During the latent phase, Kaposi's sarcoma-associated herpes virus (KSHV) maintains itself inside the host by escaping the host immune surveillance mechanism through restricted protein expression. Latency-associated nuclear antigen (LANA), the most abundantly expressed protein, is essential for viral persistence, as it plays important roles in latent viral DNA replication and efficient segregation of the viral genome to the daughter cells following cell division. KSHV evades immune detection by maintaining the levels of LANA protein below a threshold required for detection by the host immune system but sufficient to maintain the viral genome. LANA achieves this by controlling its expression through regulation of its promoters and by inhibiting its presentation through interaction with the proteins of class I and class II major histocompatibility complex (MHC) pathways. In this study, we identified a mechanism of LANA expression and restricted immune recognition through formation of G-quadruplexes in LANA mRNA. We show that the formation of these stable structures in LANA mRNA inhibits its translation to control antigen presentation, which was supported by treatment of cells with TMPyP4, a G-quadruplex-stabilizing ligand. We identified heterogenous ribonucleoprotein A1 (hnRNP A1) as a G-quadruplex-unwinding helicase, which unfolds these stable secondary structures to regulate LANA translation.IMPORTANCE LANA, the most abundantly expressed protein during latency, is a multifunctional protein which is absolutely required for the persistence of KSHV in the host cell. Even though the functions of LANA in aiding pathogenesis of the virus have been extensively studied, the mechanism of how LANA escapes host's immune surveillance is not fully understood. This study sheds light on the autoregulatory role of LANA to modulate its expression and immune evasion through formation of G-quadruplexes in its mRNA. We used G-quadruplex-stabilizing ligand to define the inhibition in LANA expression and presentation on the cell surface through MHC class I. We defined the autoregulatory role of LANA and identified a cellular RNA helicase, hnRNP A1, regulating the translation of LANA mRNA. This interaction of hnRNP A1 with LANA mRNA could be exploited for controlling KSHV latency.,Copyright © 2020 American Society for Microbiology.