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.

Cassava brown streak disease (CBSD) has major impacts on yield and quality of the tuberous roots of cassava in Eastern and Central Arica. At least two Potyviridae species cause the disease: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Cloned viral genome sequences known as infectious clones (ICs) have been important in the study of other viruses, both as a means of standardising infectious material and characterising viral gene function. IC construction is often technically challenging for Potyviridae due to sequence instability in E. coli . Here, we evaluate three methods for the construction of infectious clones for CBSD. Whilst a simple IC for in vitro transcription was made for UCBSV isolate ‘Kikombe’, such an approach failed to deliver full-length clones for CBSV isolates ‘Nampula’ or ‘Tanza’, necessitating more complex approaches for their construction. The ICs successfully generated symptomatic infection in the model host N. benthamiana and in the natural host cassava. This shows that whilst generating ICs for CBSV is still a technical challenge, a structured approach, evaluating both in vitro and in planta transcription systems should successfully deliver ICs, allowing further study into the symptomology and virulence factors in this important disease complex. Electronic supplementary material The online version of this article (10.1007/s12033-018-0139-7) contains supplementary material, which is available to authorized users.

Cassava brown streak disease (CBSD) is currently one of the major constraints on cassava production in Africa. CBSD is estimated to cause annual economic losses of over $100 million USD. CBSD is caused by at least two viral species: Cassava brown streak virus (CBSV) and the Uganda cassava brown streak virus (UCBSV). In field, CBSV and UCBSV occur in single and mixed infections with the potential to be found in mixed infections with other viruses. The interactions between CBSV and other viruses are poorly understood and many functions of CBSV genes are not fully characterised. In this study we analysed the interaction of CBSV with non-related viruses, for potential synergistic interactions, namely tobacco mosaic virus (TMV), and potato virus Y (PVY), both very well characterised for their infection and symptomatology in Nicotiana species. These interactions demonstrated to be synergistic with TMV and antagonistic with PVY. Then P1, P3, 6k1, CI, 6k2, VPg, NIa, NIb, Ham1-like and CP from CBSV were analysed separately, to determine which genes from CBSV were responsible for the direction of these interactions. For this analysis, transgenic lines expressing single CBSV genes were used, providing information about the importance of Ham1. Further functional analysis of these CBSV genes was carried out, analysing silencing suppression activity through agroinfiltration assays. This confirmed silencing suppression activity for the CBSV P1 protein and demonstrated that a functional LRRA domain is required for this activity.