Maf/ham1-like pyrophosphatases of non-canonical nucleotides are host-specific partners of viral RNA-dependent RNA polymerases

Abstract

Cassava brown streak disease (CBSD), dubbed the Ebola of plants, is a serious threat for food security in Africa caused by two viruses of the family Potyviridae: cassava brown streak virus (CBSV) and Ugandan (U)CBSV. Intriguingly, U/CBSV, along with another member of this family and one secoviridae, are the only known RNA viruses in nature encoding a protein of the Maf/ham1-like family, a group of widespread pyrophosphatase of non-canonical nucleotides (ITPase) expressed by all living organisms. Despite the socio-economic impact of CDSD, the relevance and role of this atypical viral factor has not been yet established. Here, using an infectious cDNA clone and reverse genetics, we demonstrate that UCBSV requires the ITPase activity in cassava, but not in the model plant Nicotiana benthamiana. HPLC-MS/MS experiments show that, quite likely, this host-specific constraint is due to an unexpected high concentration of non-canonical nucleotides in cassava. Finally, protein analyses and experimental evolution of mutant viruses indicate that keeping a fraction of the yielded UCBSV ITPase covalently bound to the viral RNA-dependent RNA polymerase (RdRP) optimizes viral fitness, and this seems to be a feature shared by the other members of the Potyviridae family expressing Maf/ham1-like proteins. All in all, our work (i) reveals that the over-accumulation of non-canonical nucleotides in the host might have a key role in antiviral defense, and (ii) provides the first example of an RdRP-ITPase partnership, reinforcing the idea that RNA viruses are incredibly inventive at adaptation to different host setups.

Early Detection of Plant Virus Infection Using Multispectral Imaging and Spatial-Spectral Machine Learning

“…During the timeframe of the experiment, no symptoms were seen on plants in the other treatment groups and the RT-PCR results were negative for the control and mock groups. These results indicate that inoculation was highly efficient, consistent with the original report of this infectious clone 28 , but do not guarantee that 100% of plants in the virus-inoculated group were infected. Some of the classification results presented below were therefore done using only the 10 plants with PCR-confirmed infection ("Trial 2 with PCR").…”

“…In three independent trials, we used the cassava cultivar TME204, which is susceptible to CBSD, to generate three treatment groups -control, infected and mock-inoculated (18 plants in each group, except for Trial 1 -see Table 1). The infected group was inoculated with plasmid DNA corresponding to an infectious clone for the UCBSV Kenyan isolate 125 [26][27][28] . The mock group was inoculated with an 'empty' control E. coli plasmid using the same protocol and the untreated group was not subjected to inoculation.…”

“…For each experiment, 18 plants at the 6-leaf stage were inoculated under the apical meristem area using a microsprayer and 40 psi helium to deliver gold particles coated with plasmid DNA 37 (Venganza, Inc.). Each plant was inoculated with 1.67 µg of plasmid DNA corresponding to pLX-UCBSVi (infected) 27,28 or pUC119 (mock). pLX-UCBSVi contained an E35S expression cassette driving transcription of UCBSV Ke_125 (GenBank accession KY825166 26 ).…”