Evidence that Hsc70 Is Associated with Cucumber Necrosis Virus Particles and Plays a Role in Particle Disassembly

Abstract

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 [(ϩ)ssRNA] virus having a Tϭ3 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.IMPORTANCE 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.

Hsc70-2 is required for Beet black scorch virus infection through interaction with replication and capsid proteins

“…Recently, Alam et al . reported that Hsc70-2 binds to purified CNV virions and plays a role in disassembly of virus particles 39 . Hsc70 was also reported to participate in the intercellular transport of pepino mosaic virus (PepMV) 25 .…”

“…A cytosolic Hsp70 is found to play multiple roles in TBSV replication, such as the insertion of the viral replication proteins into peroxisomal membranes and activation of TBSV replication by interaction with viral replication proteins 34 – 38 . Recent studies also indicated that Hsc70 is co-opted by CNV to functions in several aspects of the viral infection cycle, including the positive regulation of viral RNA accumulation, virus particle assembly and disassembly, and targeting of CP to the chloroplasts 27 , 39 . Additionally, a chloroplast Hsp70 isoform was reported to interact with bamboo mosaic virus (BaMV) replicase and is required for the preference of BaMV for older leaves of N .…”

Proteostasis in Viral Infection: Unfolding the Complex Virus–Chaperone Interplay

“…Preincubation of viral particles with recombinant Hsp70 or antibodies against Hsp70 prevented rotavirus internalization (Guerrero et al 2002;Zarate et al 2003;Perez-Vargas et al 2006), and Hsp70 depletion impaired reovirus disassembly into transcriptionally active particles (Ivanovic et al 2007). In cucumber necrosis virus, Hsp70 was found to directly alter capsid conformation, as determined by electron microscopy (Alam and Rochon 2017). A similar extracellular interaction with Hsp70 was reported for positive-strand RNA viruses of the flavivirus family, for example, hepatitis C virus (HCV) (Parent et al 2009;Khachatoorian et al 2014), Japanese encephalitis virus (JEV) (Das et al 2009), and dengue virus (DENV) (Reyes-Del Valle et al 2005).…”

Mechanism and Complex Roles of HSC70 in Viral Infections

“…Blocking HSC70 through specific antibodies significantly inhibits δ release and, complementing with purified HSC70, totally restores the δ-release activity (Ivanovic et al, 2007), suggesting that HSC70 plays central roles in reovirus disassembly. Apart from animal viruses, HSC70 has been reported to be associated with cucumber necrosis virus (CNV) particles and incubation of recombinant HSC70-2 with CNV results in conformational changes or partial disassembly of virus capsid and produces higher numbers of local lesions on Chenopodium quinoa, suggesting that HSC70 plays an important role in plant virus infection (Alam and Rochon, 2017).…”