A Cucumber Necrosis Virus Variant Deficient in Fungal Transmissibility Contains an Altered Coat Protein Shell Domain

Abstract

Little is currently known regarding the specific interactions that govern transmission of plant viruses by their vectors. A cucumber necrosis virus (CNV) variant (LL5) deficient in fungal transmissibility has been isolated from mechanically passaged CNV and characterized. Although LL5 accumulates to wild-type (WT) levels, is capable of rapid systemic infection, and produces stable, highly infectious particles, it is only inefficiently transmitted by Olpidium bornovanus zoospores. The LL5 coat protein (CP) gene was amplified by RT-PCR and cloned in place of the WT CNV CP gene in an infectious CNV cDNA clone. Particles derived from this construct also failed to be efficiently transmitted. The LL5 CP gene was sequenced and found to contain two amino acid substitutions relative to WT CNV CP. One substitution (Phe to Cys) occurred in the arm region and another (Glu to Lys) in the shell domain. These amino acid changes were separately introduced into the WT CNV genome through in vitro mutagenesis and it was found that the Glu to Lys change in the LL5 CP shell domain is largely responsible for the loss of transmissibility. In vitro binding assays were developed to determine if the defect in transmissibility was due to a defect in binding zoospores. LL5 particles were found to bind less efficiently than WT CNV. Furthermore, the nontransmissible tomato bushy stunt virus did not detectably bind zoospores. These binding studies suggest that the specificity of CNV transmission by O. bornovanus occurs through specific recognition of a putative zoospore receptor.

A Conserved Capsid Protein Surface Domain of Cucumber Mosaic Virus Is Essential for Efficient Aphid Vector Transmission

“…In cases where plant virus transmission has been studied in detail, the capsid protein has been shown to be the primary determinant for transmission by vectors (13,14,37,40). The interactions between virus capsid proteins and their vectors are either direct or mediated by an accessory or helper protein (22,38).…”

“…Genetic studies have revealed that specific viral capsid protein domains and amino acids are determinative for transmissibility and vector specificity. Examples can be found for aphid-transmitted viruses in the genera Potyvirus (1,2,11), Luteovirus (53), Polerovirus (3), Enamovirus (7), and Cucumovirus (35,36,44); for the nematode-transmitted viruses in the genus Tobravirus (25); and for fungus-transmitted viruses in the genera Tombusvirus (18,40) and Benyvirus (52). An emerging theme for a number of these viruses is that a minor capsid protein readthrough protein plays an important role in transmission; this protein is generated by a mechanism involving readthrough of the major capsid protein stop codon (reviewed in reference 4).…”

Identification of Specific Cucumber Necrosis Virus Coat Protein Amino Acids Affecting Fungus Transmission and Zoospore Attachment

“…However, for the most part, the exact role of these amino acids in transmission including their potential role in vector attachment, is not known. Recent work with cucumber necrosis virus (CNV) has suggested that attachment of virions to vector zoospores is an important aspect of the transmission process (24).…”

“…Transmission of CNV in nature occurs via zoospores of the Chytridiomycete fungus, Olpidium bornovanus (6,9,24). Zoospores and virus particles are released independently into the soil from the roots of infected plants.…”

“…Previous work has shown that the CNV CP contains determinants that specify its interaction with zoospores of O. bornovanus (20,24). Reciprocal exchanges between the CP gene of CNV and that of the nontransmissible cherry strain of Tomato bushy stunt virus (TBSV) in infectious full-length cDNA clones showed that particles obtained from the TBSV genome containing the CNV CP were transmissible but particles from the CNV genome containing the TBSV CP were not.…”