Supporting Information

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Fig. 7. A minor subspecies of the HSV-1 KOS_TLR2* activates TLR2. 293T/luc/TLR2/CD14 cells were cultured with 10 ml of live pooled purified HSV-1 KOS-K plaque isolates (A), single purified HSV-1 KOS-K plaque isolates (B and C), or indicated MOIs of clones of purified KOS-K plaque isolates (D) for 7 h, and luciferase activities were measured. Bulk HSV-1 KOS-K and zymosan (10 mg/ml) were included as controls. The data are representative of three independent experiments.

Fig. 8. Rare occurrence of HSV_TLR2* in human clinical isolates. 293T/luc/TLR2/CD14 cells were cultured with the indicated MOI of live HSV-1 clinical isolates or laboratory strains (A, D, and E, purified viruses; B and C, infected Vero cell lysates) or 10 mg/ml of zymosan for 7 h and luciferase activities were measured. (B) Five MOI of Ora G, Ora L, Ora M, Ora P, Vag A, and Vag B or 1 MOI of Ora B, Ora C, Ora D, Ora F, and Ora H were used. (C) Five MOI of Neo A, Neo B, Neo C, Neo D, Gen A, Gen C, Gen D, Ora E, Ora J, Ora O, CNS C, and CNS E or 1 MOI of Gen B, Ora A, Ora I, Ora K, Ora N, CNS B, CNS C, and CNS F were used. (D) Five MOI of purified clinical isolates were used. (E) Ten microliters of the indicated pooled or single plaque-purified CNS C clones were used. These experiments were repeated three times with similar results.

Fig. 9. Schematic of proposed recognition mechanisms of UV-inactivated vs. live HSV_TLR2* by DCs. (A) The results of this study are consistent with the notion that recognition of UV-inactivated HSV_TLR2* in cDCs occurs by a combination of pathways that depend on solely TLR2 (blue arrow) and one that depends on the sequential recognition of virus via TLR2 followed by TLR9 (green arrow). (B) In addition to these pathways, live HSV_TLR2* infection is recognized in a TLR2 (thick blue arrow) and TLR9 (pink arrow) single receptor pathways. The additional TLR2 signal may be a result of the engagement of the cell surface TLR2 by newly synthesized viral glycoproteins (black arrow), whereas additional viral dsDNA may trigger the TLR9-exclusive pathway.

Table 1. The source of HSV-1 laboratory isolates and TLR agonist activity

Table 2. The source of HSV-1 clinical isolates and TLR agonist activity

*Denominations for clinical isolates are as specified by the donating scientists further described in (Malmgaard L, Melchjorsen J, Bowie AG, Mogensen SC, Paludan SR (2004) J Immunol 173:6890-6898).