Virion tails of Beet yellows virus: Coordinated assembly by three structural proteins

Abstract

Filamentous virions of Beet yellows virus contain a long body formed by a major capsid protein and a short tail that is assembled by a minor capsid protein (CPm), an Hsp70-homolog (Hsp70h), a 64-kDa protein (p64), and a 20-kDa protein (p20). Using mutation analysis and newly developed in planta assays, here we investigate the genetic requirements for the tail assembly. We show that the inactivation of CPm dramatically reduces incorporation of both Hsp70h and p64. Furthermore, inactivation of Hsp70h prevents incorporation of p64 into virions and vice versa. Hsp70h and p64 are each required for efficient incorporation of CPm. We also show that the tails possessing normal relative amounts of CPm, Hsp70h, and p64 can be formed in the absence of the major capsid protein and p20. Similar to the tails isolated from the wild-type virions, these mutant tails encapsidate the approximately 700 nt-long, 5'-terminal segments of the viral RNA. Taken together, our results imply that CPm, Hsp70h and p64 act cooperatively to encapsidate a defined region of the closterovirus genome.

Fig. 1

(A) Maps of BYV genome (top) and its truncated derivative BYVΔ (bottom). Functions of BYV genes are shown above and below the diagram. L-Pro, leader proteinase; Met, Hel, and Pol, methyltransferase, RNA helicase, and RNA polymerase domains, respectively; p6, 6-kDa movement protein; Hsp70h, Hsp70 homolog; p64, 64-kDa protein, CPm, minor capsid protein; CP, major capsid protein; p20, 20-kDa protein; p21, 21-kDa protein, GFP, green fluorescent protein. (B) Comparative analysis of the virion composition using antisera specific to CP, CPm, Hsp70h, and p64; position of the corresponding protein bands is indicated by arrows. UD, undiluted protein samples, ½ and ¼, diluted samples. The mutant names are shown at the top. (C) Quantitative analysis of the CPm incorporation to mutant virions relative to that of the wild type (100%).

Fig. 2

Protein composition analysis of the virions formed by the BYV variants in which Hsp70h (A), p64 (B), or both (C) were either eliminated (NoHsp70h and Nop64) or inactivated (Hsp70hΔXho and p64R₃₈₆A) by mutations. Designations are the same as in Fig. 1B.

Fig. 3

Characterization of the viral particles formed by the mutant variant BYVΔ (A) Immunoblot detection of the CPm and Hsp70h using corresponding antisera. (B) Immunoblot detection of the CPm and p64. (C) Hybridization analysis of the RNA isolated from virions. V, wild type virions; Ts, virion tails isolated via sonication of the wild type virions as described earlier (Peremyslov et al., 2004); T1 and T2, two independent RNA isolates from the BYVΔ mutant. Arrows mark the genome-sized RNA (G) and selected RNA markers with their corresponding lengths in nucleotides.

Fig. 4

Mapping of the 3’-termini of the RNA segments encapsidated and protected in the particles formed by the BYVΔ mutant. The nucleotide sequence of the BYV cDNA starting from nucleotide 601 is shown. The 3’-terminal nucleotides detected in the cloned BYVΔ cDNAs are underlined with the number of clones whose 3’-termini are located within each area shown underneath.