Supplementary Information Simplexviruses successfully adapt to their host by fine-tuning immune responses Alessandra Mozzi, Rachele Cagliani, Chiara Pontremoli, Diego Forni, Irma Saulle, Marina Saresella, Uberto Pozzoli, Mario Clerici, Mara Biasin, Manuela Sironi Supplementary Figures: Figure S1. Positive selection in UL26, UL29, UL36 and UL55. Positively selected sites and functional domains were mapped onto HSV-1 proteins, as in Figure 2 and 3. For UL36, given the extended length of the protein sequence, positively selected sites were reported in the enlargement below. Supplementary Tables: Supplementary Table S1. List of viral genome sequences Supplementary Table S2. Herpes simplex genes excluded from the branch-sites analysis Supplementary Table S3. List of analyzed genes and dN/dS values Supplementary Table S4. Likelihood ratio test (LRT) statistics for models of variable selective pressure on the Hominin-infecting SVs branch. UL26 - Capsid scaffolding protein A379 Y374 S612 Interaction with Major Capsid protein assemblin cleavage sites UL29 - Major DNA-binding protein I878 Required for nuclear localization minimal DNA-binding region UL36 - Large tegument protein Deubiquitination Interaction with activity inner tegument protein (UL37) H2518 F2167 R2202 A2507 C1073 A1557 R1841 R2165G2198 S2488 D2506 W2573 K1052 T1103 R1702 F1746 Q1810 F1866 R2187 P2246 T2333 R2422 A2450 R2503 G2552 L2599 W2704 L3042 R3129 Tandem C-terminus repeats UL55 - Nuclear protein UL55 E99 Figure S1. Posi�ve selec�on in UL26, UL29, UL36 and UL55. Posi�vely selected sites and func�onal domains were mapped onto HSV-1 proteins, as in Figure 2 and 3. For UL36, given the extended length of the protein sequence, posi�vely selected sites were reported in the enlargement below. Supplementary Table S1. List of viral genome sequences. Accession Virus name Strain Name Virus Species Host Country ID (Common name; Strain abbreviation) Human herpesvirus 1 2016-1040 MG999886 Human alphaherpesvirus 1 Homo Sapiens USA (Herpes simplex virus 1; HSV-1 2016-1040) Human herpesvirus 1 HSV-H12113 MH999842 Human alphaherpesvirus 1 Homo Sapiens Finland (Herpes simplex virus 1; HSV-1 H12113) Human herpesvirus 1 HSV-H1412 MH999851 Human alphaherpesvirus 1 Homo Sapiens Finland (Herpes simplex virus 1; HSV-1 H1412) Human herpesvirus 1 172/2010 LT594105 Human alphaherpesvirus 1 Homo Sapiens Germany (Herpes simplex virus 1; HSV-1 172/2010) Human herpesvirus 1 HSV-1/0116209/India/2011 KJ847330 Human alphaherpesvirus 1 Homo Sapiens India (Herpes simplex virus 1; HSV-1/0116209/India/2011) Human herpesvirus 1 CJ394 JN420340 Human alphaherpesvirus 1 Homo Sapiens USA (Herpes simplex virus 1; HSV-1 CJ394) Human herpesvirus 1 CR38 HM585508 Human alphaherpesvirus 1 Homo Sapiens China (Herpes simplex virus 1; HSV-1 CR38) Human herpesvirus 1 E06 HM585496 Human alphaherpesvirus 1 Homo Sapiens Kenya (Herpes simplex virus 1; HSV-1 E06) Human herpesvirus 1 E07 HM585497 Human alphaherpesvirus 1 Homo Sapiens Kenya (Herpes simplex virus 1; HSV-1 E07) Human herpesvirus 1 E08 HM585498 Human alphaherpesvirus 1 Homo Sapiens Kenya (Herpes simplex virus 1; HSV-1 E08) Human herpesvirus 1 E13 HM585502 Human alphaherpesvirus 1 Homo Sapiens Kenya (Herpes simplex virus 1; HSV-1 E13) Human herpesvirus 1 H166 KM222726 Human alphaherpesvirus 1 Homo Sapiens USA (Herpes simplex virus 1; HSV-1 H166) Human herpesvirus 1 R11 HM585514 Human alphaherpesvirus 1 Homo Sapiens South Korea (Herpes simplex virus 1; HSV-1 R11) Human herpesvirus 1 S23 HM585512 Human alphaherpesvirus 1 Homo Sapiens Japan (Herpes simplex virus 1; HSV-1 S23) Human herpesvirus 1 CJ360 * JN420339 Human alphaherpesvirus 1 Homo Sapiens USA (Herpes simplex virus 1; HSV-1 CJ360) Human herpesvirus 2 2003-16029 KX574903 Human alphaherpesvirus 2 Homo Sapiens USA (Herpes simplex virus 2; HSV-2 2003-16029) Human herpesvirus 2 2004-4373 KX574861 Human alphaherpesvirus 2 Homo Sapiens Peru (Herpes simplex virus 2; HSV-2 2004-4373) Human herpesvirus 2 2006-30937 KX574866 Human alphaherpesvirus 2 Homo Sapiens Peru (Herpes simplex virus 2; HSV-2 2006-30937) Human herpesvirus 2 2006-49895 KX574868 Human alphaherpesvirus 2 Homo Sapiens USA (Herpes simplex virus 2; HSV-2 2006-49895) Human herpesvirus 2 2007-38120 KX574871 Human alphaherpesvirus 2 Homo Sapiens Zimbawe (Herpes simplex virus 2; HSV-2 2007-38120) Human herpesvirus 2 2007-38627 KX574874 Human alphaherpesvirus 2 Homo Sapiens Peru (Herpes simplex virus 2; HSV-2 2007-38627) Human herpesvirus 2 2007-39712 * KX574880 Human alphaherpesvirus 2 Homo Sapiens South africa (Herpes simplex virus 2; HSV-2 2007-39712) Human herpesvirus 2 2009-13458 KX574889 Human alphaherpesvirus 2 Homo Sapiens USA (Herpes simplex virus 2; HSV-2 2009-13458) Human herpesvirus 2 2009-3532 KX574892 Human alphaherpesvirus 2 Homo Sapiens South africa (Herpes simplex virus 2; HSV-2 2009-3532) Human herpesvirus 2 2009-4556 KX574894 Human alphaherpesvirus 2 Homo Sapiens Kenya (Herpes simplex virus 2; HSV-2 2009-4556) Human herpesvirus 2 2011-21761 KX574897 Human alphaherpesvirus 2 Homo Sapiens USA (Herpes simplex virus 2; HSV-2 2011-21761) Human herpesvirus 2 HSV2-H1227 KY922721 Human alphaherpesvirus 2 Homo Sapiens Finland (Herpes simplex virus 2; HSV-2 H1227) Human herpesvirus 2 HSV2-H1229 KY922722 Human alphaherpesvirus 2 Homo Sapiens Finland (Herpes simplex virus 2; HSV-2 H1229) Human herpesvirus 2 2008_15116 MF621257 Human alphaherpesvirus 2 Homo Sapiens Kenya (Herpes simplex virus 2; HSV-2 2008_15116) Human herpesvirus 2 CNS15 MK105998 Human alphaherpesvirus 2 Homo Sapiens USA (Herpes simplex virus 2; HSV-2 CNS15) Chimpanzee alpha-1 herpesvirus 105640 * NC_023677 Panine alphaherpesvirus 3 Pan troglodytes USA (Chimpanzee herpesvirus; ChHV 105640) Macacine herpesvirus 1 12930 KY628971 Macacine alphaherpesvirus 1 Macaca mulatta USA (B virus; McHV-1 12930) Macacine herpesvirus 1 1504-11 KY628969 Macacine alphaherpesvirus 1 Macaca nemestrina USA (B virus; McHV-1 1504-11) Macacine herpesvirus 1 16293 KY628972 Macacine alphaherpesvirus 1 Macaca mulatta USA (B virus; McHV-1 16293) Macacine herpesvirus 1 20620 KY628973 Macacine alphaherpesvirus 1 Macaca mulatta USA (B virus; McHV-1 20620) Macacine herpesvirus 1 24105-G KY628974 Macacine alphaherpesvirus 1 Macaca mulatta USA (B virus; McHV-1 24105-G) Macacine herpesvirus 1 26896-G KY628975 Macacine alphaherpesvirus 1 Macaca mulatta USA (B virus; McHV-1 26896-G) Macacine herpesvirus 1 31618-G KY628978 Macacine alphaherpesvirus 1 Macaca mulatta USA (B virus; McHV-1 31618-G) Macacine herpesvirus 1 32425-G KY628981 Macacine alphaherpesvirus 1 Macaca mulatta USA (B virus; McHV-1 32425-G) 7709642 KY628982 Macacine herpesvirus 1 Macacine alphaherpesvirus 1 Macaca silenus USA (B virus; McHV-1 7709642) Macacine herpesvirus 1 8100812 KY628968 Macacine alphaherpesvirus 1 Macaca silenus USA (B virus; McHV-1 8100812) Macacine herpesvirus 1 9400371 KY628983 Macacine alphaherpesvirus 1 Macaca mulatta USA (B virus; McHV-1 9400371) Macacine herpesvirus 1 E90-136 KJ566591 Macacine alphaherpesvirus 1 Macaca fascicularis USA (B virus; McHV-1 E90-136) Macacine herpesvirus 1 KQ KY628970 Macacine alphaherpesvirus 1 Macaca nemestrina USA (B virus; McHV-1 KQ) Macacine herpesvirus 1 M12-O KY628985 Macacine alphaherpesvirus 1 Macaca radiata USA (B virus; McHV-1 M12-O) Macacine herpesvirus 1 E2490 * NC_004812 Macacine alphaherpesvirus 1 Macaca mulatta USA (B virus; McHV-1 E2490) Papiine herpesvirus 2 A189164 KF908239 Papiine alphaherpesvirus 2 - USA (Herpesvirus papio 2, PaHV-2 A189164) Papiine herpesvirus 2 A951 KF908242 Papiine alphaherpesvirus 2 - USA (Herpesvirus papio 2; PaHV-2 A951) Papiine herpesvirus 2 OU2-5 KF908241 Papiine alphaherpesvirus 2 Papio anubis USA (Herpesvirus papio 2; PaHV-2 OU2-5) Papiine herpesvirus 2 OU4-2 KF908244 Papiine alphaherpesvirus 2 Papio ursinus USA (Herpesvirus papio 2; PaHV-2 OU4-2) Papiine herpesvirus 2 OU4-8 KF908243 Papiine alphaherpesvirus 2 Papio ursinus USA (Herpesvirus papio 2; PaHV-2 OU4-8) Papiine herpesvirus 2 X313 * NC_007653 Papiine alphaherpesvirus 2 Papio anubis USA (Herpesvirus papio 2; PaHV-2 X313) Cercopithecine herpesvirus 2 Cercopithecine Cercopithecus SA8 * NC_006560 - (Simian agent 8; CeHV-2 SA8) alphaherpesvirus 2 aethiops Note: * strains used for SLAC analysis. Supplementary Table S2. Herpes simplex genes excluded from the branch-sites analysis. Gene Name Motivation UL15 Overlapping ORFs UL16 Overlapping ORFs UL17 Overlapping ORFs UL26.5 Overlapping ORFs US4 Excessive divergence and length among sequences US11 Overlapping ORFs RL1 Duplicated genes/no reliable orthologs RL2 Duplicated genes/no reliable orthologs RS1 Duplicated genes/no reliable orthologs Supplementary Table S3. List of analyzed genes and dN/dS values Gene Symbol Protein Product Name dN/dS UL1 * Envelope glycoprotein L 0.219 UL2 * Uracil-DNA glycosylase 0.106 UL3 Nuclear protein UL3 0.174 UL5 * DNA replication helicase 0.0876 UL4 Nuclear protein UL4 0.182 UL6 * Capsid portal protein 0.144 UL7 * Cytoplasmic envelopment protein 1 0.188 UL9 * DNA replication origin-binding protein 0.129 UL8 * DNA helicase/primase complex-associated protein 0.226 UL10 * Envelope glycoprotein M 0.196 UL12 * Alkaline nuclease 0.206 UL11 * Cytoplasmic envelopment protein 3 0.267 UL14 * Tegument protein UL14 0.242 UL13 Serine/threonine-protein kinase UL13 0.172 UL18 * Triplex capsid protein 2 0.0933 UL20 Envelope protein UL20 0.162 UL19* Major capsid protein 0.0827 UL21 * Tegument protein UL21 0.15 UL22 * Envelope glycoprotein H 0.195 UL23 * Thymidine kinase 0.234 UL24 Nuclear protein UL24 0.153 UL25 * Capsid vertex