Molecular Mechanism Of The Interaction Between CD163 N-Terminal Domain And MYH9 C-Terminal Region In Porcine Reproductive And Respiratory Syndrome Virus Infection

Porcine reproductive and respiratory syndrome virus(PRRSV)has a highly restricted tropism for cells of the monocyte-macrophage lineage,including porcine alveolar macrophages(PAMs).PRRSV entry into permissive cells involves several mediators in addition to two required host cell receptors,CD163 and MYH9.It is unknown whether CD163 directly interacts and/or cooperates with MYH9 to facilitate PRRSV infection.In this study,we explored the interaction between CD163 and MYH9,and its role in PRRSV entry into permissive cells.The main results we got including:1.CD163 and MYH9 interacted with each other in PAMs,and this interaction was regardless of PRRSV infection status.2.CD163 N-terminal domain SRCR1-4 interacted with MYH9 C-terminal region PRA.3.The ELISA and Far-Western Blot results revealed that SRCR1-4 interacted with PRA directly without involvement of other adaptor proteins.4.Non-permissive HEK293 T cells that stably expressed CD163,truncated swine CD163 SRCR1-4 domain,and SRCR5-CT domain were generated by lentiviral transduction.The virus infection assay demonstrated that the interaction between CD163 and MYH9 was contributed to PRRSV internalization in permissive cell lines.5.The exogenous SRCR1-4 proteins incubated with PAMs could make it interacted with membrane-associated MYH9,and this recombinant SRCR1-4 proteins could decrease the PRRSV internalization in PAMs,thus lead to blocked virus infection.6.We also investigated that 111-210 aa in PRA proteins(PRA100)were responsible for its interaction with SRCR1-4.Furthermore,293 s cell lines expressing PRA100 proteins were generated successfully,and 293 s cell lines expressing the extracellular domain of CD163--SRCR1-PSTII proteins were also conducted.The generation of the two eukaryotic expression system would provide an important tool for us to study the structure biology of the interaction complexes.In summary,these results demonstrated that CD163 SRCR1-4 interacts with the MYH9 C–terminal domain to facilitate PRRSV virion internalization in permissive cells,thus expanding our understanding of PRRSV cell-invasion mechanisms.