CD163ΔSRCR5 MARC-145 Cells Resist PRRSV-2 Infection via Inhibiting Virus Uncoating, Which Requires the Interaction of CD163 With Calpain 1

Abstract

Porcine alveolar macrophages without the CD163 SRCR5 domain are resistant to porcine reproductive and respiratory syndrome virus (PRRSV) infection. However, whether the deletion of CD163 SRCR5 in MARC-145 cells confers resistance to PRRSV and interaction of which of the host proteins with CD163 is involved in virus uncoating remain unclear. Here we deleted the SRCR5 domain of CD163 in MARC-145 cells using CRISPR/Cas9 to generate a CD163 SRCR5 MARC-145 cell line. The modification of CD163 had no impact on CD163 expression. CD163 SRCR5 cells were completely resistant to infection by PRRSV-2 strains Li11, CHR6, TJM, and VR2332. The modified cells showed no cytokine response to PRRSV-2 infection and maintained normal cell vitality comparable with the WT cells. The resistant phenotype of the cells was stably maintained through cell passages. There were no replication transcription complexes in the CD163 SRCR5 cells. SRCR5 deletion did not disturb the colocalization of CD163 and PRRSV-N in early endosomes (EE). However, the interaction of the viral proteins GP2a, GP3, or GP5 with CD163, which is involved in virus uncoating was affected. Furthermore, 77 CD163-binding cellular proteins affected by the SRCR5 deletion were identified by LC-MS/MS. Inhibition of calpain 1 trapped the virions in EE and forced then into late endosomes but did not block viral attachment and internalization, suggesting that calpain 1 is involved in the uncoating. Overall, CD163 SRCR5 MARC-145 cells are fully resistant to PRRSV-2 infection and calpain 1 is identified as a novel host protein that interacts with CD163 to facilitate PRRSV uncoating.

Mutations within scavenger receptor cysteine-rich (SRCR) protein domain 5 of porcine CD163 involved in infection with porcine reproductive and respiratory syndrome virus (PRRS)

“…Different studies demonstrated that the viral envelope proteins GP2a, GP3, GP4 and GP5 specifically interact with CD163 [28,43]. Additionally, one recent report showed that the CD163 SRCR5 domain is required for CD163 to associate with GP2a, GP3, GP5, but not GP4 (28). To assess the effect of PR insertions with a high impact on PRRSV infection on the interaction of CD163 with individual viral glycoproteins, we analysed the biochemical ability of the different CD163 mutant variants containing a FLAG tag to interact with viral glycoproteins containing an HA tag.…”

Structural comparison of CD163 SRCR5 from different species sheds some light on its involvement in porcine reproductive and respiratory syndrome virus-2 infection in vitro

“…CD163 contains nine class B SRCR domains (SRCR1-9) in its large ectodomain [ 34 , 35 ]. SRCR5 has been shown to play a crucial role during PRRSV infection in vitro [ 36 – 38 ]. Additionally, a significantly reduced permissiveness to PRRSV-2, particularly HP-PRRSV, was shown in the PAMs with pCD163 SRCR5 substitution by homologous hCD163L1 SRCR8 [ 31 , 39 ].…”

Recent Advances in PRRS Virus Receptors and the Targeting of Receptor–Ligand for Control

“…Using a CRISPR/Cas9 gene editing strategy, the entire exon 7, which codes for SRCR5, was removed in MARC-145 cells. The modified cells showed complete resistance to PRRSV-2 infection [109]. Interestingly, localization of the virions in the early endosome was visualized at the beginning stage of infection in both wild-type and modified MARC-145 cells, but in the CD163 modified MARC-145 cells, localization of virions was only observed in the late endosome.…”

“…Interestingly, localization of the virions in the early endosome was visualized at the beginning stage of infection in both wild-type and modified MARC-145 cells, but in the CD163 modified MARC-145 cells, localization of virions was only observed in the late endosome. Further examination of the interaction of CD163 and viral proteins shows that SRCR domain 5 deletion from CD163 inhibits the PRRSV uncoating in the early endosome by affecting the interaction of CD163 with GP2a, GP3, and GP5 [109].…”