The precursors of class I fusion proteins are synthesized in the ER, where they receive initial N-glycosylation and undergo protein folding and trimerization.  However, the majority of these viral proteins are misfolded,  and the elevation of misfolded proteins not only induces ER stress but also triggers their degradation by different mechanisms.  We reported that these viral glycoproteins are new clients for class I α-mannosidases such as MAN1B1, which select substrates for ER-associated protein degradation (ERAD). We also reported that ER chaperones such as calnexin, calreticulin, and PDIA3 target Ebola virus glycoproteins to reticulophagy via K27-polyubiquitination. Currently, we are investigating the molecular mechanisms of how reticulophagy selectively targets these viral glycoproteins.

Properly folded class I fusion proproteins exit the ER and enter the Golgi to undergo maturation. High-mannose type N-glycans are processed into complex-type and hybrid-type N-glycans, and O-glycans are further added to these proteins. Furin further cleaves these proproteins into the surface receptor-binding and the transmembrane fusion subunits. We reported that after removal of the prodomain, furin is subjected to K33-polyubiquitination by MARCHF2, MARCHF8, and MARCHF9 to egress the TGN, which is counteracted by USP32. Polyubiquitinated furin undergoes exocytosis via exosomes, where it is cleaved by an unknown cellular protease for shedding, producing extracellularly active fuin. Polyubiquitinated furin also migrates to the cell surface, which is phosphorylated by CKII for endocytosis. Phosphorylated furin undergoes retrograde transport from early endosomes to late endosomes, where it binds PACS1 and is retrieved to the TGN by AP1 and Rab9. Furin is also dephosphorylated by PP2A, which directly retrieves furin from early endosomes to the TGN. Retrieved furin is re-targeted by M2, M8, and M9 for the next round of cycling. Alternatively, furin is activated by another unknown cellular protease in the TGN to produce intracellularly active furin that processes proproteins such as class I fusion proteins for maturation.

Serine incorporator 5 (SERINC5) is a multipath transmembrane host restriction factor, which is incorporated into virions from the plasma membrane and inhibits viral entry. This SERINC5 activity is antagonized by different retroviral accessory proteins including HIV-1 Nef, MLV glycoGag, and EIAV S2. We reported that SERINC5 disrupts HIV-1 Env trimer formation to inactivate the Env activity, and these accessory proteins degrade SERINC5 via the endocytic pathway. However, the precise SERINC5 antiviral mechanism and the viral antagonism are still unclear. Currently, we are investigating how SERINC5 travels to the cell surface from the TGN for incorporation. We reported that SERINC5 undergoes K33-polyubiquitination by Cul3/KLHL20 E3 ubiquitin ligase, which is required for its post-Golgi trafficking. We are also investigating how SERINC5 is internalized from the plasma membrane, which excludes SERINC5 from incorporation into virions. We reported that SERINC5 is phosphorylated by CCNK/CDK13, which is required for its endocytic degradation by Nef. We will continue to address these outstanding questions to elucidate the important role of SERINC5 in retroviral infection.

SARS-CoV-2 entry requires not only the cell surface receptor ACE2, but also two cellular proteases TMPRSS2 and CTSL  to further cleave its spike proteins on the cell surface or in late endosomes. However, the precise entry mechanism still needs to be elucidated. Recently, we reported the identification of Tubeimosides as potent Ebola virus entry inhibitors by targeting its receptor NPC1 from screening a natural compound library. Surprisingly, we found that Tubeimosides also inhibit SARS-CoV-2 entry at the same potency, which led us to investigate the role of NPC1 in SARS-CoV-2 entry. After knocking out NPC1 by CRISPR/Cas9, we found that NPC1 is required for SARS-CoV-2 entry and infection in both TMPRSS2(-) and TMPRSS(+) cells. These results demonstrate that the endocytic entry route plays a predominant role in SARS-CoV-2 infection. Currently, we are investigating how SARS-CoV-2 spike proteins interact with NPC1 and how NPC1 triggers the membrane fusion in late endosomes for infection.