Exploring the role of heat shock proteins in foot-and-mouth disease virus infection

Foot-and-mouth disease virus (FMDV) infection poses significant challenges to livestock health and induces economic loss. Because of its high mutation rate and temperature sensitivity, maintaining vaccine stability and developing effective antiviral strategies are crucial for disease control. This thesis explores how heat shock proteins (HSPs) influence FMDV infection, aiming to provide new insights into viral adaptation and host defense responses. FMDV is more temperature-sensitive than other Picornaviridae members, making vaccine stability a major concern for disease control. HSP60 plays an essential role in protein folding and is involved in FMDV replication. Therefore, our first study investigates how FMDV adapts to HSP60 inhibition to enhance its thermal stability, which is critical for improving vaccine performance. While HSP60 promotes viral stability, HSPA1 has an antiviral function in FMDV infection. Previous studies have shown that HSPA1 is important in the replication cycles of other Picornaviridae members. However, the function of HSPA1 in FMDV infection remains unclear. The second study examines the role of HSPA1 in restricting FMDV replication.
To investigate FMDV adaptation under HSP60 inhibition, we selected resistant strains using an HSP60 inhibitor. Sequencing and reverse genetics identified a key mutation, T171P in the VP1 protein, which enhanced viral stability. Structural analysis revealed that the T171P mutation improved VP1 integrity, increasing the resistance of the virus to heat stress. Thermostability assays, including particle stability (PaSTry assay) and heat treatment evaluations, confirmed that the T171P-VP1 strain exhibited significantly higher heat resistance compared to the wild-type (WT) strain. Furthermore, animal experiments showed that the T171P mutation improved immunogenicity after heat treatment, suggesting it could help enhancing vaccine stability and effectiveness. These findings provide insights into viral evolution under host-induced stress and may contribute to the development of more stable inactivated FMD vaccines.
In the second part, we demonstrated that HSPA1 inhibits FMDV replication by selectively degrading the viral RNA-dependent RNA polymerase (RdRp, 3D protein) through the chaperone-mediated autophagy (CMA) pathway. The pathway activation and inhibition experiments confirmed that this degradation occurs specifically through CMA. Further mutation analysis identified the 421QEKLI425 motif in 3D as the key motif responsible for HSPA1-mediated degradation. Suppression of HSPA1 led to increased viral replication, while its activation restricted FMDV RNA synthesis. These findings suggest that HSPA1 plays a crucial role in host antiviral defense and may serve as a potential target for antiviral intervention.
In conclusion, this thesis identifies two distinct yet interconnected aspects of how HSPs influence FMDV infection. First, FMDV adapts to HSP60 inhibition through the T171P mutation in VP1, enhancing viral thermal stability and offering insights for vaccine optimization. Second, HSPA1 restricts FMDV replication by degrading the viral 3D polymerase via CMA, revealing a host-driven antiviral mechanism. These findings provide a deeper understanding of host-virus interactions and may help to develop new strategies for FMDV prevention and control.