Modification also permits viral membrane fusion and penetration (Shukla et al., 1999; Xia et al., 2002; Chen et al., 2008). Unique sites within HS chains generated by 3-OSTs produce 3-OS HS, the newest family of HSV-1 glycoprotein D (gD) receptors, (Liu et al., 2002; Borst et al., 2013). The 3-OSTs act to modify HS further along in its biosynthesis, and each isoform recognizes glucosamine residues, as substrates, within regions of the HS chain having specific and unique, prior modifications, including epimerization and sulfation at other positions (Liu et al., 1999; Shworak et al., 1999). Thus, each 3-OST is capable of producing potentially unique protein-binding sites within HS. To date, seven different isoforms of human 3-OSTs (3-OST-1, 3-OST-2, 3-OST-3 A, 3- OST-3 B, 3-OST-4, 3-OST-5, 3-OST-6, and 3OST-7) are known. Although all the isoforms modify HS to generate 3-OS HS, it remains a mystery as to why seven isoforms are present. All, forms of 3-OST except 3-OST-1, produce HSV-1 entry receptors (Liu et al., 1999; Shukla et al., 1999). Interestingly, zebrafish embryo encodes 3-OST isoforms that are functionally similar in terms of recognizing HSV-1 glycoprotein D (gD) to promote viral entry. The gD receptors generated by other isoforms of 3-OST are very similar in structure, but likely not identical (Liu et al., 1999; Shukla et al., 1999; Chen et al., 2008; Borst et al., 2013). The isoform 3-OST-1 creates binding sites for antithrombin (Shworak et al., 1999; Mochizuki et al., 2003) but are unable to produce receptors for HSV-1 gD (Borst et al., 2013). Additionally, one or more isoforms of 3-OSTs have been found to be expressed in both human and mouse tissues that are relevant to HSV-1 infection examined thus far (Liu et al., 1999; Mochizuki et al., 2003; Lawrence et al., 2007) suggesting the significant role 3-OST play in contributing to disease development. The general distinction between plain-type of HS and the HS chain containing 3-O sulfation regarding their usage in viral entry is well-documented. Although the vast jungle of HS in the host cell assists in the capture and concentration of pathogens at a given site, it may not guarantee successful infection since HS mainly serves as an attachment receptor. Overall interactions between HS and pathogens are considered to be non-specific and may influence pathogenesis indirectly. On the other hand, target cells expressing 3-O or 6-O sulfated moieties in HS chains may guarantee that certain pathogens will be able to cross the host cell membrane and therefore the presence of the later form of specialized-HS may serve as a “Wi-Fi” zone for the targeting pathogens to easily invade the host cell. That is, pathogens in the vicinity of cells expressing modified HS can connect to “the internet” (host organism) by using modified HS as a means and signal to literally connect to host cells. To further the analogy, pathogens may even be able to detect modified HSPGs similar to how one can scan for Wi-Fi signals with a computer or a cellphone. Nonetheless, it remains to be investigated whether such “Wi-Fi” zones can be distinctly equipped with a particular signature of the 3-O-sulfated isoform that influences or contributes to establishing HSV tropism. Moreover, a HSV-1 gD-type 3-O HS specific to neurons or to ocular cells are well-documented (Clement et al., 2006; Deligny et al., 2010).

A series of evidence support the role of HS in viral infection as indicated in Figure 3. For example, enzymatic removal of specific regions in the HS chain in target cells by using heparanase significantly impairs the viral infection (Tiwari et al., 2004). Likewise, use of soluble heparin or HS-mimetic analogs inhibits viral infection through direct competition for cell surface HS (Gangji et al., 2018). Furthermore, a higher degree of viral entry was noticed in the cells that expressed HS compared to the mutant cells that were devoid of or weakly expressed HS. Lastly, the expression of viral envelop proteins which interact with HS in target cells, resulted in a resistance to viral entry by sequestering cell surface HS (Tiwari et al., 2006). As in the case of HSV-1, the expression of viral envelop glycoprotein B (gB) in target cells, which is known to bind HS, significantly impairs viral entry- a process also known as gB-mediated interference. Profoundly, our initial work made a unique discovery that viruses do in fact utilize HS beyond cell attachment and binding. Using cDNA library screening, direct evidence was presented for a modified version of HS; 3-OS HS generated by 3-OST-3 isoform as a receptor which promotes virus-cell membrane fusion (Shukla et al., 1999). Expression of the HS-modifying 3-OST-3 enzymes confers susceptibility for HSV-1 infection in previously HSV-1 resistant Chinese hamster ovary (CHO-K1) cells (Shukla et al., 1999). Additionally, an event with the protein receptors (nectin and HVEM) was observed that supports HSV-1 entry (Shukla and Spear, 2001). After identifying the significance of 3-OST-3 in HSV-1 entry, our group has cloned an additional six individual human and zebrafish isoforms of 3-OST enzymes and have characterized them against HSV-1 entry. Given the fact, that these enzymes are tightly regulated and are expressed in specific cells and tissues, it is tempting to speculate that 3-OST isoforms might be prime candidates for HSV localized infection either in brain or ocular cells and tissues, essentially contributing toward viral tropism. In this regard, our previous work has demonstrated the significance of one 3-OST-3 isoform type in primary cultures of human corneal stroma (CF) derived from eye donors- a natural target cell for HSV infection. Using si-RNA approaches together with the use of heparanase enzyme in cultured CF, we found a significant reduction in HSV-1 entry (Tiwari et al., 2006). Additional support for HSV use of the 3-OS HS receptor in CF was gained by using phage display-derived peptide targeting 3-OS HS receptor, which further resulted in a compelling inhibition in HSV-1 entry (Tiwari et al., 2011). To further strengthen our work, we used anti-3-OS HS peptides to successfully prevent HSV-1 infection in a mouse corneal model (Tiwari et al., 2011). With the above encouraging results, we have tested the clinical usage of anti-3-OS HS peptides by using them on a commercially available contact lens as a delivery vehicle for extended release as a potential and effective way to control corneal herpes infection (Jaishankar et al., 2016). Interestingly, a laboratory produced 3-O-sulfonated HS octasaccharide, was demonstrated to inhibit HSV-1 and host-cell interactions, suggesting the application of HS derived molecules as potential therapeutic tools against viral pathogens.

In the case of 3-OS HS, the ligands that are documented to interact are HSV-1 glycoprotein D (gD), antithrombin III, Fibroblast growth factor (FGF), Cyclophlin-B, and Neuropilin-1 (Liu et al., 1999; Datta et al., 2013; Baldwin et al., 2015; Zhang et al., 2015a). Emerging evidence suggests that multiple viruses have evolved with the interaction of O-sulfated domains in HS to promote infection. For instance, 3-OS HS although first recognized to be HSV-1 gD receptor, is now reported to be utilized by other members of the herpes virus family as well. A recent study by Baldwin et al. (2015) demonstrated that human cytomegalovirus interacts with 3-OS HS generated by 3-OST-3 isoforms to mediate virus spread (Zhang et al., 2010). This result emphasizes the symbolic role of 3-OS HS in co-infection or in superinfection models especially since herpes viruses are opportunistic by nature. On the other hand, it is also possible that viruses keep a secret and independent life in the infected cell by down-regulating the expression of 3-OS HS to make supportive habitats in the infected cells for their own distinct advantage. This question remains unresolved and needs to be further addressed. Similarly, 3-OS HS is exploited by viruses as an entry receptor, but at the line of duty, it primarily exists as a signaling transduction molecule. Therefore, it is possible that viruses intelligently sense the 3-OS HS environment and as such, they have no control over their turnover. Clearly, all the above questions are extremely vital as HS/3-OS HS widely participates in various steps of the virus life cycle.

Interestingly among non-herpes viruses, expression of human 3-OST-3a may suppress Hepatitis B virus replication in hepatocytes (Hallak et al., 2000), while 6-O sulfation in HS chain potentially supports entry of human cytomegalovirus (Zautner et al., 2006). In addition, 6-OS HS has been shown to mediate internalization of coxackievirus B3 (Connell and Lortat-Jacob, 2013). Interestingly, 2-O sulfation is recognized by HIV glycoprotein gp120 during cell entry (Figure 2) (Makkonen et al., 2013; Matos et al., 2014). Conversely, N-sulfation of heparan is necessary for inhibition of Respiratory Syncytial Virus (RSV) infection (Fechtner et al., 2013) and is required for CHIKV infection (Tanaka et al., 2017). In case of Hepatitis C Virus (HCV), envelope glycoprotein E1/E2 requires both N and 6-O sulfate group to interact with HS (Trottein et al., 2009).

Besides viruses, other pathogens can also interact with the modified product of HS. For instance, an outer membrane protein of Chlamydia trachomatis known as OmcB interacts with 6-OS HS during infection (Tanaka et al., 2012). Seemingly, HS/3-OS HS becomes an ideal target for both viral and bacterial infection- a possibility that exists in many sexually transmitted infections. In the case of human T-cell leukemia virus type 1 (HTLV), it has been demonstrated that the combination of the number and the length of HS chains containing heparin-like regions is a critical factor in determining cell tropism of HTLV-1 (Monneau et al., 2016). It is hypothesized that shorter HS chains may be able to induce receptor complexes (HTLV-1 Env-HS-NRP-1-GLUT1) more efficiently than their longer counterparts by attracting HTLV-1 particles to the target cell surface. Nonetheless, the findings obtained in this study may provide the foundation for the development of effective therapies against HTLV-I infection and aid in the development of a metric for assessing the prognosis of HTLV-1 induced diseases. Whether the length of the HS chain influences virus infection in general still requires further investigation. Furthermore, whether the virus infection subverts the length of HS or effects HS-polymerization is also unrecognized. However, recent progress made in glycobiology has shown that glycosaminoglycan structural properties such as length, sulfation, and epimerization patterns, are cell, tissue, and developmental stage specific (Patel et al., 1993).

It is clear that a single type of sulfation in a HS chain can serve as an attachment point for multiple types of viruses. For instance, 3-O sulfation in HS generated by 3-OST-3 is utilized by both HSV and cytomegalovirus (CMV) to trigger virus entry. Moreover, an individual virus can exhibit the preference to two entirely different sulfation patterns in HS chains (Shukla et al., 1999; Baldwin et al., 2015). According to recent studies, CMV can facilitate both 6-OS HS and 3-OS HS for infectious entry (Borst et al., 2013; Baldwin et al., 2015). Though it is still unknown for many viruses, what specific sulfonation pattern is most preferred.

An exponential amount of evidence suggests that HSPGs play a significant role in the HIV lifecycle and disease development. Initially, it was demonstrated that viruses enter multiple T cells by using HS chains of HSPGs to facilitate entry (Roderiquez et al., 1995). Concomitantly, the presence of heparanase has been shown to competitively inhibit HIV entry into the target cells (Roderiquez et al., 1995; Ohshiro et al., 1996). In addition, multiple studies demonstrated that the presence of HSPG in various cell types enhances HIV entry (Ibrahim et al., 1999; Saphire et al., 2001; Guibinga et al., 2002; Zhang et al., 2002; Bobardt et al., 2004). For instance, primary human endothelial cells which are loaded with HSPG are extremely efficient in capturing viruses on the cell surface (Argyris et al., 2003). The overexpression of HS in primary endothelial cells near the blood-brain barrier and in the microvasculature, in addition to their ability to capture HIV-1, has been proposed as a novel mechanism to facilitate the invasion of the brain by HIV-1 (Argyris et al., 2003). Further prime target cells like DCs, macrophages, epithelial, and endothelial cells provide docking sites for HIV by expressing HS (Guibinga et al., 2002; Wu et al., 2003; Gallay, 2004; de Witte et al., 2007; Ceballos et al., 2009).

Additionally, HS-expressing spermatozoa play a crucial role in virus transformation into DCs, macrophage and T cells during HIV transmissions (de Parseval et al., 2005). In fact, four HS-binding domains have been identified in the V2 and V3 loops, the C-terminal domain, and the CD4-induced bridging sheet of the HIV gp120 (Crublet et al., 2008; Herrera et al., 2016). In contrast, one recent study depicts that HS combines with the innate protein human beta-defensin (hBD) and reduces HIV trans-epithelial transmission through inactivation of virus. Using an adult oral epithelial cell which expresses hBD, the authors showed the HSPG-mediated internalization of hBD and HIV-gp 120 in the endosome results in oligomerization and reduced infectivity of HIV (Vivès et al., 2005).

Remarkably, it has been demonstrated that HS not only facilitates viral attachment to host cells via HIV gp120 (Cladera et al., 2001) but also that HS mediates viral-host fusion by interacting with the fusion domain of gp41 (Teixé et al., 2008). On permissive cells, HIV binding to HS is thought to increase infectivity by concentrating viral particles at the cell surface (in the cis process). Additionally, for some cell types such as macrophages, HS may compensate for low CD4 expression (Guibinga et al., 2002). It has also been proven that the expression of HSPG on the CD4+ cell surface is regulated by virus infections and immune activation (Ibrahim et al., 1999; Alvarez Losada et al., 2002). HS may be directly involved in the infection of CD4⁺ and/or CD4⁻ cells, making it an ideal target for new anti-HIV therapies (Wu et al., 2003; Somiya et al., 2016). Beside HS interaction to gp120, it also co-localizes with matrix protein p17 on activated human CD4⁺ T cells. Additionally, single particle tracking confirms that multivalent Tat protein transduction results in a domain-induced heparan sulfate proteoglycan cross-linkage, which activates Rac1 for virus internalization. HIV-1 Tat and HSPG interaction have been proposed as a novel mechanism of lymphocyte adhesion and migration across the endothelium (Xu et al., 2011).

Besides the symbolic role of HS for viral entry, HS plays other extensively documented roles in the viral lifecycle. Using a recombinant Hepatitis B virus (HBV) surface antigen L protein particles (bio-nanocapsules, BNCs), a HS-dependent mechanism of HBV uptake has been proposed (Dong et al., 2013). To study the hepatitis C virus (HCV), a gene silencing approach was used against enzymes involved in HS-biosynthesis to investigate structural determinants required by HCV during infection. The study found the N- and 6-O-sulfation, but not 2-O-sulfation, is required for HCV infection. It is also known that the minimum HS oligosaccharide length required for HCV infection is a decasaccharide. Overall, previous discoveries highlighted that HCV uses specific HS structure to initiate infectious cycle. Concurrently, another study implemented a different approach by using heparanase I, II, and III enzymes to cleave specific structures in HS to reach the same conclusion. The findings indicate that the heterogeneity of HS especially for rich N-sulfation and iduronic acids heavily participate in Respiratory Syncytial virus (RSV) infection in some mammalian cells (Cooper et al., 2005).

Besides the role of HS in viral entry, it is justified that HS is also involved in the induction of cytokines by HBV capsid in macrophages via TLR2 signaling (Tsidulko et al., 2015). A recent study has proposed that within various bovine tissues used for HS preparation, only liver-based HS can strongly bind to both E1 and E2 of Hepatitis C Virus (HCV). This study is very significant since HCV, which displays liver tropism, contains highly sulfated structures compared to HS from other tissues. This concludes that HS-proteoglycan on liver cell surfaces appear to be one of the molecules that define liver-specific tissue tropism of HCV (Trottein et al., 2009). Later, a study by Lawrence et al. (2007), Deligny et al. (2010) supports the notion that HS structures vary depending on the cell and tissue, and this can influence virus tropism. For instance, HS-modifying enzymes 3-OST-2 and 3-OST-4 are the predominant forms expressed in the neurons of the trigeminal ganglions while 3-OST-2 and 3-OST-4 generated HS have been shown to be of the principal gD-receptor type for HSV-1. Ultimately, all of these subtleties reinforce the presence of a selective interaction between HS and viruses. Although multiple viruses use HS proteoglycans which are ubiquitously expressed GAGs for binding and or attachment, it remains to be ruled out if virus interactions with HS are random or if they require structural specificity.

A unique pattern of HS expression in Epstein Barr Virus (EBV) infected cells is established. Lymphoblastoid cell lines infected with EBV have shown specific proteoglycan expression with down-regulation of CD44 and ECM components and up-regulation of serglycin and perlecan/HSPG2. Nevertheless, in Burkitt's lymphoma cells (BL), serglycin was shown to be down-regulated. Fundamentally, the biosynthetic machinery for HS construction and modification was active in all cell lines, with the cavate that this machinery was down-regulated in BL cells (Shannon-Lowe and Rowe, 2011). A separate study documents the involvement of sulfated-HS during B cell/epithelial cell interactions during Epstein—Barr virus (EBV) transfer (Woodard et al., 2016). In fact, understanding HS-virus association has been investigated for many, entirely different reasons to directly help patients. In ocular gene therapy, a novel chimeric adenovirus-associated vector (AAV) has been developed, which binds to HS and hence displays better transduction ability. This result has generated AAV with higher accumulation and penetration to the retina and therefore offers a less invasive intravitreal injection route for ocular gene therapy (Kaever et al., 2016). Similarly, the significance of HS in developing antibodies against vaccinia virus A27, human papillomavirus (HPV) has been advocated (Silva et al., 2014; Xia et al., 2016).

In addition, a study tested two different strains (vaccine strain and clinical isolates) of Chikungunya virus (CHIKV) for GAG binding generated efficient virus replication. Although GAGs are necessary for efficient binding of both strains, they exhibit differential requirements for GAGs. However, the study delineated the critical amino acid residue 82 in the envelope glycoprotein E2 is a primary determinant of GAG utilization. A critical outcome for the future development of viral entry inhibitors against CHIKV infection (Richards et al., 2013). Additionally, the importance of HS structure for CHIKV infection was demonstrated in a study by Tanaka et al. (2017), Following genome screening the CRISPR/Cas9 system was deployed to create knockout models of haploid human cells (HAP1). These knockout models were created to assess the importance of various genes that modify HS in efficient CHIKV infection. Infectivity was not affected following disruption of genes encoding proteins that act sequentially after NDST1, an enzyme that catalyzes the N-sulfation step of HS (Tanaka et al., 2017). GLCE is one such protein that acts after the N-sulfation step. GLCE catalyzes the C5 epimerization of GlcA to IdoA which is a necessary step required to later produce the 2 and 3-O-sulfated forms of HS. This phenomenon stands in contrast to the mechanism of HSV infection, where these modifications have been shown to be necessary for infection. Concerning CHIKV infection, HS in the 2 and 3-O-sulfated forms are not required for productive infection but rather it is the N-sulfated HS that facilitates viral binding and host cell entry.

In the case of HPV, multiple HS-binding sites were found on the capsid protein L1 which bring sequential confirmation shifts during virus-receptor engagement assisting virus attachment and internalization. For instance, primary binding is regulated by site 1 (Lys278, Lys361). It can cause confirmation shift in L2, which mediates the display of the amino terminus. In the step after primary attachment, Site 2 (Lys54, Lys356) and site 3 (Asn57, Lys59, Lys442, Lys443) are essential for viral entry (Richards et al., 2013). Overall, the HS-binding mediates exposure of secondary binding sites aiding in the virus engulfment.

There is a study comparing the amino acid sequences of the E2 glycoprotein from natural North American eastern equine encephalitis virus (NA-EEEV) isolates that shows that the neurovirulence of EEEV is supported by the interaction with HS (Voss et al., 2010). For instance, mutations from lysine to glutamine at E2 71, from lysine to threonine at E2 71, or from threonine to lysine at E2 72 were found to have an altered virulence and interaction with HS. Using an electrostatic map, the HS-binding regions in the EEEV E1/E2 heterotrimer were predicted at the apical surface of E2. Basing this method upon the recent Chikungunya virus crystal structure, variants were detected to affect the electrochemical nature of the binding site (Ryman et al., 2007). The EEEV sylvatic cycle could be the starting point for these natural variations in the EEEV HS-binding domain, which may influence receptor interaction as well as the severity of EEEV disease (Voss et al., 2010).

Similarly, HS-binding has been proposed to be an important factor in neurovirulence of neuroadapted and non-neuroadapted Sindbis Viruses (SV) (Milho et al., 2012). Substantially, HS on olfactory neuroepithelium offers an essential and multifaceted site for HS-dependent murid herpesvirus-4 uptake. Therefore, olfactory neuroepithelium poses a critical entry point for heparan-dependent herpesvirus (Antoine et al., 2014).

Decades ago in 1964, it was discovered that heparin, a glycosaminoglycan related to heparan sulfate, inhibited HSV infection, which was possibly the origin for the idea that heparan sulfate is a viral receptor. Inhibition by heparin is a competitive process and, therefore, is most effective when the inhibitor is present during the attachment phase of viral entry. Heparin has been shown to bind to soluble forms of gB, gC, and gD (Nahmias and Kibrick, 1964; Herold et al., 1995; Tal-Singer et al., 1995; Feyzi et al., 1997; Trybala et al., 2000). Interestingly, whereas 6-O- and 3-O-sulfation is the primary determinant for HSV-1 infection, they appear to have a little role in HSV-2 infection, suggesting differences between the two types of viruses (Herold et al., 1996). Likewise, O-sulfation was found to be more important for binding to gB from HSV-1 than gB from HSV-2. Fractionation of polymeric heparin into discrete sulfated oligosaccharide mixtures suggests that nearly 10–12 monosaccharides are necessary for effective binding to gC (Feyzi et al., 1997).

Numerous other sulfated polysaccharides have been explored as mimics of HS in the inhibition of herpesvirus entry into cells. A principal source of these sulfated polysaccharides is sea algae, which biosynthesizes these molecules to retain K⁺ and Ca⁺² ions from seawater and for enhanced resistance to desiccation. These polysaccharides include κ and λ carrageenans (Marchetti et al., 1995; Carlucci et al., 1997, 1999a,b; Zacharopoulos and Phillips, 1997), galactan sulfate (Damonte et al., 1996; Mazumder et al., 2002), galactofucan sulfate (Thompson and Dragar, 2004), fucan sulfate (Preeprame et al., 2001), spirulan (Hayashi K. et al., 1996; Hayashi T. et al., 1996), fucoidan (Ponce et al., 2003; Lee et al., 2004a), rhamnan sulfate (Lee et al., 1999), chitin sulfate (Ishihara et al., 1993), and other uncharacterized polymers (Hasui et al., 1995; Witvrouw and De Clercq, 1997; Lee et al., 2004b; Zhu et al., 2004). In addition, other polysaccharides investigated as mimics of heparan sulfate include chemically modified heparins (Herold et al., 1995, 1997; Feyzi et al., 1997), non-anticoagulated heparin (Herold et al., 1997), pentosan polysulfate (Herold et al., 1997), and dextran sulfate (Marchetti et al., 1995; Neyts et al., 1995; Dyer et al., 1997; Herold et al., 1997).

A growing a class of small molecules these days is called non-saccharide glycosaminoglycan (GAG) mimetics (NSGMs). NSGMs are much smaller than polymeric HS mimetics, which makes them more drug-like in nature in comparison to the oligomeric and polymeric HS mimetics. NSGMs are also much easier to prepare (synthesize) in comparison to oligomeric HS mimetics and they are highly water soluble. Most importantly, NSGMs are functional mimetics of polymeric GAGs, which arises from their ability to bind to sites on proteins that interact with GAGs. Many NSGMs have been discovered so far that modulate various processes in addition to viral infection (Desai, 2013). For example, sulfated flavonoids and xanthones have been found to work as anticoagulant and antiplatelet agents (Al-Horani et al., 2011; Correia-da-Silva et al., 2011); sulfated benzofurans have been found as inhibitors of thrombin (Sidhu et al., 2013); and sulfated benzylated glycosides have been discovered as inhibitors of human factor Xia (Al-Horani et al., 2013; Al-Horani and Desai, 2014).

In a recent work, Gangji et al. (2018) show for the first time NSGMs present an excellent alternative to polymeric HS agents for inhibiting HSV (Gangji et al., 2018). These authors screened a small library of synthetic NSGMs (MW in range of 500–2500) and identified a distinct group of NSGMs that bind glycoprotein D with high affinity (10 nM or so). More importantly, one specific NSGM, called SPGG, inhibited cellular entry of HSV-1 with IC₅₀ in the range of 430 nM to 1.0 μM. This is greater than a 10-fold lower response than that reported for 3-O-sulfate containing heparin/heparan sulfate-derived octasaccharides (Liu et al., 2002; Copeland et al., 2008; Hu et al., 2011).

Overall, competitive inhibition of viral entry through the use of either oligosaccharidic or non-saccharidic agents is very exciting. Several poly/oligosaccharides and small aromatic agents have been discovered that present major opportunities for development of anti-virals. It appears that the category of NSGMs represents excellent agents for targeting viral glycoproteins.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.