Interaction of species A rotavirus VP4 with the cellular proteins vimentin and actin related protein 2 discovered by a proximity interactome assay

Rotavirus (RV) is one of the most significant pathogens in humans and animals with diarrhea worldwide. Cell entry is the first step in viral infection, and the outer capsid protein VP4 is crucial for RV attachment and internalization. In order to discover novel candidate host factors involved in RV cell entry, a proximity labeling method was applied to systematically investigate the VP4 and host protein interactions. A total of 174 high-confidence host proteins were identified using proximity labeling. Further analysis showed that 88 proteins were located in the cytoskeleton, plasma membrane, and extracellular region, which could be involved in RV entry. Importantly, vimentin (VIM) and actin-related protein 2 (ACTR2) were identified to promote RV infection at an early step. The results of co-immunoprecipitation assay showed that VIM or ACTR2 physically interacted with VP4. Blocking VIM or ACTR2 function by silencing with small interfering RNA or inhibition by specific antibodies significantly restricted RV infection. Furthermore, increasing the amounts of VIM or ACTR2 by overexpression from transfected recombinant proteins or incubation with recombinant proteins promoted RV infection. Collectively, this study revealed that RV VP4 interacted with host proteins and demonstrated that interaction with VIM and ACTR2 promoted RV replication, providing valuable resources and potential drug targets for better understanding and treating this disease. IMPORTANCE Rotavirus (RV) is an important zoonosis virus, which can cause severe diarrhea and extra-intestinal infection. To date, some proteins or carbohydrates have been shown to participate in the attachment or internalization of RV, including HGBAs, Hsc70, and integrins. This study attempted to indicate whether there were other proteins that would participate in the entry of RV; thus, the RV VP4-interacting proteins were identified by proximity labeling. After analysis and verification, it was found that VIM and ACTR2 could significantly promote the proliferation of RV in intestinal cells. Through further viral binding assays after knockdown, antibody blocking, and recombinant protein overexpression, it was revealed that both VIM and ACTR2 could promote RV replication.