ARF1 with Sec7 Domain-Dependent GBF1 Activates Coatomer Protein I To Support Classical Swine Fever Virus Entry

Abstract

Classical swine fever virus (CSFV), a positive-sense, enveloped RNA virus that belongs to the Flaviviridae family, hijacks cell host proteins for its own replication. We previously demonstrated that Golgi-specific brefeldin A-resistance factor 1 (GBF1), a regulator of intracellular transport, mediates CSFV infection. However, the molecular mechanism by which this protein regulates CSFV proliferation remains unelucidated. In this study, we constructed a series of plasmids expressing GBF1 truncation mutants to investigate their behavior during CSFV infection and found that GBF1 truncation mutants containing the Sec7 domain could rescue CSFV replication in BFA (brefeldin A)- and GCA (Golgicide A)-treated swine umbilical vein endothelial cells (SUVECs), demonstrating that the effect of GBF1 on CSFV infection depended on the activity of guanine nucleotide exchange factor (GEF). Additionally, it was found that ADP ribosylation factors (ARFs), which are known to be activated by the Sec7 domain of GBF1, also regulated CSFV proliferation. Furthermore, we demonstrated that ARF1 is more important for CSFV infection than other ARF members with Sec7 domain dependence. Subsequent experiments established the function of coatomer protein I (COP I), a downstream effector of ARF1, which is also required for CSFV infection by mediating CSFV invasion. Mechanistically, inhibition of COP I function impaired CSFV invasion by inhibiting cholesterol transport to the plasma membrane, and regulating virion transport from early to late endosomes. Collectively, our results suggest that ARF1, with domain-dependent GBF1 Sec7, activates COP I to facilitate CSFV entry into SUVECs. Importance Classical swine fever (CSF), a highly contact infectious disease, caused by the classical swine fever virus (CSFV) infecting domestic pigs or wild boars, has caused huge economic losses to the pig industry. Our previous studies have revealed that GBF1 and class I and II ARFs are required for CSFV proliferation. However, a direct functional link between GBF1, ARF1, and COP I, and the mechanism of the GBF1-ARF1-COP I complex in CSFV infection is still poorly understood. Here, our data support a model in which COP I supports CSFV entry into SUVECs in two different ways, depending on the GBF1-ARF1 function. On the one hand, the GBF1-ARF1-COP I complex mediates cholesterol trafficking to the plasma membrane to support CSFV entry. On the other hand, the GBF1-ARF1-COP I complex mediates CSFV transport from early to late endosomes during the entry steps.

Attachment, Entry, and Intracellular Trafficking of Classical Swine Fever Virus

“…COP I and ARF1 form a complex with Golgi-specific brefeldin A resistance factor 1 (GBF1) [ 152 ]. Zhang et al first demonstrated that the GBF1–ARF1–COP I axis mediates CSFV transportation from early to late endosomes, promoting viral entry into swine umbilical vein endothelial cells (SUVECs) [ 153 ]. GBF1 was previously validated as being important in CSFV replication in SUVECs [ 154 ].…”

“…Meanwhile, CSFV infection promoted GBF1, ARF, and COP I mRNA expression. These results indicate that CSFV infection induces the expression of the GBF1–ARF–COP I complex and mediates the distribution of COP I-coated vesicles [ 153 ].…”

Host protein ARF1 is a proviral factor for SARS-CoV-2 and a candidate broad-spectrum therapeutic target

“…ARF1 can recruit soluble cytosolic effectors to the membranes, such as coat protein complex I coatomer, clathrin cargo adaptors, and lipid metabolism enzymes, to regulate the vesicular assembly or remodel the membranes [63][64][65][66] . Previous work has shown that ARF1 might be involved in multiple virus infections with unknown or several proposed mechanisms [31][32][33][34][35][67][68][69][70][71] , while whether and how ARF1 has a role in SARS-CoV-2 infection were unknown before our study. Particularly, Faure et al have reported that the HIV-I Nef protein can interact with both cluster of differentiation 4 (CD4) and ARF1, and hijack ARF1 onto the endosomal membranes to downregulate CD4 by accelerated endocytosis and lysosomal degradation 31 .…”

Cholesterol Biosynthesis Modulates CSFV Replication

“…Studies have shown that cellular cholesterol is essential for CSFV infection, and cholesterol depletion significantly blocks virus internalization and thus inhibits CSFV infection at the initial stage [ 39 ]. Moreover, inhibiting coatomer protein I (COP I) function and Niemann-pick C1 (NPC1) expression, which impairs cholesterol transport and leads to cholesterol and virion accumulation in early endosomes, thus suppresses CSFV invasion and replication [ 40 , 41 ]. To our knowledge, our study is the first detailed report on alterations to intracellular cholesterol in PK-15 cells during CSFV infection.…”