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...