# Structural and Functional Analysis of Porcine CR1-like Proteins in C4b-Mediated Immune Responses

## TL;DR

This study explores how porcine CR1-like proteins interact with C4b to help the immune system respond to PRRSV, a major swine disease.

## Contribution

The study identifies specific CR1-like fragments and key amino acids involved in C4b binding, offering new insights into porcine immune mechanisms.

## Key findings

- PRRSV triggers C4 cleavage, and C4b facilitates adhesion of porcine red blood cells to the virus.
- Three CR1-like fragments bind C4b, with 18 key amino acids identified through structural simulations.
- The 118I mutation reduces the affinity between CR1-like CCPs 1-3 and C4b, as confirmed by SPR.

## Abstract

The complement system is crucial for innate immunity, but its role against Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a significant swine pathogen, is not well understood. This study investigated the interaction between porcine CR1-like protein and C4b in PRRSV immunity. ELISA showed PRRSV triggers C4 cleavage, and immunoelectron microscopy indicated C4b facilitates porcine red blood cell adhesion to PRRSV. Yeast two-hybrid assays identified three CR1-like fragments binding C4b, structural simulations pinpointed 18 key amino acids, and SPR confirmed that the 118I mutation reduced affinity between CCPs 1-3 and C4b. These findings reveal that porcine CR1-like protein aids immune adhesion to PRRSV through C4b binding, clarify the structural basis, improve understanding of porcine complement defense, and lay the groundwork for PRRSV treatments.

The complement system is crucial for immune defense, linking innate and adaptive immunity. In the classical and lectin pathways, C4 is split into C4b, triggering opsonization, lysis, and the removal of pathogens and damaged cells. Dysregulated activation of C4 and other components of the classical pathway can lead to tissue damage and heightened inflammation, whereas appropriate regulation of C4b activity serves to mitigate excessive inflammation and prevent injury. ELISA analysis demonstrated C4 activation and cleavage during the co-incubation of PRRSV with fresh porcine serum. Immunoelectron microscopy revealed that porcine red blood cells could immunologically adhere to PRRSV, and C4b was involved in this adhesion process. BLAST (NCBI BLAST+ 2.14.1) analysis revealed that porcine CR1-like CCPs 1-3, CR1-like CCPs 12-14, and CR1-like CCPs 19-21 share high similarity with the CCP 1-3 region of human CR1, which mediates C4b binding. Yeast two-hybrid assays confirmed that all three CR1-like fragments bind C4b. To elucidate the interaction mechanism, homology models of C4b and CR1-like fragments were constructed, followed by molecular docking and dynamics simulations, identifying 18 key amino acids in porcine CR1-like involved in C4b binding. Surface plasmon resonance further validated the binding affinity of CR1-like CCPs 1-3, its mutant 118I, and C4b. These results enhance our understanding of complement regulation and provide a foundation for developing therapeutic strategies targeting complement-related diseases.