Closterovirus encoded HSP70 homolog and p61 in addition to both coat proteins function in efficient virion assembly

Assembly of the viral genome into virions is a critical process of the viru s life cycle often defining the ability of the virus to move within the pla nt and to be transmitted horizontally to other plants. Closteroviridae viri ons are polar helical rods assembled primarily by a major coat protein, but with a related minor coat protein at one end. The Closteroviridae is the o nly virus family that encodes a protein with similarity to cellular chapero nes, a 70-kDa heat-shock protein homolog (HSP70h). We examined the involvem ent of gene products of Citrus tristeza virus (CN) in virion formation and found that the chaperone-like protein plus the p61 and both coat proteins w ere required for efficient virion assembly. Competency of virion assembly o f different CTV mutants was assayed by their ability to be serially passage d in Nicotiana benthamiana protoplasts using crude sap as inoculum, and com plete and partial virus particles were analyzed by serologically specific e lectron microscopy. Deletion mutagenesis revealed that p33, p6, p18, p13, p 20, and p23 genes were not needed for virion formation. However, deletion o f either minor- or major-coat protein resulted in formation of short partic les which failed to be serially transferred in protoplasts, suggesting that both coat proteins are required for efficient virion assembly. Deletion or mutation of HSP70h and/or p61 dramatically reduced passage and formation o f full-length virions. Frameshift mutations suggested that the HSP70h and p 61 proteins, not the RNA sequences, were needed for virion assembly. Substi tution of the key amino acid residues in the ATPase domain of HSP70h, Asp(7 ) to Lys or Glu(180) to Arg, reduced assembly, suggesting that the chaperon e-like ATPase activity is involved in assembly. Both HSP70h and p61 protein s appeared to contribute equally to assembly, consistent with coordinate fu nctions of these proteins in closterovirus virion formation. The requiremen t of two accessory proteins in addition to both coat proteins for efficient assembly is uniquely complex for helical virions. (C) 2000 Academic Press.