Comprehensive Analysis of Herpes Simplex Virus 1 (HSV-1) Entry Mediated by Zebrafish 3- O -Sulfotransferase Isoforms: Implications for the Development of a Zebrafish Model of HSV-1 Infection

Abstract

Here, we performed a comprehensive analysis of the role of ZF 3-OST isoforms (3-OST-1, 3-OST-5, 3-OST-6, and 3-OST-7) in HSV-1 entry. We found that a group of 3-OST gene family isoforms (3-OST-2, -3, -4, and -6) with conserved catalytic and substrate-binding residues of the enzyme mediates HSV-1 entry and spread, while the other group (3-OST-1, -5, and -7) lacks these properties. These results demonstrate that HSV-1 entry can be recapitulated by certain ZF 3-OST enzymes, a significant step toward the establishment of a ZF model of HSV-1 infection and tissue-specific tropism. Herpes simplex virus 1 (HSV-1) entry into the host cells requires interactions between viral envelope glycoprotein D (gD) and host cell receptors (1). An important gD receptor is 3-O-sulfated heparan sulfate (3-OS HS) (2, 3). Synthesis and modifications of HS is a multistep process (4-8), of which the last step involves O-sulfation at different positions of the molecule, catalyzed by various sulfotransferases (4). 3-O-Sulfotransferases (3-OST-1, -2, -3A, -3B, -4, -5, and -6 isoforms; also called Hs3st1, -2, -3A, -3B, -4, -5, and -6, respectively) catalyze sulfation at the C-3 of the glucosamine units of HS, generating specific proteinbinding sites for HSV-1 gD (2, 3, 9, 10).Recent studies of zebrafish (ZF) provided a functional analysis of 3-OST-generated HS (11-15). Out of the characterized eight 3-OST family members in ZF, seven genes show homology to known 3-OST genes in mice and humans (11). These ZF enzyme isoforms are differentially expressed in a tissue-specific manner, suggesting the possibility of creating a tool to study HSV tissuespecific tropism, whose study is hampered by the lack of available convenient tools. Our previous preliminary studies suggested that ZF 3-OST may mediate HSV entry (15). In this study, we investigated the ability of ZF 3-OST-1, -5, -6, and -7 isoforms, comparatively to their human analogue, to facilitate HSV-1 entry and cell-cell fusion. Our previous studies have shown that ZF-encoded 3-OST isoforms (3-OST-2, -3, and -4) allow HSV-1 entry into host cells (14,16,17). Here, we provide a comprehensive analysis of the differential abilities of the remaining ZF 3-OST isoforms to mediate HSV-1 entry and propose a unique model for future studies of HSV-1 tropism using ZF as a convenient tool.Analysis and cloning of zebrafish-encoded 3-OST enzymes. To characterize ZF 3-OST isoforms relative to their human counterparts, we first performed an amino acid sequence alignment of the ZF-encoded 3-OST enzymes to the previously characterized human 3-OST-3A and 3-OST-3B isoforms (2, 11). Interestingly, we found that the amino acid sequences of the ZF 3-OST enzymes show various degrees of homology to the human 3-OST-3 isoforms (Fig. 1). Comparing the conservation of the catalytic residues of the enzymes characterized for human 3-OST-3, we found that ZF enzymes 3-OST-2, -3, -4, and -6 show 100% conservation of the catalytic residues; these were designated group I members ( Fig. 1A and B). The other isoforms (3-OST-1, -5, and...

Diversity of Heparan Sulfate and HSV Entry: Basic Understanding and Treatment Strategies

“…More direct and visual evidence for HSV-1 entry was demonstrated by using green fluorescent protein (GFP)-tagged HSV-1 (K26GFP) virions infecting zebrafish 3- O ST-3 expressing CHO-K1 cells [ 88 ]. In recent years our group has cloned and characterized all the 3- O ST isoforms expressed in zebrafish embryos [ 49 , 50 , 96 , 97 ]. Interestingly 3- O ST enzymes are uniquely expressed in different cells and tissues during zebrafish embryonic development [ 91 ].…”

“…Interestingly 3- O ST enzymes are uniquely expressed in different cells and tissues during zebrafish embryonic development [ 91 ]. To date our results with zebrafish clones to study HSV infection are very encouraging as they complement human 3- O ST isoform in terms of supporting HSV entry and spread [ 50 ]. The result from the study promotes the usage of zebrafish as a new model to address the role of 3- O ST generated HS in viral tropism, tissue specific damages in cornea and central nervous system (CNS) and associated inflammation and also for 3- O S HS structure-function studies for other systems.…”

A DN-mda5 transgenic zebrafish model demonstrates that Mda5 plays an important role in snakehead rhabdovirus resistance

“…The zebrafish ( Danio rerio ) model has provided novel insights into pathogenesis (Allen and Neely, 2010; Briolat et al, 2014; Gabor et al, 2014; Ludwig et al, 2011; Meijer and Spaink, 2011; Phelan et al, 2005b; Ramakrishnan; Sullivan and Kim, 2008; Tobin and Ramakrishnan, 2008; Yakoub et al, 2014; Zou et al, 2014b). Recently, a zebrafish MDA5 homolog and splice variant were described as transcriptionally induced upon virus infection and capable of conferring viral resistance when overexpressed in vitro (Zou et al, 2014b).…”

Cell entry mechanisms of HSV: what we have learned in recent years

“…It is known to mediate HSV-1 entry into primary cultures of corneal fibroblasts [43], which lack nectin-1 expression and demonstrate very low levels of HVEM on the cell surface. Recent studies have shown that zebrafish expresses multiple 3-OST isoforms, and many of those generate HSV-1 entry receptors [44]. To add further credence to its role as an HSV-1 receptor, a soluble form of 3-OS HS is known to trigger entry in CHO cells, an infection-resistant cell line [45].…”