Both structural and non-structural forms of the readthrough protein of cucurbit aphid-borne yellows virus are essential for efficient systemic infection of plants

Abstract

Cucurbit aphid-borne yellows virus (CABYV) is a polerovirus (Luteoviridae family) with a capsid composed of the major coat protein and a minor component referred to as the readthrough protein (RT). Two forms of the RT were reported: a full-length protein of 74 kDa detected in infected plants and a truncated form of 55 kDa (RT*) incorporated into virions. Both forms were detected in CABYV-infected plants. To clarify the specific roles of each protein in the viral cycle, we generated by deletion a polerovirus mutant able to synthesize only the RT* which is incorporated into the particle. This mutant was unable to move systemically from inoculated leaves inferring that the C-terminal half of the RT is required for efficient long-distance transport of CABYV. Among a collection of CABYV mutants bearing point mutations in the central domain of the RT, we obtained a mutant impaired in the correct processing of the RT which does not produce the RT*. This mutant accumulated very poorly in upper non-inoculated leaves, suggesting that the RT* has a functional role in long-distance movement of CABYV. Taken together, these results infer that both RT proteins are required for an efficient CABYV movement.

Figure 1. Schematic representation of CABYV mutants.

A) Genetic organization of CABYV RNA with the position of the encoded structural proteins (CP and RT*) and the full-length RT protein (RT) (arrows). The restriction sites AgeI, EagI and SalI used to obtain the PCS mutants are shown. The wild-type amino acid sequence (amino acid 430 to 470) of the region on the CABYV RT protein targeted by alanine scanning is shown below with the corresponding changes in the ten PCS mutants. The underlined R residue represents the last C-terminal amino acid identified by mass spectrometry on the CABYV-RT* protein . The C-terminal amino acid on RT* is tentatively positioned at amino acid 461 (grey-shaded serine). B) Genetic map of CABYV-RTΔC_ter deletion mutant. NheI and MluI restriction sites originally introduced in CABYV-NM3 to obtain CABYV-RTΔC_ter mutant are positioned.

Figure 2. RT protein synthesis by CABYV mutants and RT* incorporation into purified virions.

A) Western blot analysis on proteins extracted from non-infiltrated leaves of M. perfoliata inoculated with wild-type CABYV (WT), PCS1, PCS3+, NM3 and RTΔC_ter mutants. The analysis was performed on several plants for each mutant but one sample for NM3 and RTΔC_ter or two samples for PCS1 and PCS3+ are shown. Two blots corresponding to two independent experiments have been juxtaposed. The right panel is a fusion a lanes originating from the same blot. The blots were incubated with a mixture of two antisera, one directed against CABYV virions and one against the C-terminal part of CABYV-RT protein. N.I.: non-inoculated plant; the white triangle (ca. 60 kDa) indicates a cross reaction of the antibodies with plant proteins. B) Western blot analysis of proteins extracted from infiltrated leaves of M. perfoliata inoculated with PCS1, PCS3+, RTΔC_ter, NM3 or WT. Immunodetection was done with antibodies directed against the CP. The right panel corresponds to a prolonged exposure of a blot from an independent experiment. Black circle indicates an additional viral product of about 35 kDa present in PCS1 infected plants. Notice that because of the different antisera used, the cross reactions (white triangles in Fig. 2A and Fig. 2B) are different. C) Western blot analysis of capsid proteins in CABYV mutant particles (1 μg) prepared from agroinfected M. perfoliata. The whole blot was incubated with antibodies directed against CABYV-virions but the upper panel was overexposed. Black star in Fig. 2C: viral protein of 30 kDa present in PCS3+ purified particles; Positions of the molecular markers (in kDa) are indicated on the left. In brackets, apparent molecular weight of the different forms of the RT proteins.

Figure 3. Detection of CABYV proteins and virions in infected C. sativus and M. perfoliata.

A) Immunodetection by western blot of CABYV proteins in extracts prepared from infected C. sativus or M. perfoliata or from purified virus; B) Immunodetection by western blot of CABYV proteins in phloem exudate collected from infected C. sativus. Two blots corresponding to two independent collections of phloem exudate are presented. For (A) and (B), a CABYV polyclonal antiserum was used to detect the major coat protein (CP) and the RT* protein (lower panels) whereas a CABYV-RT-C_ter specific antiserum detected only the complete RT protein (upper panels). Positions of the molecular markers (in kDa) are indicated on the left. The white triangles (ca. 60 kDa and 35 kDa) indicate major cross reactions of the antibodies with plant proteins. Inf.: infected plant; N.I.: non-infected plant; Pur. Virus: Purified virus. C) Observation by transmission electron microscopy of virus-like particles from phloem exudate collected from infected cucumber plants. The grids were coated with a CABYV-polyclonal antiserum before addition of phloem exudate. The bar corresponds to 50 nm.

Figure 4. Viral particles of CABYV-WT, PCS1, PCS3+, CABYV-NM3 and CABYV-RTΔC_ter observed by transmission electron microscopy (TEM).

A CABYV polyclonal antiserum was used to capture virus particles on the grids before TEM. Bars correspond to 50

Figure 5. Hypothetical model for the mode of action of the RT proteins in CABYV movement.

Because of the intrinsic disorder of the C-terminal part of the RT protein, the pool of RT proteins (in black) may adopt different conformations in infected cells. A fraction of them could be processed to give RT* proteins (in grey) and further incorporated into virions. In the case of CABYV, these virions decorated with the RT* protein cannot move systemically without the assistance of the complete RT protein (1). The free RT protein could bind to the RT* proteins on the surface of virions, or could interact with plant components involved in virus transport (on the figure only plasmodesmata are presented) to promote efficient virus transport into sieve elements (2). Virions devoid of RT* proteins could follow another transport pathway independent of the RT protein leading to inefficient systemic transport (3). CC: companion cells; PPC: phloem parenchyma cells.