Cassava brown streak virus Ham1 protein hydrolyses mutagenic nucleotides and is a necrosis determinant

Abstract

Summary Cassava brown streak disease (CBSD) is a leading cause of cassava losses in East and Central Africa, and is currently having a severe impact on food security. The disease is caused by two viruses within the Potyviridae family: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), which both encode atypical Ham1 proteins with highly conserved inosine triphosphate (ITP) pyrophosphohydrolase (ITPase) domains. ITPase proteins are widely encoded by plant, animal, and archaea. They selectively hydrolyse mutagenic nucleotide triphosphates to prevent their incorporation into nucleic acid and thereby function to reduce mutation rates. It has previously been hypothesized that U/CBSVs encode Ham1 proteins with ITPase activity to reduce viral mutation rates during infection. In this study, we investigate the potential roles of U/CBSV Ham1 proteins. We show that both CBSV and UCBSV Ham1 proteins have ITPase activities through in vitro enzyme assays. Deep‐sequencing experiments found no evidence of the U/CBSV Ham1 proteins providing mutagenic protection during infections of Nicotiana hosts. Manipulations of the CBSV_Tanza infectious clone were performed, including a Ham1 deletion, ITPase point mutations, and UCBSV Ham1 chimera. Unlike severely necrotic wild‐type CBSV_Tanza infections, infections of Nicotiana benthamiana with the manipulated CBSV infectious clones do not develop necrosis, indicating that that the CBSV Ham1 is a necrosis determinant. We propose that the presence of U/CBSV Ham1 proteins with highly conserved ITPase motifs indicates that they serve highly selectable functions during infections of cassava and may represent a euphorbia host adaptation that could be targeted in antiviral strategies.

Viral inosine triphosphatase: A mysterious enzyme with typical activity, but an atypical function

“…As RNA viruses encode very few genes and will not maintain unnecessary genetic information within their genomes, is highly likely that these viral‐encoded ITPases carry out an important function. Evidence from analysis of mutant viruses shows changes to the disease phenotype, suggesting that ITPases modulate the plant response to the virus (Tomlinson et al, 2019). Understanding their function may assist in the development of virus‐resistant cassava, an essential quest to improve food security across sub‐Saharan Africa (Tomlinson et al, 2018).…”

“…Possibly, incorporation of the noncanonical nucleotide into the RNA genome has a greater effect on RNA structure and thus the rate of translation (Sakumi et al, 2010; Thomas et al, 1998). The study by Tomlinson et al (2019) did find a delayed increase in transcript abundance for mutant viruses lacking ITPase compared to wild‐type viruses, which could potentially be a result of reduced translation efficiency of viral proteins involved in viral RNA replication.…”

“…Removal of Ham1 from the CBSV genome or mutation of a conserved substrate‐binding motif resulted in striking changes in symptom development in the indicator plant N. benthamiana , including a lack of necrosis normally seen in wild‐type infection and leaf curling and chlorotic mottling normally absent from wild‐type infected plants (Tomlinson et al, 2019). Similarly, replacing the CBSV ITPase with the UCBSV ITPase‐encoding sequence also resulted in the same change in phenotype.…”

“…Virus‐induced necrosis is associated with pathways involved in the hypersensitive response (Komatsu et al, 2010; Mandadi & Scholthof, 2013), suggesting viral ITPase activity may be triggering these signalling pathways. Intriguingly, plants infected with wild‐type UCBSV virus showed a similar phenotype to plants infected with CBSV Ham1 mutant viruses, suggesting the two viruses encode ITPases with different roles in infection; however, no mutant UCBSV virus was tested so any attenuation of symptoms is unknown (Tomlinson et al, 2019). The UCBSV and CBSV Ham1 sequences show low homology, which is probably responsible for the observed differences in symptomatologies (Tomlinson et al, 2018).…”

Application of Reverse Genetics in Functional Genomics of Potyvirus

“…Reverse genetics of RNA viruses refers to the generation of recombinant viruses through site-directed mutagenesis such as substitution, deletion or insertion [ 1 ]. This consequently made various phenotypic studies possible, apart from providing a powerful tool to enhance our knowledge on life cycles and pathogenic mechanisms of RNA viruses as well as the structure or function of individual viral genes [ 2 , 3 ]. Besides that, reverse genetics approaches have largely contributed to the development of antiviral therapeutics, vaccines [ 4 , 5 ], and vectors [ 6 ].…”