ABSTRACT: Marek’s disease virus (MDV) establishes latency in CD4⁺ T cells and can reactivate to initiate productive replication and viral dissemination.To define host and viral mechanisms governing the transition from latency to lytic replication, we developed recombinant MDV strains expressing RLORF4-mRFP and UL47-eGFP reporters. These reporters were used to isolate latent (Lo; mRFP⁻), early lytic (Hi; mRFP⁺), and late lytic (DP; mRFP⁺eGFP⁺) populations from MDV-induced lymphoblastoid cell lines (LCLs) following temperature-induced reactivation. Fluorescence-activated cell sorting-purified populations from two independent LCLs (Lines 82 and 62) were subjected to Illumina RNA sequencing. Viral transcription increased progressively from Lo to Hi to DP populations; however, initiation of viral gene expression differed markedly between cell lines. During the early transition (Lo to Hi), Line 82 exhibited robust induction of 17 viral genes, including US3, UL16, UL51, and UL34, whereas Line 62 showed only two differentially expressed viral genes. In contrast, both lines converged on a highly conserved late-lytic transcriptional program during Lo-to-DP transitions, sharing 118 viral transcripts, dominated by structural and assembly-associated genes. Among the host transcriptional responses, the Line 82 exhibited activation of TP53-associated stress signaling, chromatin remodeling factors, RNA biogenesis pathways, consistent with a permissive cellular state. In contrast, Line 62 displayed enhanced inflammatory, metabolic and proteostasis-associated signatures, suggesting a restrictive environment that limits early viral induction. Integrated pathway analysis revealed coordinated remodeling of cytoskeletal trafficking, RNA processing, proteostasis, and lipid metabolism during reactivation, while TP53, MTOR, XBP1, NFKB1/RELA, EIF2AK2, DDX58, and STUB1 emerged as candidate host determinants of lytic permissiveness. Collectively, these findings demonstrate that MDV reactivation is a host-gated process in which cellular state governs initiation of the lytic switch, whereas the downstream reproductive replication proceeds through a conserved viral program.
