Chicken Organic Anion-Transporting Polypeptide 1A2, a Novel Avian Hepatitis E Virus (HEV) ORF2-Interacting Protein, Is Involved in Avian HEV Infection

Abstract

Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease in chickens. Due to the absence of a highly effective cell culture system, there are few reports about the interaction between avian HEV and host cells. In this study, organic anion-transporting polypeptide 1A2 (OATP1A2) from chicken liver cells was identified to interact with ap237, a truncated avian HEV capsid protein spanning amino acids 313 to 549, by a glutathione S-transferase (GST) pulldown assay. GST pulldown and indirect enzyme-linked immunosorbent assays (ELISAs) further confirmed that the extracellular domain of OATP1A2 directly binds with ap237. The expression levels of OATP1A2 in host cells are positively correlated with the amounts of ap237 attachment and virus infection. The distribution of OATP1A2 in different tissues is consistent with avian HEV infection in vivo. Finally, when the functions of OATP1A2 in cells are inhibited by its substrates or an inhibitor or blocked by ap237 or anti-OATP1A2 sera, attachment to and infection of host cells by avian HEV are significantly reduced. Collectively, these results displayed for the first time that OATP1A2 interacts with the avian HEV capsid protein and can influence viral infection in host cells. The present study provides new insight to understand the process of avian HEV infection of host cells. IMPORTANCE The process of viral infection is centered around the interaction between the virus and host cells. Due to the lack of a highly effective cell culture system in vitro, there is little understanding about the interaction between avian HEV and its host cells. In this study, a total of seven host proteins were screened in chicken liver cells by a truncated avian HEV capsid protein (ap237) in which the host protein OATP1A2 interacted with ap237. Overexpression of OATP1A2 in the cells can promote ap237 adsorption as well as avian HEV adsorption and infection of the cells. When the function of OATP1A2 in cells was inhibited by substrates or inhibitors, attachment and infection by avian HEV significantly decreased. The distribution of OATP1A2 in different chicken tissues corresponded with that in tissues during avian HEV infection. This is the first finding that OATP1A2 is involved in viral infection of host cells.

The Hepatitis E Virus Open Reading Frame 2 Protein: Beyond Viral Capsid

“…Early report employing HEV-VLPs suggested that attachment of HEV-VLP to cells surface depends on heparin sulfate proteoglycans (HSPGs) and pre-incubation of HEV with heparan sulfate blocks HEV infectivity (Kalia et al, 2009;Yin et al, 2016), whereas sequential research identified asialoglycoprotein receptor (ASGPR) and retinol-binding protein 4 (RBP4) as putative HEV capsids interacting receptors (Shen et al, 2011;Zhang et al, 2016). Meanwhile, a recent study based on avian HEV suggested that organic anion-transporting polypeptide 1A2 (OATP1A2) may be an avian HEV-specific receptor (Li et al, 2019), but whether mammalian counterpart of OATP1A2 acts as potential receptor for mammalian HEV requires further investigation.…”

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“…Due to the lack of highly effective culture systems in vitro, the life cycle of HEV remains unclear. Using HEV-like particles (HEV-LPs), several host factors have been determined to participate in naked HEV infection, including cell attachment, entry, and/or trafficking (Kamar et al, 2009;Yu et al, 2011;Kapur et al, 2012;Holla et al, 2015;Ahmed et al, 2016;Zhang et al, 2016;Li et al, 2019;Shiota et al, 2019). For example, during clathrinand dynamin-dependent endocytosis, membrane cholesterol, the PI3K pathway, and actin have been shown to participate in cell entry and membrane trafficking of naked HEV particles (Kapur et al, 2012;Holla et al, 2015).…”

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“…The complete HEV genome consists of three open reading frames (ORF): ORF1, ORF2, and ORF3. The ORF2-encoded viral capsid protein generally includes 660 amino acids (aa), although in avian HEV it is only 606 aa in length (Zhao et al, 2015;Fu et al, 2019), it plays a crucial role during viral infection by interacting with host factors (Kamar et al, 2009;Shukla et al, 2011;Kapur et al, 2012;Ahmed et al, 2016;Yin et al, 2016Yin et al, , 2017Li et al, 2019;Sayed et al, 2020). Due to the lack of a highly efficient in vitro cell culture system, two truncated HEV capsid proteins spanning 112-606 aa (Kamar et al, 2009) and 368-606 aa (designated p239) were universally used to screen the host proteins interacting with HEV based on both truncated capsid proteins mimic non-enveloped HEV particles (Yamashita et al, 2009).…”

“…Previously, a truncated avian HEV capsid protein spanning aa 313-549 (designated ap237) with sequence homology to HEV p239 served as a bait protein to screen for host interaction partners of avian HEV capsid protein (Li et al, 2019). Among screened host factors, organic anion transporting polypeptide 1A2 (OATP1A2) was shown to enhance avian HEV infection of host cells, prompting the creation of a cell line (designated LMH OATP1A2 ) that expressed OTAP1A2 (Li et al, 2019). Meanwhile, another host factor, a small Rho family GTPase, known as cell control protein 42 (CDC42), was also pulled down by ap237 (Li et al, 2019).…”

“…Among screened host factors, organic anion transporting polypeptide 1A2 (OATP1A2) was shown to enhance avian HEV infection of host cells, prompting the creation of a cell line (designated LMH OATP1A2 ) that expressed OTAP1A2 (Li et al, 2019). Meanwhile, another host factor, a small Rho family GTPase, known as cell control protein 42 (CDC42), was also pulled down by ap237 (Li et al, 2019). CDC42 is known to moderate cellular actin dynamics and is the primary upstream triggering molecule of CDC42 signaling pathways (Doherty and McMahon, 2009;Swaine and Dittmar, 2015;Antkowiak et al, 2016).…”