Interaction between HSP70 Homolog and Filamentous Virions of the Beet Yellows Virus

Abstract

An HSP70 homolog (HSP70h), encoded by the Closterovirus Beet yellows virus (BYV), functions in viral movement from cell to cell. A previous study revealed that in infected cells, HSP70h colocalizes with the masses of BYV filamentous virions. Here we demonstrate that HSP70h forms a physical complex with BYV virions. This conclusion is based on both the comigration of HSP70h with BYV virions in sucrose density gradients and the coimmunoprecipitation of the HSP70h and BYV capsid protein using anti-HSP70h serum. The HSP70h-virion complex is stable at high concentrations of sodium chloride; its dissociation using sodium dodecyl sulfate, lithium chloride, or alkaline pH was accompanied by virion disassembly. However, the complex formation does not involve covalent bonds between HSP70h and virion components. Each BYV virion contains approximately 10 molecules of HSP70h. The possible role of HSP70h interaction with the virions in cell-to-cell movement of BYV is discussed.

Complex molecular architecture of beet yellows virus particles

“…Wild-type virions of the BYV-California isolate were purified from Nicotiana benthamiana plants as detailed (16). Mutation Nop20, which inactivated the start codon of the p20 ORF (27), was introduced into the binary vector p35S-BYV-GFP harboring a full-length cDNA copy of the BYV genome tagged by insertion of the gene encoding the green f luorescent protein (BYV-GFP) (18).…”

“…The fragmented virions were then loaded on the top of 20 -60% sucrose gradients prepared in buffer 1 and centrifuged at 100,000 ϫ g for 16 h in a Beckman SW40 rotor at 4°C. Gradients were separated into 15 fractions; those fractions containing the peak of CPm were pooled and diluted in buffer 1; virion fragments were recovered by ultracentrifugation for 6 h at 150,000 ϫ g. Immunoblot analysis was performed according to standard procedures (16 Northern Hybridization Analyses. Total RNA was extracted from virion tail pellets or from intact BYV particles by using TRIzol (Invitrogen) according to the manufacturer's protocol, and the hybridization analysis was carried out as described (27).…”

“…1A). Recent studies demonstrated that BYV virions incorporate at least five of these proteins (15)(16)(17)(18). The major CP forms a long virion body that is analogous to ''ordinary'' filamentous virions.…”

“…Perhaps the most remarkable component of BYV virions is the virus-encoded homolog of cellular Hsp70 molecular chaperones (Hsp70h) (16,20). So far, only closteroviruses have been found to harbor a Hsp70 gene, although papovaviruses and a baculovirus code for Hsp70 cochaperones called J-domain proteins (10,21).…”

Helical capsids of plant viruses: architecture with structural lability

“…The main virion part (approximately 95 % of its length) is made of the CP, while the remaining segment, which is located at the virion end encapsidating the 5¢-terminal region of genomic RNA, is formed by the CPm (Agranovsky et al, 1995;Peremyslov et al, 2004;Satyanarayana et al, 2004). Additionally, the CPm-formed region of BYV virions contains three other viral proteins, namely an Hsp70 homologue (Hsp70h), a p64 protein with a CP-like structural domain in its C-terminal region and p20 (Kiss et al, 2013;Napuli et al, 2000Napuli et al, , 2003Tian et al, 1999). The latter is dispensable for virion formation and virus cell-to-cell movement, but is essential for the viral systemic transport through the plant vascular system (Prokhnevsky et al, 2002).…”