Construction of a Three-Dimensional Culture Model of HSV-1 Based on the Nano-Self-Assembling Peptide RADA16-I and Preliminary Exploration of the Relationship Between HSV-1 and Autophagy

Herpes simplex virus type 1 (HSV-1) is a neurotropic alphaherpesvirus that interacts dynamically with host cells within structured tissue environments. Conventional two-dimensional (2D) cultures do not fully recapitulate these spatial and microenvironmental features. In this study, we established a three-dimensional (3D) culture system using the self-assembling peptide RADA16-I to generate an extracellular matrix-mimetic hydrogel scaffold. This platform supported the formation of stable Vero cell spheroids that remained viable for more than 30 days. Following HSV-1 infection, viral spread initiated at the spheroid periphery and progressively extended toward the core. Sustained viral replication was detected for up to 22 days, indicating long-term maintenance of infection within the 3D structure. Ultrastructural examination identified viral particles and vesicular compartments consistent with autophagy-related organelles. Comparative analysis of autophagy-associated markers revealed distinct temporal patterns between 2D monolayer cultures and 3D spheroids. In the 3D system, LC3B-II levels progressively increased, accompanied by a reduction in p62, suggesting altered regulation of autophagic flux relative to conventional 2D conditions. These findings demonstrate that the RADA16-I-based 3D culture model supports prolonged HSV-1 infection and reproduces key spatial features of viral dissemination. The differential autophagic responses observed between 2D and 3D systems highlight the influence of cellular architecture on host-virus interactions and support the application of 3D culture platforms for mechanistic studies of HSV-1 pathogenesis.