Effects of Point Mutations in the Major Capsid Protein of Beet Western Yellows Virus on Capsid Formation, Virus Accumulation, and Aphid Transmission

Abstract

Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected ϳ90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/ N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a "reverse" substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid.Beet western yellows virus (BWYV; genus Polerovirus), like all members of the family Luteoviridae, is transmitted in a circulative, nonpropagative manner by aphids (see reference 7 for a review). Poleroviruses and luteoviruses (the other major genus of the family) are transmitted vector specifically, implying that determinants on the viral capsid selectively interact with specific structures within vector aphids. The particles of poleroand luteoviruses are composed of two types of protein, the major capsid protein (P3) of 22 to 23 kDa and a minor species of ϳ75 kDa known as readthrough (RT) protein (see reference 13 for a review). The RT protein is a C-terminally extended form of P3 produced by episodic suppression of P3 translation termination so that translation continues into the downstream open reading frame (ORF) 5, which encodes the RT domain (12). The RT protein is believed to be incorporated into the capsid via its P3 moiety, with the RT domain protruding from the virion surface (1, 4).Nicotiana clevelandii can be agroinfected with full-length BWYV cDNA (10), and the resulting plants can serve as a virus source in aphid transmission experiments (1). Site-directed mutagenesis of the viral cDNA prior to agroinfection has been used to map sequences in the RT domain important for virus movement in planta and for aphid transmission of BWYV (1-3). In this paper, w...

A Surface Loop of the Potato Leafroll Virus Coat Protein Is Involved in Virion Assembly, Systemic Movement, and Aphid Transmission

“…Based on sequence alignments with other icoahedral viruses whose structures were known, two of the surface domains were located on loops known to be important antigenic sites on structurally related viruses. More recently, independent structure models were developed for the CP of two members of the genus Polerovirus in the Luteoviridae, PLRV and Beet western yellows virus (BWYV) (2,31). The models support the data from the epitope-mapping studies and locate the surface epitopes on the loops between ␤-strands G and H and between ␤-strands B and C. Although the best-fit structure models for PLRV and BWYV both locate epitope 10 (32) on the G-H loop, the structural features of the loop differed between these closely related viruses (2).…”

Transmissão por afídeos e afinidade a Buchnera sp. GroEL de um mutante deletério da proteína de RTD do Potato leafroll virus

“…Negative controls (sample buffer) showed absorbance values of less than 0.015. Brault et al, 1995;Brault et al, 2000). However, the molecular aspects of luteovirids-vector interactions and the involvement of RTD in the transmission process are essentially based on work with BYDV and BWYV.…”

“…Previous studies on the role of the viral capsid-associated proteins of luteoviruses have shown that the BWYV RTD protein is indispensable for transmission by Myzus persicae (Sulz. ), and harbour determinants which mediate circulation of the virus in the aphid (Brault et al, 1995). The recognition and movement of BWYV into the aphid vector hemocoel is greatly reduced in RTD deficient mutants, suggesting an important role for this protein in virus recognition and the trans-cellular transport process (Reinbold et al, 2001).…”

“…Viral structural proteins of the virions purified from protoplasts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), in 10% gels, as previously described (Brault et al, 1995) and detected by Western blot analysis using a specific antiserum raised against PLRV.…”

“…The recognition and movement of BWYV into the aphid vector hemocoel is greatly reduced in RTD deficient mutants, suggesting an important role for this protein in virus recognition and the trans-cellular transport process (Reinbold et al, 2001). It is known that BWYV and BYDV virions lacking the RTD protein are stable in the aphid's intestinal lumen and able to cross the gut epithelium (Brault et al, 1995). Nevertheless, these particles are not able to cross the ASG basal lamina, and therefore are not transmissible by the aphid .…”