mock! A3AC106S! A3BE255Q! 86.7! 90.1! 88.0! 89.8! 89.0!

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a A3A A3Bi7 A3Btok A3Bwh A3Blan mock V5 DAPI merge 5 µm b edited A3A A3Bi7 A3Btok A3Blan A3Bwh A3BF38L A3BW359L mock A3AC16S A3BE255Q 79.5 79.7 8.4 81.3 82.5 83.9 85.3 86.7 88. 89. 89.8 9.1 HBV 3DPCR Td/ C Supplementary Figure 1. Cellular localization of A3B proteins and HBV editing. a) Confocal microscopy of V5 tagged A3A proteins performed in HeLa cells 24 hours post transfection. Nuclei are stained using DAPI. b) 3D-PCR analysis of HBV DNA editing by A3B proteins.

Supplementary Figure 2. A3B nudna editing and 5-methylcytidine deamination. a) Quail cmyc specific 3DPCR gels after QT6 cells transfections with A3 proteins along with UGI coding plasmid. Asterisks denote the samples that were molecularly cloned and sequenced. b) Mutation matrices of cmyc hyperedited sequences. The numbers below the matrices indicate the number of bases sequenced. c) 5 Dinucleotide analysis of the deamination context performed on DNA minus strand for cloned PCR products. * denote significant deviation from expected values (χ2 test, p<.5, n=18 for A3A, n=23 for A3Bi7). d) Selection of hypermutated 5Me-dC substituted HIV-1 sequences after co-transfection along with A3 coding plasmids in QT6 cells. e) Mutation matrices of HIV-1 hyperedited sequences. The numbers below the matrices indicate the number of bases sequenced. Data presented correspond to 3DPCR products obtained at 81.3 C. a b quail Myc Td/ C A3A A3Bi7 87.4 87.6 88. 88.7 89.6 9.6 91.7 92.7 93.6 94.4 94.9 95.1 M * * A3A T C G A T 2 C 45 1 1 G 9 A A3Bi7 T C G A T 1 C 391 1 G 1 3 A d A3Btok A3Bwh A3Blan A3AC16S A3BE255Q A3A A3Bi7 A3Btok A3Blan mock 5Me-dC HIV-1 : 1 bp from 276 bp c Deamination context (%) 5 4 3 2 1 n= 4864 bp n= 5888 bp % * * TpC CpC GpC ApC A3A A3Bi7 expected GAGCAGTTTTTTATCTCTCCTTTCTCCATCATCATTTCCCCGCTACTACTATTGGTATTACTACTATTGGTATTAGTAGCATTCCCCAAATCAGTGCACT...T.T.TT...T.TT..T..T...TTTT.T...T...T..T...TTTT...T......T.T.TT...T.TT..T..T...TTTT...T...T... A.A...A...AA...A......T.T.TT...T.TT..T..T...TTTT.T...TTTT...T...T......T.T.TT...T.TT..T..T...TT...T......T...T...T...T..T...T.TT...T.TT...T......T.T.TT...T.TT..T..T...TTTT...T...TTTT...T......T...T...T...TTT...TTTT...T...T.T....T...T.T.TT...T...TTTT...T...T......T...T...T...TTT...T.T......T...T...T..T..T...TT.T...TTT......T...T.T..T...T..T..T..T...TTTT...T...T...TT.T...T...T... * * e A3A T C G A A3Bi7 T C G A A3Btok T C G A A3Blan T C G A T 8 1 2 T 14 3 1 T 4 2 T 8 C 763 C 539 C 346 C 412 1 G 27 G 63 G 17 G 1 18 A 1 4 A 5 1 1 A 3 A 2 1 4 n= 6825 bp n= 9955 bp n= 3864 bp n= 5796 bp

25K.44 25K 1 Mock 2K 15K 1K A3Btok 2K 15K 1K 44.5 8 6 4 5K 1 2 1 3 1 4 1 5 V5 5K 1 2 1 3 1 4 1 5 2 1 2 1 3 1 4 1 5 V5 γh2ax 25K 1 25K 1 A3AC16S 2K 15K 1K 37.8 8 6 4 A3Blan 2K 15K 1K 57 8 6 4 5K 2 5K 2 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 V5 γh2ax 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 V5 γh2ax 25K 1 25K 1 A3A 2K 15K 1K 34.1 8 6 4 A3Bwh 2K 15K 1K 42.7 8 6 4 5K 2 5K 2 1 2 1 3 1 4 1 5 V5 1 2 1 3 1 4 1 5 γh2ax 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 V5 γh2ax 25K 1 25K 1 A3Bi7 2K 15K 1K 37.5 8 6 4 A3BE255Q 2K 15K 1K 42.3 8 6 4 5K 2 5K 2 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 V5 γh2ax 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 V5 γh2ax Supplementary figure 3a. FACS plots of γh2ax staining on V5 positive HeLa cells gated on V5 (pink) after A3 (2µg) transfection performed at 48 hours. Cells. Blue histograms represent γh2ax staining for A3AC16S catalytic mutant transfection used as negative control.

25K 25K 1 Mock 2K 15K 1K.149 A3Btok 2K 15K 1K 4.47 8 6 4 5K 1 2 1 3 1 4 1 5 V5 5K 1 2 1 3 1 4 1 5 2 1 2 1 3 1 4 1 5 V5 γh2ax A3AC16S 25K 2K 15K 1K 5.28 1 8 6 4 A3Blan 25K 2K 15K 1K 6.62 1 8 6 4 5K 2 5K 2 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 V5 γh2ax 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 V5 γh2ax 25K 1 25K 1 A3A 2K 15K 1K 4.76 8 6 4 A3Bwh 2K 15K 1K 5.92 8 6 4 5K 2 5K 2 1 2 1 3 1 4 1 5 V5 1 2 1 3 1 4 1 5 γh2ax 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 V5 γh2ax 25K 1 25K 1 A3Bi7 2K 15K 1K 7.6 8 6 4 A3BE255Q 2K 15K 1K 8.12 8 6 4 5K 2 5K 2 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 V5 γh2ax 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 V5 γh2ax Supplementary figure 3b. FACS plots of γh2ax staining on V5 positive QT6 cells gated on V5 (pink) after A3 (2µg) transfection performed at 48 hours. Cells. Blue histograms represent γh2ax staining for A3AC16S catalytic mutant transfection used as negative control.

a A3AC16S A3A A3Bi7 A3Btok 1 1 1 1 8 8 8 8 6 4 6 4 6 4 6 4 2 2 2 2 1 2 1 3 1 4 1 5 γh2ax 1 2 1 3 1 4 1 5 γh2ax 1 2 1 3 1 4 1 5 γh2ax 1 2 1 3 1 4 1 5 γh2ax A3Blan A3Bwh A3BE255Q 1 1 1 8 8 8 6 4 6 4 6 4 2 2 2 b % γh2ax in V5 2 15 1 5 1 2 1 3 1 4 1 5 * γh2ax 1 2 1 3 1 4 1 5 γh2ax A3 + mock A3 + UGI 1 2 1 3 1 4 1 5 γh2ax Supplementary Figure 4. FACS analysis of γh2ax-positive HeLa cells gated on V5 after A3 (1µg) cotransfection with mock (1µg) or UGI (1µg) coding plasmids at 48 hours. a) FACS plots of γh2ax staining on V5 positive cells. Red histograms represent γh2ax staining for transfections performed with 1µg of mock plasmid, blue histograms represent γh2ax staining for transfections performed with UGI coding plasmid. Error bars represent standard deviation from four independent transfections. Difference between mock transfected cells and UGI transfected cells for A3A was calculated using student test (*p=.375).

exon 5/8 AsnGlnGlyAsn*** A3A-UTR AATCAGGGAAACTGAAGGATGGGCCTCAGTCTCTAAGGAAGGCAGAGACCTGGGTTGAGCAGCAGAATAAAAGATCTTCTTCCAAGAAATGCAAACAGAC 1 A3B-UTR AATCAGGGAAACTGAAGGATGGGCCTCAGTCTCTAAGGAAGGCAGAGACCTGGGTTGAGCAGCAGAATAAAAGATCTTCTTCCAAGAAATGCAAACAGAC 1 **************************************************************************************************** A3A-UTR CGTTCACCACCATCTCCAGCTGCTCACAGACGCCAGCAAAGCAGTATGCTCCCGATCAAGTAGATTTTTAAAAAATCAGAGTGGGCCGGGCGCGGTGGCT 2 A3B-UTR CGTTCACCACCATCTCCAGCTGCTCACAGACACCAGCAAAGCAATGTGCTCCTGATCAAGTAGATTTTTTAAAAATCAGAGT------------------ 182 ******************************* *********** * ****** **************** ************>>> ALU element A3A-UTR CACGCCTGTAATCCCAGCACTTTGGAGGCCAAGGCGGGTGGATCACGAGGTCAGGAGATCGAGACCATCCTGGCTAACACGGTGAAACCCTGTCTCTACT 3 A3B-UTR ---------------------------------------------------------------------------------------------------- 182 A3A-UTR AAAAATACAAAAAATTAGCCAGGCGTGGTGGCGGGCGCCTGTAGTCCCAGCTACTCTGGAGGCTGAGGCAGGAGAGTAGCGTGAACCCGGGAGGCAGAGC 4 A3B-UTR ---------------------------------------------------------------------------------------------------- 182 A3A-UTR TTGCGGTGAGCCGAGATTGCGCTACTGCACTCCAGCCTGGGCGACAGTACCAGACTCCATCTCAAAAAAAAAAAAACCAGACTGAATTAATTTTAACTGA 5 A3B-UTR -----------------------------------------------------------------------------------CAATTAATTTTAATTGA 199 Alu element <<< ************ *** A3A-UTR AAATTTCTCTTATGTTCCAAGTACACAATAGTAAGATTATGCTCAATATTCTCAGAATAATTTTCAATGTATTAATGAAATGAAATGATAATTTGGCTTC 6 A3B-UTR AAATTTCTCTTATGTTCCAAGTGTACAAGAGTAAGATTATGCTCAATATTCCCAGAATAGTTTTCAATGTATTAATGAAG-----TGATTAATTGGCTCC 294 ********************** **** ********************** ******* ******************* **** * ****** * NM_12746, U3891 < A3A-UTR ATATCTAGACTAACACAAAATTAAGAATCTTCCATAATTGCTTTTGCTCAGTAACTGTGTCATGAATTGCAAGAGTTTCCACAAACACTAGCAAATGTGT 7 BC3183, AK24854 < BC53859 < U6183, U6184 < A3B-UTR ATATTTAGACTAATAAAACATTAAGAATCTTCCATAATTG-----------------------------------TTTCCACAAACACTAGCAAATGTGT 359 **** ******** * ** ********************* ************************* A3A-UTR AGATGTCTTTCCTTGTGTAGCGGACCTGTAGCGGGGAAAGGTCACACAACATCCCTCTGGATCCAGAAAACTCAGCTAAACCTCACAGGAGAGGAACCTA 8 A3B-UTR AGATGTCTTTCCTTGTGTAGCGGACCTGTAGCTGGGAAAGGTCACACAACATCCCTCTGGATCCAGAAAACTCAGCTAAACCACACAGGAGAGGAACCTA 459 ******************************** ************************************************* ***************** A3A-UTR AATGCAGACCCCACCCTCACTCACAGAGCCCCGCCCACCCTCACTTACAGAGCCCCGGGCGCTGATTGG 869 A3B-UTR AATGCAGACCCCACCCTCACTCACAGAGCCCCGCCCACCCTCACTCACAGAGCCCCGGGCGCTGATTGG 528 ********************************************* *********************** Supplementary Figure 5. Sequence comparison of the 3 UTR encoded by exon 5 of A3A and exon 8 of A3B genes used in the study. An imperfect repeated sequence flanking the Alu element is underlined. A3A and A3B transcripts are identified by their accession numbers and their 3 ends denoted by arrowheads (<).

a Mock A3A- UTR A3A-UTR A3A A3A-UTR A3B 25K 25K 48.5 25K 38.4 25K 5.2 2K 15K 1K.173 2K 15K 1K 2K 15K 1K 2K 15K 1K 5K 5K 5K 5K 1 2 1 3 1 4 1 5 HA 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 HA HA HA 1 1 1 8 8 8 6 4 6 4 6 4 2 18.8 2 11.3 2 17.7 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 γh2ax γh2ax γh2ax b DMSO PMA Etoposide 1 1 1 8 8 8 6 4 6 4 6 4 2 2 2 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 γh2ax γh2ax γh2ax Supplementary figure 6 a) FACS plots of γh2ax staining on HA positive HeLa cells gated on HA (pink) after A3 (2µg) transfection performed at 48 hours. Cells. Blue histograms represent γh2ax staining for A3AC16S catalytic mutant transfection used as negative control. b) FACS plots of γh2ax staining of SKBR3 cells after treatment. Blue histograms represent γh2ax staining for DMSO treated cells used as negative control.

Plasmid Matrix Primers For : 5 -CACCATGAATCCACAGATCAGAAATCCGATGGAGC A3Blan pcdna3.1-a3b-ha Rev : 5 -GTTTCCCTGATTCTGGAGAAT For : 5 -CACCATGAATCCACAGATCAGAAATCCGATGGAGC A3Bi7 Puc57-A3Bi Rev : 5 -GTTTCCCTGATTCTGGAAGAGCAGGGGGGTTG A3A UTR A3AC16S A3A UTRA A3A UTRB Luc UTR Luc UTRA Luc UTRB A3Ai4 For : 5'-CACCATGGCTTATCCTTACGACGTGCCTGACTACGCCATGGAAGCCAGCCCAGCATCCGGGCCCAGACACTTG A3AC16S Rev : 5'-ACTGAGGCCCATCCTTCAGTTTCCCTGATTCTGGAAGAGCAG For : 5'-CACCATGGCTTATCCTTACGACGTGCCTGACTACGCCATGGAAGCCAGCCCAGCATCCGGGCCCAGACACTTG A3Ai4 Rev : 5'-ACTGAGGCCCATCCTTCAGTTTCCCTGATTCTGGAAGAGCAG For : 5'-AATCAGGGAAACTGAAGGATGGGCCTCAGTCTCTAAGGAAGG 293T gdna Rev : 5'-CCAATCAGCGCCCGGGGCTCTGTRAGTGAGGGTGGGCGGGGC For : 5'-CACCATGGCTTATCCTTACGACGTGCCTGACTACGCCATGGAAGCCAGCCCAGCATCCGGGCCCAGACACTTG A3Ai4 Rev : 5'-ACTGAGGCCCATCCTTCAGTTTCCCTGATTCTGGAAGAGCAG For : 5'-AATCAGGGAAACTGAAGGATGGGCCTCAGTCTCTAAGGAAGG 293T gdna Rev : 5'-CCAATCAGCGCCCGGGGCTCTGTRAGTGAGGGTGGGCGGGGC pgl4.5 For : 5'-CACCATGGAAGATGCCAAAAACATTAAGA [luc2/cmv/hygro] Rev : 5'-TTACACGGCGATCTTGCCGCCCTTCTTGGCCTT pgl4.5 For : 5'-CACCATGGAAGATGCCAAAAACATTAAGA [luc2/cmv/hygro] Rev : 5'-CCTTCCTTAGAGACTGAGGCCCATCCTTTACACGGCGATCTTGCCGCCCTTCTTGGCCTT For : 5'-AGGATGGGCCTCAGTCTCTAAGGAAGG A3A UTRA Rev : 5'-GTTTCCCTGATTCTGGAAGAGCAGGGGGGTTG pgl4.5 For : 5'-CACCATGGAAGATGCCAAAAACATTAAGA [luc2/cmv/hygro] Rev : 5'-GTTTCCCTGATTCTGGAAGAGCAGGGGGGTTG For : 5'-AGGATGGGCCTCAGTCTCTAAGGAAGG A3A UTRB Rev : 5'-CCTTCCTTAGAGACTGAGGCCCATCCTTTACACGGCGATCTTGCCGCCCTTCTTGGCCTT Supplementary table 1. Compendium of primers used for PCR cloning

Plasmid Matrix Primers A3Btok (T146K) A3Bi7 For : 5'-GCAGGAGCCCGCGTGAAGATCATGGACTATGAA Rev : 5 -TTCATAGTCCATGATCTTCACGCGGGCTCCTGC A3BiE255Q A3Bi7 For : 5 -TACGGCCGCCATGCGCAGCTGCGCTTCTTGGAC Rev : 5 -GTCCAAGAAGCGCAGCTGCGCATGGCGGCCGTA A3BF38L A3Bi7 For : 5'-GTGAGACTGCGCATCTTAGCTGCCCGCATCTAT Rev : 5'-ATAGATGCGGGCAGCTAAGATGCGCAGTCTCAC A3BW359L A3Bi7 For : 5'-TGTCCCTTCCAGCCCTTGGATGGACTAGAGGAG Rev : 5'-CTCCTCTAGTCCATCCAAGGGCTGGAAGGGACA Supplementary table 2. Compendium of primers used for mutagenesis

Primer Application Sequence Cycling conditions HBV out fwd HBV out rev HBV PCR HBV PCR 5'-AAGAGTYYTYTTATGTAAGACYTT 5'-CGCAAATATACATCGTATCCAT 95 C 5 min 35x (95 C 1 min, 5 C 3 sec, 72 C 5 min), 72 C 1 min HBV in fwd HBV in rev HBV 3DPCR HBV 3DPCR 5'-AAGTGCACACGGTYYGGCAGAT 5'-ATGGCTGCTARGCTGTGCTGCCAA 8-9 C 5 min 35x (8-9 C 1 min, 5 C 3 sec, 72 C 5 min), 72 C 1 min TP53 out fwd TP53 out rev TP53 PCR TP53 PCR 5 GAGCTGGACCTTAGGCTCCAGAAAGGACAA 5 -GCTGGTGTTGTTGGGCAGTGCTAGGAA 95 C 5 min 42x (95 C 3 sec, 6 C 3 sec, 72 C 2 min), 72 C 1 min TP53 in fwd TP53 in rev TP53 3DPCR 5 -TTCTCTTTTCCTATCCTGAGTAGTGGTAA TP53 3DPCR 5 -AAAGGTGATAAAAGTGAATCTGAGGCATAA 84-92 C 5 min 4x (84-92 C 1 min, 58 C 3 sec, 72 C 2 min), 72 C 1 min HIV-1 out fwd HIV-1 out rev HIV-1 PCR HIV-1 PCR 5 -TGTACCCACAGACCCCAACCCACAA 5 -TTCCATTGAACGTCTTATTATTACA 95 C 5 min 3x (95 C 45 sec, 54 C 45 sec, 72 C 2 min), 72 C 1 min HIV-1 in fwd HIV-1 in rev HIV-1 3DPCR 5 -ATCAAAGCCTAAAGCCATGTGTAA HIV-1 3DPCR 5 -CAATAATGTATGGGAATTGGCTCAA 78-9 C 5 min 45x (78-9 C 1 min, 56 C 3 sec, 72 C 5 min), 72 C 1 min QT6 cmyc out fwd QT6 cmyc out rev cmyc PCR cmyc PCR 5 -GATGCCGCTCAGCGCCAGCCTCCCCAGCAA 5 -GCAGTCCTGGATGATGATGGATTTGACGAA 95 C 5 min 4x (95 C 1 min, 58 C 45 sec, 72 C 2 min), 72 C 1 min QT6 cmyc in fwd QT6 cmyc in rev cmyc 3DPCR 5 -TTCGAGGAGGAGGAGGAGAACTTCTA cmyc 3DPCR 5 -GATTCGTCGTCCGGGTCGCAGATGAA 88-95 C 5 min 42x (88-95 C 1 min, 6 C 1 min, 72 C 2 min), 72 C 1 min Supplementary table 3. Compendium of primers and PCR conditions used for nested PCR/3DPCR amplifications

b 293T A3A- UTR A3A-UTRA3A A3A-UTRA3B Mock 293T A3A- UTR A3A-UTRA3A A3A-UTRA3B Mock kda Mock A3A A3AC16S A3Bi7 A3Btok A3Blan A3Bwh A3BE255Q a 55-35- Ac$n 7-55- 35-25- Mock A3A A3AC16S A3Bi7 A3Btok A3Blan A3Bwh A3BE255Q kda 7-55- 35-25- V5 kda Ac$n HA Supplementary data 1. Uncropped scans of westerblots from a) Figure 1B, b) Figure 3C.

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