Figure 1. Mast cells support KSHV infection in vitro and in patient lesions.

(A-D) HMC-1 and LAD2 cells were infected with KSHV and cultured for indicated times. (A) Mast cells express abundant KSHV genes. GAPDH was used as a loading control. BCBL-1 cells induced with valproic acid were used as a positive control for viral lytic gene expression. NTC- no template control. Data are representative of two separate experiments. (B) QRT-PCR gene expression indicated MCs express LANA in growing cultures over the entire culture period. Data are expressed as CT values normalized to GAPDH. Data are representative of three independent experiments performed in triplicate. (C-D) Limiting dilution qPCR analysis of infected (C) HMC-1 and (D) LAD2 demonstrated approximately 1 in 7 MCs were KSHV genome⁺ at 24 hpi.. Data are expressed as mean ±SEM, n=3. (E-G) HIV/KS tissue double stained for KSHV LANA (nucleus-brown) and MC-specific tryptase (cytoplasm-red) demonstrate: (E) LN shows (left upper inset) two tryptase⁺ MCs, one LANA⁻ (blue nucleus, arrow head) and the other LANA⁺ (brown nucleus, black arrow), (right lower inset) tryptase+ LANA⁺ MC (brown nucleus, black arrow) paired with a LANA+ plasma cell (brown nucleus); (F) dermal lesion shows tryptase+ LANA⁺ MCs (black arrows) and tryptase+ LANA⁻ MCs (arrow heads); (G) HIV-KS LN double stained for MC-specific tryptase (cytoplasm-red) and KSHV lytic antigen K8.1a (cytoplasm-brown) shows (left upper inset) a tryptase⁺ K8.1a⁺ MC paired with a KSHV K8.1a⁺ stained plasma cell cytoplasm and (right lower inset) a solo tryptase⁺ K8.1⁺ MC cytoplasm (brown). Scale 25 μm (inset scale, 10 μm). (H-I) HMC-1 cells were infected as described in the methods. (H) KSHV-infected MCs produce encapsulated, DNAse resistant, virus during infection. Data are from two independent experiments with three experimental replicates and two technical replicates each and expressed as mean ± SEM. Mock infected cultures gave no CTs. (I) Mast cell-derived-KSHV establishes latency in primary human endothelial cells. Cell-free supernatants were isolated from MCs uninfected or infected with KSHV at 24h p.i. and used to treat primary human ECs. 48 h post-treatment, primary ECs were fixed and stained as indicated in the Methods; LANA positive nuclei-localized staining demonstrated establishment of latency. Dapi was used to visualize nuclei. Magnification x630. Data are representative of 3 independent experiments.