestion. This is the first report suggesting that a cellular Hsc70 chaperone is involved in disassembly of a plant virus.


OBJECTIVE
Virus particles must disassemble and release their nucleic acid in order to establish infection in a cell. Despite the importance of disassembly in the ability of a virus to infect its host, little is known about this process, especially in the case of nonenveloped spherical RNA viruses. Previous work has shown that host HSP70 homologs play multiple roles in the CNV infection cycle. We therefore examined the potential role of these cellular components in the CNV disassembly process. We show that the HSP70 family member Hsc70-2 is physically associated with CNV virions and that HSP70 antibody reduces the ability of CNV to establish infection. Statistically significantly fewer lesions are produced when virions having undetectable HSc70-2 are used as an inoculum. Finally incubation of Hsc70-2 with CNV particles results in conformational changes in particles. Taken together, our data point to an important role of the host factor Hsc70-2 in CNV disassembly.
Faculty of Land and Food Systems, Plant Science Program, University of British Columbia, Vancouver, British Columbia, Canada.
Syed Benazir
SB
Alam
Faculty of Land and Food Systems, Plant Science Program, University of British Columbia, Vancouver, British Columbia, Canada dann.rochon@agr.gc.ca.|Summerland Research and Development Center, Agriculture and Agri-Food Canada, Summerland, British Columbia, Canada.
D'Ann
D
Rochon
D002213:Capsid / Q000378:metabolism; D036022:Capsid Proteins / Q000378:metabolism; D018553:Cucumis sativus / Q000378:metabolism* / Q000821:virology*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D010641:Phenotype; D010935:Plant Diseases / Q000821:virology*; D011485:Protein Binding; D017864:Tombusvirus / Q000502:physiology* / Q000648:ultrastructure; D014771:Virion / Q000302:isolation & purification / Q000502:physiology* / Q000648:ultrastructure; D057646:Virus Uncoating
D036022:Capsid Proteins; D018840:HSP70 Heat-Shock Proteins
Cucumber necrosis virus; Hsc70; Hsp70; Tombusviridae; Tombusvirus; coat protein; heat shock 70-kDa protein; heat shock cognate 70-kDa protein; virus disassembly; virus uncoating