Closterovirus Encoded HSP70 Homolog and p61 in Addition to Both Coat Proteins Function in Efficient Virion Assembly

Abstract

Assembly of the viral genome into virions is a critical process of the virus life cycle often defining the ability of the virus to move within the plant and to be transmitted horizontally to other plants. Closteroviridae virions 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 only virus family that encodes a protein with similarity to cellular chaperones, a 70-kDa heat-shock protein homolog (HSP70h). We examined the involvement of gene products of Citrus tristeza virus (CTV) in virion formation and found that the chaperone-like protein plus the p61 and both coat proteins were required for efficient virion assembly. Competency of virion assembly of different CTV mutants was assayed by their ability to be serially passaged in Nicotiana benthamiana protoplasts using crude sap as inoculum, and complete and partial virus particles were analyzed by serologically specific electron microscopy. Deletion mutagenesis revealed that p33, p6, p18, p13, p20, and p23 genes were not needed for virion formation. However, deletion of either minor- or major-coat protein resulted in formation of short particles 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 of full-length virions. Frameshift mutations suggested that the HSP70h and p61 proteins, not the RNA sequences, were needed for virion assembly. Substitution 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 chaperone-like ATPase activity is involved in assembly. Both HSP70h and p61 proteins appeared to contribute equally to assembly, consistent with coordinate functions of these proteins in closterovirus virion formation. The requirement of two accessory proteins in addition to both coat proteins for efficient assembly is uniquely complex for helical virions.

Evidence of Recombinant Citrus tristeza virus Isolate Occurring in Acid Lime cv. Pant Lemon Orchard in Uttarakhand Terai Region of Northern Himalaya in India

“…The ORFs 1a and 1b encode replication related proteins, whereas, the 3 0 end ten ORFs (ORFs 2-ORF11) encodes proteins p33, p6, p65, p61, p27, p25, p18, p13, p20 and p23 [11,22]. Analysis of 19 complete genomes CTV isolates available in GenBank data base revealed extensive variation in sequence among the CTV isolates and enabled them to be grouped into six genotypes, recognized as T36 (Florida), T30 (Florida), VT (Israel), B165 (India), HA16-5 (Hawaii) and RB genotypes (New Zealand) [6,10,16,19].…”

The Complex Genetics of Citrus tristeza virus

“…Additionally, as indicated above, the 10 ORFs located at the 3' half of the CTV genome are expressed by the synthesis of ten 3' co-terminal sgRNAs (Figure 4). Each 3' sgRNA serve as RNA messenger for the translation of its 5' proximal ORF [13,46] and the Current Issues in Molecular Virology -Viral Genetics and Biotechnological Applicationsexpression of each of the ten 3' proximal ORFs is regulated independently both in amount and timing [46,47].…”

“…About the 97% of the CTV genome is coated by CP, while the remainder 3% is coated by CPm resulting in viral particles with the emblematic tail of the members of the Closteroviridae family [11,24]. The coordinate action of HSP70h and p61, in addition to the CP and CPm coat proteins, are required for proper assembly of CTV particles [13]. The previously described CTV 5´NTR conserved SL structures contains the origin of assembly of CPm, overlapping the sequences that function as a cis-acting element required for gRNA synthesis [24].…”

“…In fact, CTV evolved ending up with three proteins, CP, p20 and p23, which are suppressors of the plant RNA silencing mechanism in N. benthamiana and N. tabacum plants [18]. Unexpectedly, the ORFs that encode proteins p33, p18 and p13 are not required either for replication or assembly [6,13] or for systemic infection of Mexican lime [C. aurantifolia (Christm.) Swing.]…”

Complex molecular architecture of beet yellows virus particles

“…In contrast, p20 is dispensable for virion assembly and cell-to-cell movement, but is necessary for BYV transport through the plant vascular system (18). A CPm, Hsp70h, and p64 ortholog were also found in the virions of lettuce infectious yellows virus (25) and were implicated in assembly of citrus tristeza virus (26). Both these viruses belong to the family Closteroviridae and are distantly related to BYV.…”