The polerovirus minor capsid protein determines vector specificity and intestinal tropism in the aphid

Abstract

Aphid transmission of poleroviruses is highly specific, but the viral determinants governing this specificity are unknown. We used a gene exchange strategy between two poleroviruses with different vectors, Beet western yellows virus (BWYV) and Cucurbit aphid-borne yellows virus (CABYV), to analyze the role of the major and minor capsid proteins in vector specificity. Virus recombinants obtained by exchanging the sequence of the readthrough domain (RTD) between the two viruses replicated in plant protoplasts and in whole plants. The hybrid readthrough protein of chimeric viruses was incorporated into virions. Aphid transmission experiments using infected plants or purified virions revealed that vector specificity is driven by the nature of the RTD. BWYV and CABYV have specific intestinal sites in the vectors for endocytosis: the midgut for BWYV and both midgut and hindgut for CABYV. Localization of hybrid virions in aphids by transmission electron microscopy revealed that gut tropism is also determined by the viral origin of the RTD.

FIG. 1.

Schematic representation of ORFs 3 and 5 of mutated and recombinant viruses. The genetic organization of polerovirus genome encoding the five ORFs is presented. The encoded major (CP) and minor (RT) coat proteins are indicated by arrows. Positions of the unique NheI (▾) and MluI (▿) restriction sites introduced up- and downstream of the RTD gene (ORF5) to obtain the recombinant viruses are indicated. Intermediate mutated viruses obtained during the cloning procedure (BWn and CAn) carry only the nucleotide changes creating the NheI site downstream of the ORF3 sequence. Nucleotide changes introduced to create the two restriction sites are shown in the boxes in italic letters with reference to the wild-type sequences of CABYV and BWYV. The amino acid replacement (VD to LA) is indicated.

FIG. 2.

Accumulation in protoplasts of RNA of mutated and recombinant viruses. Shown is Northern blot analysis of total RNA extracted from protoplasts inoculated with transcripts corresponding to the different mutated and chimeric viruses. RNA was extracted from mock-inoculated protoplasts (Healthy). Positions of genomic (g) and subgenomic (sg) RNAs are indicated. ³²P-labeled probes specific for each virus (BWYV or CABYV probe) were used. rRNAs were stained with ethidium bromide.

FIG. 3.

Accumulation of recombinant viruses in agroinfected M. perfoliata. (A) Northern blot analysis of total RNA extracted 5 weeks p.i. RNA was extracted from noninoculated plants (Healthy). Positions of genomic (g) and subgenomic (sg) RNAs are indicated. Virus-specific digoxigenin-labeled probes were used. rRNAs were stained with ethidium bromide. (B) Immunodetection of RT proteins. Protein extracts were prepared from ELISA-positive plants 5 weeks p.i. and from noninoculated (Healthy) plants. The volume of protein extract from BW(RTD_CA)- and CA(RTD_BW)-infected plants loaded onto the gel was doubled compared to extracts from CAwt- and BWwt-inoculated plants. Membranes were probed with the indicated specific antiserum. The square to the left indicates a background band due to cross-reaction of anti-RT-CABYV antibodies with an unidentified host protein. Positions of 91- and 113-kDa marker proteins are indicated.

FIG. 4.

Distribution of second-site mutations detected in viral RNA progeny of CAnm and BWnm. Analysis of viral progeny (A) following agroinfection of M. perfoliata with BWnm, CAnm, or CAwt or (B) after aphid transmission to M. perfoliata is shown. Positions of mutations introduced to create NheI and MluI sites are indicated. For each virus, the horizontal line represents the sequenced RTD domain. At the right, the number of clones obtained from different plants that were analyzed and the number of mutations found per 1,000 nt sequenced are indicated. Open circles denote silent mutations, and filled circles represent mutations which provoke an amino acid change. Small triangles refer to insertions or deletions of nucleotides.

FIG. 5.

Immunodetection of capsid proteins in virus purified from M. perfoliata. The upper and lower parts of the left- and right-hand panels are each from one single membrane split into two before incubation with the indicated antibodies. The upper part of the membrane was probed with an anti-CABYV antiserum (upper left panel) or with antibodies raised against the BWYV RTD (anti-RT-BWYV, upper right panel). The position of C-terminally-truncated RT protein (RT*) is indicated. The lower part of the membrane was incubated with either the anti-CABYV antiserum (lower right panel) or antiserum specific for BWYV CP (anti-CP-BWYV, lower left panel). The positions of CABYV and BWYV CP are indicated with apparent molecular masses in brackets.

FIG. 6.

Observation of recombinant virions in the gut of M. persicae. (a to c) BW(RTD_CA) virions in posterior midgut and hindgut cells. (d to f) CA(RTD_BW) virions in posterior midgut cells and in hindgut lumen. lu, lumen; mv, microvilli; mi, mitochondrium; tv, tubular vesicle; cv, coated vesicle; end, endodome-like vesicle; apl, apical plasmalemma; bpl, basal plasmalemma; bl, basal lamina; he, hemolymph; v, virions; r, ribosomes. Bar, 100 nm.