# 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

## TL;DR

Researchers created a 3D cell culture model to study HSV-1 infection and found that it better mimics real tissue interactions and autophagy responses compared to traditional 2D methods.

## Contribution

A novel 3D culture model using RADA16-I peptide supports long-term HSV-1 infection and reveals distinct autophagy dynamics.

## Key findings

- HSV-1 spreads progressively in 3D spheroids with sustained replication over 22 days.
- 3D cultures show altered autophagy regulation with increased LC3B-II and reduced p62 compared to 2D cultures.

## Abstract

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.