Figure 1.

LUNA Is Required for Efficient Reactivation in CD34⁺-Derived DCs

(A) HFFs were infected at an MOI of 0.1 with Merlin, LUNA_SHORT, or revertant virus, and the growth was measured over 14 days by titration of supernatants for infectious virus production every 2 days.

(B) Viral gene expression was also assessed by western blotting of mock infected cells (M) or cells infected with LUNA_SHORT (KO), wild-type (WT), or revertant (Rev) virus at 24 and 72 hrs post-infection (hpi).

(C) CD34⁺ cells infected with WT, revertant, or LUNA_SHORT were analyzed using qPCR for viral and cellular DNA levels at 3 dpi and then at 10 dpi after differentiation to an immature CD34⁺-derived DC phenotype.

(D) RNA isolated from WT revertant or LUNA_SHORT infected CD34⁺ cells at 7 dpi were analyzed for UL138 gene expression using qRT-PCR. All samples were normalized to GAPDH and then expressed relative to WT virus.

(E) CD34⁺ cells infected for 3 days to establish latency with WT, revertant, and LUNA_SHORT viruses were differentiated and matured into CD34⁺-derived DCs to induce reactivation and then co-cultured with fibroblasts. At 15 days, co-cultures were harvested and assayed for infectious virus production (IE forming units) on fresh fibroblasts. In (A) and (C)–(E), data are mean ± SD and represent triplicate analyses performed in two independent experiments. ^∗p < 0.05.

(F) Cell surface phenotyping for E-cadherin, CD1a, and CD207/Langerin expression was performed on DCs derived from CD34⁺ cells latently infected with mock, revertant, or LUNA_SHORT virus. Isotype (shaded) and specific antibody (open) staining are shown.

(G) Cell surface phenotyping for CD83 expression was performed on immature (−LPS) and mature (+LPS) DCs derived from CD34⁺ cells latently infected with mock, revertant, or LUNA_SHORT virus. Isotype (shaded) and specific antibody (open) staining are shown.