Catherine Reinbold

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.

Studies on the role of the minor capsid protein in transport of Beet western yellows virus through Myzus persicae

Beet western yellows virus (BWYV), family Luteoviridae, is an icosahedral plant virus which is strictly transmitted by aphids in a persistent and circulative manner. Virions cross two cellular barriers in the aphid by receptor-based mechanisms involving endocytosis and exocytosis. Particles are first transported across intestinal cells into the haemolymph and then across accessory salivary gland cells for delivery to the plant via saliva. We identified the midgut part of the digestive tract as the site of intestinal passage by BWYV virions. To analyse the role in transmission of the minor capsid component, the readthrough (RT) protein, the fate of a BWYV RT-deficient non-transmissible mutant was followed by transmission electron microscopy in the vector Myzus persicae. This mutant was observed in the gut lumen but was never found inside midgut cells. However, virion aggregates were detected in the basal lamina of midgut cells when BWYV antiserum was microinjected into the haemolymph. The presence of virions in the haemolymph was confirmed by a sensitive molecular technique for detecting viral RNA. Thus, transport of the mutant virions through intestinal cells occurred but at a low frequency. Even when microinjected into the haemolymph, the RT protein mutant was never detected near or in the accessory salivary gland cells. We conclude that the RT protein is not strictly required for the transport of virus particles through midgut cells, but is necessary for the maintenance of virions in the haemolymph and their passage through accessory salivary gland cells.

Discovery of a Small Non-AUG-Initiated ORF in Poleroviruses and Luteoviruses That Is Required for Long-Distance Movement.

Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5’ end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement.

Transcriptomic analysis of intestinal genes following acquisition of pea enation mosaic virus by the pea aphid Acyrthosiphon pisum

Viruses in the family Luteoviridae are strictly transmitted by aphids in a non-propagative, circulative and persistent mode. Virions ingested by aphids successively cross the gut and the accessory salivary gland epithelia before being released, together with saliva, into the plant vasculature. Virion transport through aphid cells occurs by a transcytosis mechanism. This study conducted a transcriptomic analysis of intestinal genes of the pea aphid Acyrthosiphon pisum following uptake of pea enation mosaic virus. Among the 7166 transcripts analysed, 128 were significantly regulated (105 genes downregulated and 23 upregulated). Of these genes, 5 % were involved in intracellular trafficking, endocytosis and signal transduction, three important steps in the internalization and transport of virions. The limited levels of downregulation (maximum of 3.45-fold) and upregulation (maximum of 1.37-fold) suggest that the virus hijacks a constitutive endocytosis-exocytosis mechanism without heavily perturbing cell metabolism. Although limited to about 20 % of the pea aphid genes, this work represents the first large-scale analysis of aphid gene regulation following virus acquisition. A better knowledge of this virus-vector interaction will be possible only when tools representing the complete genomic capacity of the aphid become available.

Characterization of a new densovirus infecting the green peach aphid Myzus persicae

A new icosahedral DNA virus was isolated from aphids (Myzus persicae) that showed abnormal growth and development. The purified virus particles have a diameter of 20 nm and contain a single-stranded DNA molecule of approximately 5.7 kb. The viral particles are composed of five structural proteins (92, 85, 68, 64, and 57 kDa). As the main biophysical properties of this virus are similar to those of the members of the genus Densovirus it was tentatively named Myzus persicae densovirus (MpDNV). A PCR-based detection method and a polyclonal antiserum raised against MpDNV allowed the detection of the virus in a single-infected aphid. MpDNV is immunologically related to Junonia coenia densovirus, but not to other members of the subfamily Densovirinae. Biological assays showed that MpDNV could be both transmitted transovarially and horizontally via honeydew and saliva. MpDNV was able to infect whiteflies but not other aphid species tested.