Figure S1: Relevant sections of a T-coffee alignment of 8 CBSV and 8 UCBSV Ham1 amino acid sequences showing differences in proteolytic cleavage sequences. N’ Ham1 proteolytic cleavage sequences between the NIb – Ham1 peptides are different in CBSV isolates (green) UCBSV isolates (pink), indicating potential differences in proteolytic processing. Whereas the C’ Ham1 proteolytic cleavage sequence between the Ham1 – CP peptides is conserved in CBSV and UCBSV sequences (blue). Highly conserved regions (>90%) are highlighted in black. Sequences were obtained from the NCBI database; accession numbers are provided for each sequence.

1

CBSV_gu563326 1 IDLQVVDRPQLSSMNKREEEVTSKIRMGIEAPITFVTGNAQKLKEVKQIFCBSV_gq329864 1 IDLQVVDRPQSLNVAKREEEVTSKFRMGIEAPITFVTGNAQKLKEVKQIFCBSV_fn434436 1 IDLQVVDRPQLLNVAKREEEVTSKFRMGIEAPITFVTGNAQKLKEVKQIFCBSV_fn434437 1 IDLQVIDKPQPSKVAKREEEVTSRIRMGIEAPITFVTGNAQKLKEVKQIFCBSV_gu563320 1 IDLQVIDRPQSSNMTKREEEVTSKIRMGIEAPITFVTGNAQKLKEVKQIFCBSV_gu563323 1 IDLQVVDRPQLSSMTKREEEVTSKVRMGIEAPITFVTGNAQKLKEVKQIFCBSV_gu563325 1 IDLQVVDRSQSTNVAKREEEVTSKIRMGIEAPITFVTGNAQKLKEVKQIFCBSV_mg570022 1 IDLQVVDRSQPSNVAKREEEVTSKIRMGIEAPITFVTGNAQKLKEVKQIFUCBSV_fj039520 1 VDTQTEDLREKEKPELRIESHDGTSRMQMKFPVTFVTGNLGKLAEVRSILUCBSV_fj185044 1 VDTQTEDLRGREKLELRTESHDRISQLQMKFPVTFVTGNLGKLAEVKSILUCBSV_fn433930 1 VDTQTKDLRGREKLELRTESHDGTLQMQMKFPVTFVTGNFGKLAEVKSILUCBSV_fn433932 1 VDTQIKDLRERDEPELRVGSHDGVPRMQMKFPVTFVTGNLGKLAEVKSILUCBSV_fn434109 1 VDTQK-DLRGGEKPELRTESHDGTPQMQMKFPVTFVTGNFGKLAEVKSILUCBSV_hm181930 1 VDTQTEDLRGREKLELRTESHDRISQLQMKFPVTFVTGNLGKLAEVKSILUCBSV_kx753357 1 VDTQTKDLRGREKPELRIESHDGVPQMQMKFPVTFVTGNLGKLAEVKSILUCBSV_gq169761 1 VDTQTKDLREKEEPELRIESHDGISRMQMKFPVTFVTGNLGKLEEVRSIL

CBSV_gu563326 201 ALSLVRDFLKDSSYFSFAKGVDRDFFIDVQACBSV_gq329864 201 ALSLVRDFLKSSSYFSFAKGLDRDIFIDVQACBSV_fn434436 201 ALSLVRDFLKSSSYFSFAKGLDRDIFIDVQACBSV_fn434437 201 ALSLVRDFLKSSSYFSFAKGLDRDIFIDVQACBSV_gu563320 201 ALSLVRDFLKNSSYFNFAKGVDRDFFIDVQACBSV_gu563323 201 ALSLVRDFLKDSSYFSFAKGVDRDFFIDVQACBSV_gu563325 201 ALSLVRDFLKNSSYFSFAKGVDRDLFIDVQACBSV_mg570022 201 ALSLVRDFLKDSSYFSFAKGVDRDFFIDVQAUCBSV_fj039520 201 ALEKVKLFLDNLVVRQEEKRASMALTIDVQAUCBSV_fj185044 201 ALEKVKLFLDNLVVKQEKKKAGVALTIDVQAUCBSV_fn433930 201 ALEKVKLFLDNLMVKQEKNKASVALTIDVQAUCBSV_fn433932 201 ALEKVKLFLDNLVVKQEEKRAKVALTIEVQAUCBSV_fn434109 200 ALEKVKSFLDNLVVKQEEKKARVALTIDVQAUCBSV_hm181930 201 ALEKVKLFLDNLVVKQEKKKAGVALTIDVQAUCBSV_kx753356 201 ALEKVKLYLDNLMVKQEEKKAKVALTIDVQAUCBSV_gq169761 201 ALEKVKLFLDNLVVRQEEKRASVALTIDVQA

Figure S2: Relevant section of T-coffee alignment of 12 CBSV and 20 UCBSV Ham1 amino acid sequences. The ITPase signature Serine-Histidine-Arginine (SHR) motif is highly conserved and was found in all sequences at positions 192 – 194 (yellow). Highly conserved regions (>90%) are highlighted in black. Sequences were obtained from the NCBI database; accession numbers are provided for each sequence.

2

CBSV_GU563327 181 AEMMAEEKNMISHRFRALSLVRDFLKDSSYFSFAKGVDRDFFIDVQCBSV_GQ169758 181 AEMMIEEKNMISHRFRALSLVRDFLKSSSYFSFAKGLDRDIFIDVQCBSV_GU563320 181 AEMMAEEKNMISHRFRALSLVRDFLKNSSYFNFAKGVDRDFFIDVQCBSV_GU563324 181 AEMMAEEKNMISHRFRALSLVRDFLKDSSYFNFAKGVDRDCFIDVQCBSV_N434436 181 AEMMTEEKNMISHRFRALSLVRDFLKSSSYFSFAKGLDRDIFIDVQCBSV_GQ329864 181 AEMMTEEKNMISHRFRALSLVRDFLKSSSYFSFAKGLDRDIFIDVQCBSV_FN434437 181 AEMMTEEKNMISHRFRALSLVRDFLKSSSYFSFAKGLDRDIFIDVQCBSV_GQ169759 181 AEMMTEEKNMISHRFRALSLVRDFLKSSSYFSFAKGLDRDIFIDVQCBSV_MG570022 181 AEMMTEEKNMISHRFRALSLVRDFLKDSSYFSFAKGVDRDFFIDVQCBSV_LT577537 181 AEMMTEEKNMISHRFRALSLVRDFLKSSSYFSFAKGLDRDIFIDVQCBSV_GU563323 181 AEMMAEEKNMISHRFRALSLVRDFLKDSSYFSFAKGVDRDFFIDVQCBSV_GU563325 181 AEMMPEEKNILSHRFRALSLVRDFLKNSSYFSFAKGVDRDLFIDVQUCBSV_FN433930 181 AEMPSSIKNDFSHRRRALEKVKLFLDNLMVKQEKNKASVALTIDVQUCBSV_FN433931 181 AEMPSSIKNDFSHRRRALEKVKLFLDNLMVKQEEKKTRVALTIDVQUCBSV_GU205820 181 AEMSSNIKNDFSHRRRALEKVKLFLDNLVVKQEKKKARVALTIDVQUCBSV_KX753356 181 AEMSSNIKNDFSHRRKALEKVKLYLDNLMVKQEEKKAKVALTIDVQUCBSV_FN433932 181 AEMSSSIKNDFSHRRRALEKVKLFLDNLVVKQEEKRAKVALTIEVQUCBSV_FN433933 181 AEMSSSIKNDFSHRRRALEKVKLFLDNLVVKQEEKRAKVALTIEVQUCBSV_EU916825 181 AEMPSGIKNEFSHRRRALEKVKLFLDNLVVRQEEKRASMALTIDVQUCBSV_EU916826 181 AEMPSGIKNEFSHRRRALEKVKLFLDNQVVRQEKKRASVALTIDVQUCBSV_GU205818 181 AEMSSSIKNEFSHRRRALEKVKLYLDNLVVKQEEKKAKVALTIDVQUCBSV_GQ169760 181 AEMPSGIKNEFSHRRRALEKVKLFLDNLVVRQEEKRASMALTIDVQUCBSV_KR108836 181 AEMPSSIKNDFSHRRRALEKVKLYLDNLVVKQEEKKAKVALTIDVQUCBSV_KR108835 181 AEMPSNIKNDFSHRRKALEKVKLYLDNLMVKQEEKKAKVALTIDVQUCBSV_FN434109 181 AEMSSSMKNDFSHRRRALEKVKSFLDNLVVKQEEKKARVALTIDVQUCBSV_GU205819 181 AEMSSSMKNDFSHRRRALEKVKSFLDNLVVKQEEKKARVALTIDVQUCBSV_EU916831 181 AEMSSSIKNDFSHRRRALEKVKLFLDNLVVKQEKKKARVALTIDVQUCBSV_EU916832 181 AEMPSSFKNDFSHRRRALEKVKLFLDDLVVKQEKKEARVALTIDVQUCBSV_EU916830 181 AEMSSGMKNDFSHRRRALEKVKSFLDNLVVKQEEKKARVALTIDVQUCBSV_EU916829 181 AEMSSSMKNDFSHRRRALEKVKSFLDNLVVKQEEKKARVALTIDVQUCBSV_EU916827 181 AEMSSSMKNDFSHRRRALEKVKSFLDNLVVKQEEKKARVALTIDVQUCBSV_EU916828 181 AEMSSSMKNDFSHRRRALEKVKSFLDNLVVKQEEKKARVALTIDVQ

Figure

S3: SDS-PAGE of purified CBSV_Tanza (gel A) and UCBSV_Kikombe (gel B) Ham1 proteins (25 kDa). Lanes in gel A correspond separate fractions B4 – B13 that were eluted from the AKTA machine during protein purification at a range of imidazole concentrations. Lane D in gel B refers to UCBSV Ham1 protein which had been dialysed into the storage buffer. To prepare the protein samples for loading, 10 μL of loading buffer (4% SDS, 0.25 M Tris-HCl - pH 6.8, 20% glycerol, 0.004% bromophenol blue, 10% β-mercaptoethanol), 1 μL of protein sample and 9 μL of water were mixed and heated at 95ᵒC for 5 mins. A TruPAGE Precast Gel (Sigma Aldrich) was loaded with 10 μL of each prepared sample and 10 μL of PageRuler Protein Ladder (Thermo Fisher Scientific). The gel was run at 220V for 40 mins. The gel was stained with 20 ml InstantBlue Protein Stain (Sigma Aldrich) and analysed under white light using the ChemDoc Bio-Rad System and images were taken using the Quantity One 1D software (Bio-Rad).

3

No heat 70°C - 10mins

95°C - 10mins

95°C - 1 hour

BSA Neg. 1 Neg. 2 Neg. 30

20

40

60

80

100

120

140Pi

(µM)

Figure S4: Enzyme assay results from the heat inactivation experiment to test for loss of CBSV Tanza Ham1 ITPase activity. Incubation of 0.2 mM dITP with active CBSV Tanza Ham1 protein (1.3 μg) resulted in a phosphate concentration of 136 μM. Heating the CBSV Tanza Ham1 at 95°C for 10 mins – 1 hour resulted in a 41 – 43% reduction in phosphate concentration, indicating inactivation of its pyrophosphohydrolase activity. Control assays were set where BSA protein (1.3 μg) was added, which produced a comparable phosphate concentration to assays where CBSV Tanza Ham1 had been heat inactivated, indicating that BSA could be used as a control for addition of protein to assay samples. Low background phosphate concentrations were found in negative controls: 1) containing 0.2 mM dITP in reaction buffer with the addition of 0.1 units of yeast inorganic pyrophosphatase, 2) 0.2 mM dITP in reaction buffer and 3) water.

4

Control (-) 5-FU Test (+) 5-FU

pYES2

pYES2-HAM1(CBSV-

Nampula)

pYES2-HAM1 (CBSV-Tanza)pYES2-HAM1

(UCBSV-Kikombe)

pYES2-HAM1

(S. cerevisia

e)

10-1 10-2 10-3 10-4 10-5

10-1 10-2 10-3 10-4 10-5

S5: 5-FU resistance agar plate growth assays. The wild-type yeast strain BY4742 was transformed with pYES2 plasmids containing Ham1 sequences from CBSV Nampula, CBSV Tanza, UCBSV Kikombe and yeast. Transformant yeast were cultured and plated onto SD media as ten-fold serial dilutions onto test plates containing 2% galactose and 10 µg/ml 5-FU or control plates containing 2% galactose only. Colony growth was imaged after 72 hours. Results were consistent in three separate experiments.

5

4 hours 9 hours 24 hours 48 hours 72 hours0.0

0.5

1.0

1.5

2.0

2.5

pYES2 CBSV_Nampula UCBSV_KikombeCBSV_Tanza Yeast

OD6

00

S6: 5-FU resistance liquid growth assays. The wild-type yeast strain BY4742 was transformed with pYES2 plasmids containing Ham1 sequences from CBSV Nampula, CBSV Tanza, UCBSV Kikombe and yeast. Transformant yeast were cultured liquid SD media containing 2% galactose and 10 µg/ml 5-FU. The cell density (OD600) was taken at 4, 8, 12, 24, 48 and 72 hours. Yeast transformed with yeast Ham1 sequence demonstrated relatively high levels of growth. Whereas as yeast transformed with U/CBSV Ham1 sequences showed low levels of growth, comparable to the negative control (empty pYES2 plasmid). This indicates that unlike the yeast Ham1 sequence, the U/CBSV Ham1 sequences were unable to protect against mutagenic 5-FU. Each result is the mean OD600 value from three replicate samples (n = 3) ± S.E. Results were consistent in three separate experiments.

6

Figure S9: Schematic showing the replacement of CBSV Tanza Ham1 sequence (blue) with UCBSV Ham1 sequence (red) in the CBSV_UHam1 IC. To ensure proteolytic cleavage of UCBSV Ham1 sequence from the CBSV Tanza polyprotein the NIb – Ham1 protease cleavage sequence T-L-Y-V-V-D was fused to the start of the UCBSV Ham1 sequence: T-K-D.

7

Table S1: Games-Howell one-way ANOVA tests to compare mean phosphate concentration in enzyme assay reactions with CBSV Tanza Ham1 incubated with the non-canonical nucleotides XTP and dITP and a range of canonical nucleotides.

Protein CBSV Ham1

Non-canonical nucleotides

XTP dITP

Canonical nucleotides

Mean Pi (μM)198 172

Mean Pi (μM)

dGTP134 Difference

(μM)  64  38

Sig. p value  0.828  0.979

GTP90 Difference

(μM)  108   82

Sig. p value   0.062  0.139

UTP

48 Difference (μM)  150   124

Sig. p value  0 .030 * 

 0.040 *

dTTP

27 Difference (μM)  171  145

Sig. p value  0.008 **  0.011 *

dATP

19 Difference (μM)  179  152

Sig. p value  0.016 *  0.019 *

dCTP

12 Difference (μM)  186  160

Sig. p value  0.009 **  0.011 *

CTP

13 Difference (μM)  14  158

Sig. p value  0.013 *  0.016 *

ATP 9 Difference (μM)  189   163

Sig. p value 0.019 *  0.023

8

*

Table S2: Games-Howell one-way ANOVA tests to compare mean phosphate concentration in enzyme assay reactions with UCBSV Kikombe Ham1 incubated with the non-canonical nucleotides XTP and dITP and a range of canonical nucleotides.

Protein UCBSV Ham1

Non-canonical nucleotides

XTP dITP

Canonical nucleotides

Mean Pi (μM)190 178

Mean Pi (μM)

dGTP100 Difference

(μM) 90 79

Sig. p value 0.180 0.227

GTP83 Difference

(μM) 106 95

Sig. p value 0.095 0.107

UTP59 Difference

(μM) 131 119

Sig. p value 0.074 0.073

dTTP20 Difference

(μM) 70 157

Sig. p value 0.051 * 0.049 *

dATP24 Difference

(μM) 165 154

Sig. p value 0.052 * 0.053 *

dCTP26 Difference

(μM) 164 153

Sig. p value 0.054 * 0.052 *

9

CTP32 Difference

(μM) 158 147

Sig. p value 0.063 0.062

ATP 9 Difference (μM) 180 169

Sig. p value 0.050 * 0.048 *

Table S3: Primers used to amplify CBSV and UCBSV Ham1 sequences during cloning into the POPINF vector. Sequences overlapping with the POPINF vector are shown in red.Primer name Sequence 5’3 POPINF_CBSV_Ham1_Fw AAGTTCTGTTTCAGGGCCCGGTGGTGGACAGGTCTCAGCC

POPINF_CBSV_Ham1_Rv ATGGTCTAGAAAGCTTTAGCTTGAACATCAATAAAGAAATCACG

POPINF_UCBSV_Ham1_Fw AAGTTCTGTTTCAGGGCCCGACAAAGGATTTGAGAGGAAGAGAG

POPINF_UCBSV_Ham1_Rv ATGGTCTAGAAAGCTTTACTGCACATCAATTGTTAGAGCCAC

Table S4: Primers used to amplify PCR fragments to construct the CBSV_mutHam and CBSV_UHam ICs. The nucleotide sequence encoding the SHR to SAA mutation are shown in blue. The nucleotide sequence encoding the UCBSV Ham1 are shown in red.

Infectious clone

Primer Sequence 5’ – 3’ Size (bp)

Target

CBSV_mutHam

SHR_mut_F1_Fw

GAAATATAATGAACCTGTTCGAGTGGGGTTGGTAAAC

1916 NIb – Ham1

SHR_mut_F1_Rv

TCCTTCAAAAAGTCTCTCACTAATGACAGAGCCCGAAAGGCAGCAGATATCATATTCTTC

SHR_mut_F2_Fw

GAAGAATATGATATCTGCTGCCTTTCGGGCTCTGTCATTAGTGAGAGACTTTTTGAAGGA

1810 Ham1 – 3’UTR

SHR_mut_F2_Rv

GGCTGGCTGGTGGCAGGATATATTGTGGTGTAAAC

CBSV_UHam UCBSV_HF1_Fw

GAAATATAATGAACCTGTTCGAGTGGGGTTGGTAAAC

1336 CBSV NIb

UCBSV_HF1_Rv

CAGGCTTCTCTCTTCCTCTCAAATCCTTTGTGTCCACCACTTGTAAGTCAATGTAACAAT

UCBSV_HF2_Fw

ACAAAGGATTTGAGAGGAAGAGAGAAGCCTGAGTTGAGAATTGAGAGCCAT

677 UCBSV Ham1

UCBSV_HF2_Rv

CTGCACATCAATTGTTAGAGCCACCTTTGCCTTCTTCTCCTCTTGTTTCACCATC

UCBSV_HF3_Fw

GAAGGCAAAGGTGGCTCTAACAATTGATGTGCAGGCAATTGACAAGGA

1729 CBSV CP –

10

TGAGATTGA 3’UTRUCBSV_HF3_Rv

GGCTGGCTGGTGGCAGGATATATTGTGGTGTAAAC

11