KSHV infection in MSCs correlates with the amount of viral input and persists for several weeks. (A) Coculture of lytically induced BCBL-1 cells with MSCs resulted in ratio-dependent expression of LANA, with maximal LANA reactivity of approximately 34%, 48 hours after infection at a BCBL-1/MSC ratio of 9:1. Error bars represent the SD of 4 individual fields of at least 100 cells. Higher BCBL-1/MSC ratios in otherwise identical experiments resulted in even higher infection rates (not shown). (B) Incubation of cell-free KSHV with MSCs resulted in concentration-dependent rates of LANA expression, with maximal infection (> 96%) 48 hours after exposure of the culture to approximately 9300 KSHV genome equivalents/cell. Error bars as per panel A. In parallel experiments, identical aliquots of input KSHV (and virus-to-cell ratios) achieved similar rates of LANA expression within T4 TIME cells (96% LANA reactivity), although LANA expression decreased more rapidly over time in these cells compared to MSCs (40% reactivity for MSC at 3 weeks versus 15% for TIME cells, data for TIME cells discussed in Tomescu et al¹⁴ ). (C) Time course following LANA IFA reactivity of MSCs infected with cell-free KSHV (with a genome equivalents to target cell ratio of 5900:1). Data reflect LANA reactivity (mean ± range) in 2 settings: (1) infection of MSCs from a single donor in 2 separate experiments using KSHV isolated from separate BCBL-1 stocks (dotted line); and (2) infection of MSCs isolated from 3 separate donors and infected with KSHV from the same viral stock (solid line). Note: after a 7- to 9-day lag in the growth of the MSC culture following infection, expansion of the population resumed with a constant doubling time of approximately 30 hours through week 6 (not shown).