key: cord-0815752-63y9pweq
authors: Feng, Tingting; Zhang, Jinyu; Chen, Zhiqian; Pan, Wen; Chen, Zhengrong; Yan, Yongdong; Dai, Jianfeng
title: Glycosylation of viral proteins: Implication in virus–host interaction and virulence
date: 2022-04-18
journal: Virulence
DOI: 10.1080/21505594.2022.2060464
sha: 8eccc145aa0f492917bbdb79d24749d749033839
doc_id: 815752
cord_uid: 63y9pweq

Glycans are among the most important cell molecular components. However, given their structural diversity, their functions have not been fully explored. Glycosylation is a vital post-translational modification for various proteins. Many bacteria and viruses rely on N-linked and O-linked glycosylation to perform critical biological functions. The diverse functions of glycosylation on viral proteins during viral infections, including Dengue, Zika, influenza, and human immunodeficiency viruses as well as coronaviruses have been reported. N-linked glycosylation is the most common form of protein modification, and it modulates folding, transportation and receptor binding. Compared to N-linked glycosylation, the functions of O-linked viral protein glycosylation have not been comprehensively evaluated. In this review, we summarize findings on viral protein glycosylation, with particular attention to studies on N-linked glycosylation in viral life cycles. This review informs the development of virus-specific vaccines or inhibitors.

The high number of infectious diseases caused by viruses has posed a major threat to global public health [1] . Moreover, there are no vaccines and antiviral drugs for most of the newly emerged viruses. Therefore, there is an urgent need to develop antiviral strategies to control the spread of these viral pathogens.

Viruses are obligate intracellular infectious agents that exploit host machinery to modify their viral proteins for survival. One of the key modifications is protein glycosylation. Glycosylation is the attachment of glycans to proteins and is a critical post-translational modification (PTM). To date, various glycosidic linkages such as N-, O-, and C-linked glycosylation, glypiation (GPI anchor attachment), and phosphoglycosylation have been reported [2] . Among them, N-linked glycosylation, where glycans are attached to the amide nitrogen of asparagine (Asn), is the most common and extensively studied form of protein glycosylation [3] .

Recent studies have shown that many viral proteins, especially structural proteins are glycosylated during viral infection cycles. The N-glycans of the viral glycoproteins have multiple functions, which include promotion of expression, transport, fusion, binding to cell surface receptors, and prevention of antibody neutralization. For instance, glycosylation of envelope proteins of the human immunodeficiency virus (HIV), Japanese encephalitis virus (JEV) and West Nile virus (WNV) is essential for membrane fusion and virus infectivity [4] [5] [6] . Similarly, Influenza virus (IAV), lymphocytic choriomentingitis virus (LCMV), Lassa virus (LFV), Severe acute respiratory syndrome virus (SARS), Zika virus (ZIKV), Dengue virus (DENV) and Ebola virus (EBOV) also have their extensively glycosylated envelope proteins [7] [8] [9] [10] [11] [12] [13] (Table 1) . Furthermore, some host cell-derived glycans have diverse effects in the life cycle of viruses, including glycan shield mediated immune evasion and enhancement of immune cell infection [14] . Here, we analyze recent findings on the importance of glycosylation to viral infection and host immune response.

arthropod-borne viral diseases in humans, and poses a huge threat to the public social, health, and economic status [15] . DENV has four different serotypes (DENV-1, DENV-2, DENV-3, and DENV-4), all of which can cause disease [16, 17] . The viral particles are composed of three structural proteins, namely capsid (C), envelope (E), and (pre) membrane protein (prM/M) [18] [19] [20] . In addition, DENV has seven nonstructural (NS) proteins, which include NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 [21] . Numerous studies have shown that the E protein plays an essential role in host receptor attachment, cellular uptake of viral particles, and membrane fusion [17, 21] . The E protein has two potential N-linked glycosylation sites at Asn-67 and Asn-153. The Asn-153 glycosylation site is conserved in most flaviviruses, while the Asn-67 glycosylation site is unique to DENV [11] (Figure 1 ). The glycosylation of envelope E protein at Asn-67 has an important role in the transmission of DENV [11] . Absence of Asn-67 glycosylation significantly suppresses replication of DENV and reners the virus unable to produce new infectious particles [11] . Besides, Asn-67 glycosylation of the DENV E protein was also shown to significantly enhance the entry of DC-SIGN-mediated dengue virus [22, 23] . On the other hand, N-glycans located at Asn-153 of the DENV E protein are beneficial in the survival of DENV in both mammalian and mosquito cells [11, 21, 22] . In DENV lifecycle, the prM/M protein was also shown to be N-glycosylated at amino acid positions 7, 31, 52, and 69 [21, 24] . However, the functions of prM/M glycosylation have not been extensively studied [21] . Assembly,Trafficking, Reproduction, Replication, virion-cell, cell-cell fusion, Cell-to-cell spread and infectivity [129] [130] [131] [132] [133] Zika virus (ZIKV) is an emerged mosquitoe-borne flavivirus that causes severe human diseases, which include neurodevelopmental malformations (congenital Zika syndrome) and Guillain-Barre syndrome [25] [26] [27] . The prM and E proteins of the ZIKV virus have glycosylation sites. The N-linked glycosylation at Asn-154 of the ZIKV E protein was shown to affect virus assembly and infectivity in vitro [28] , and also mediate invasion of the ZIKV into the mosquito midgut [29] (Figure 1 ). The N-linked glycosylation of the ZIKV E protein may enhance viral infectivity through cell surface lectins [28, 30] . Fontes-Garfias et al demonstrated that in type 1 interferon-deficient mice and mosquito cells, N-linked glycosylation at Asn-154 was essential for ZIKV infection but does not affect the neurovirulence in mice [30] . However, in Asian ZIKV strain ZKC2P6, the N154Q mutation or deletion at the N154 position of E protein facilitated the ZIKV replication and enhances neurotoxicity and apoptosis in newborn mice [31] (Figure 1 ).

N-linked glycosylation of the prM protein is also essential for the life cycle of the ZIKV. The prM protein of all the ZIKV strains contains a single N-linked glycosylation site at Asn-69 12 . A previous study showed that loss of prM N-glycans leads to protein aggregation and induce CHOP nuclear translocation [12] . Furthermore, prM and E N-glycans in the ZIKV are essential for effective secretion of the ZIKV E protein, and both are indispensable [12] . Lack of prM N-glycosylation leads to impaired expression and secretion of the E protein, which causes accumulation of the E protein in the endoplasmic reticulum (ER), thus triggering ER stress response [12] , a phenomenon that is detrimental to the ZIKV life cycle.

West Nile virus (WNV) is also an arthropodtransmitted virus that belongs to the Flaviviridae family. Although the prM proteins of all the WNV strains contain N-linked glycosylation sites, but not all strains contain an N-linked site in the E protein [6] . The N-linked glycosylation motif (NYS/T) of the WNV E protein is located between amino acid positions 154-156 [32] [33] [34] [35] . Martina et al showed that the WNV strains containing E glycosylation at the N154 can use DC-SIGN as an attachment factor to enhance virus infection [36] . In other studies, WNV lacking N-linked glycans in the E protein could not efficiently replicate and spread in mosquito cells [37, 38] . Once the WNV E protein is glycosylated, virus assembly is enhanced and the viral infectivity is significantly increased [6] . On the other hand, when mice were infected with the WNV, only the virus with glycosylated E protein showed neuroaggressiveness, suggesting that glycosylation of the E protein is a molecular determinant of the WNV neuroaggressiveness [39] . The prM protein of WNV has a potential N-linked glycosylation site at amino acid 15 of the extracellular domain [6] . Studies have shown that N-linked glycosylation sites in the WNV prM play a role in regulating virus assembly and release, but have little effect on virus infectivity. Removal of glycosylation on the prM or E protein results in reduction of the release of subviral particles [6] . Nevertheless, the specific role(s) of glycosylation in WNV pathogenesis needs further evaluation.

The two membrane glycoproteins of Japanese encephalitis virus (JEV), prM and E, contain an N-linked glycosylation site at positions N15 and N154, respectively [5, 40] . In animal model studies, removal of prM N-glycosylation resulted in a sharp decline in viral virulence in mice after virus inoculation [5] . With mutation of N-linked glycosylation site of prM, the growth of JEV is inhibited in a cell-typespecific manner [5] . In a DNA vaccine study of JEV, immunization of proteins with both prM and E glycosylation site mutations were shown to significantly enhance the antibody response, increase IL-4 secretion, and provide complete protection against lethal attack of the JEV [40] . In addition, Liang et al. employed three types of JEV mutants to determine the role of glycosylation of JEV E protein in viral pathogenesis [41] . Compared with the N154 glycosylated wild-type JEV, strains with no glycosylation or N67 glycosylation showed less growth fitness, and decreased neurovirulence/neuroinvasion in mice. Furthermore, compared with the wild-type viruses, viruses with both N67 and N154 E glycosylation showed effective replication and neurovirulence in cell culture, but reduced neuroinvasiveness in vivo ( Figure 1 ). Therefore, the JEV E protein with a single glycosylation site at N154 was likely to have the selection advantage in crossing the bloodbrain barrier during evolution [41] . This study also supports the role of flavivirus E protein glycans on viral infections and cell tropism.

Hepatitis C virus (HCV) encodes two envelope glycoproteins E1 and E2. There are 4 and 11 N-linked glycosylation sites on E1 and E2, respectively [42, 43] . They play an important role in protein folding, virus entry, and regulation of immune responses [42] [43] [44] [45] . Mutations in glycosylation sites E1N1, E2N3, E2N7, E2N8, E2N10, and E2N11 have a negative impact on virus particle assembly and/or secretion as well as virus entry [42] . Moreover, the E2N7 mutation greatly reduces the infectivity of HCV. In addition, the glycans at the E2N2 and E2N4 positions are essential for the entry function of the HCV envelope glycoproteins. Glycans at positions E2N1, E2N2, E2N4, E2N6, and E2N11 reduced the accessibility of neutralizing antibodies to their E2 glycoprotein epitopes. Besides, the glycans at E2N1, E2N2, E2N4, and E2N6 could regulate binding between CD81 and E2, which may partly explain how HCV evades the immune response [42] .

For most flaviviruses, glycosylation of the envelope protein affects the adaptability, infectivity, replication, and virulence of the virus [6, 11, [46] [47] [48] [49] [50] [51] . To date, the structure and functions of these glycans in virus transmission, infection and pathogenesis are not fully understood yet, and further exploration is needed.

The human immunodeficiency virus (HIV) envelope glycoprotein (Env) is composed of surface subunit gp120 and transmembrane subunit gp41 [4] . It is reported that the N-linked glycans on the surface of gp120 are essential for proper protein folding. HIV gp120 contains nine disulfide bonds and is highly glycosylated, carrying an average of 24 N-linked glycans. Among them, the Asn260 glycosylation site defines correct expression of gp120 and gp41 [4] . Studies have shown that N260Q mutation could affect the folding and lysosomal degradation of the gp120, leading to loss of viral infectivity. Further studies have shown that the reduction of virus infectivity is caused by the deletion of glycans in the gp120 V1/V2 domain [4, 52] . Moreover, several highly conserved N-linked glycans of the gp120 are preferentially located near the disulfide bridge. Removal of disulfide bonds has been shown to significantly affect the ability of the DC-SIGN to bind to HIV. On the other hand, in a variety of virus strains lacking N-glycans, introduction of new N-glycans upstream of several disulfide bonds could result in complete loss of viral infectivity [53] . The ability to bind to the CD4 receptor was significantly reduced in the gp120 strain bearing the N260Q mutation, suggesting that N260 glycosylation affects the virus entry process [54] . In addition, N-linked glycans could affect protein function and neutralizing antibody response [55] . Given the pivotal role of N-glycans in HIV-1 gp120 V1/V2 domains, most studies have focused on neutralizing antibody response for gp120 N-linked glycans, which could be a new target for specific therapeutic drug intervention.

Influenza A virus (IAV) encodes two main viral surface glycoproteins, hemagglutinin (HA), and neuraminidase (NA). The HA protein determines the antigenicity of the IAV. The HA of most human H1N1 has multiple N-glycosylation sites ranging from 5 to 11, most of which are located in the globular head of the HA molecule [14, 56] . Glycosylation of the HA stem region is important for protein folding, transport, and pH stability [57] [58] [59] . Other studies have shown that change of glycosylation near HA receptor binding site would change its affinity to receptors [60] [61] [62] . Glycosylation near the cleavage HA cleavage site also regulates the pathogenicity of the virus [63, 64] . Furthermore, the loss of a single N-linked glycan in HA was associated with the related to the resistance to collection and increased virulence in mice [65] . N-linked glycosylation is also important in the NA functions [66] . Lack of NA glycosylation could increase the neurovirulence of mouse A/ WSN/33 IAV strain [67] .

SARS-CoV-2 is a novel virus that has caused the global pandemic "Coronavirus Disease2019" (COVID-19) , a severe acute respiratory disease [68] . The viral Spike protein, its receptor binding domain (RBD) and its receptor ACE2 are all extensively glycosylated. The Spike protein contains 22 N-glycosylation sites, while ACE2 contains 7 N-glycosylation sites [69] [70] [71] . The O-linked and N-linked glycans of the SARS-CoV-2 Spike protein are less important in regulating the direct binding of the Spike and ACE2, but inhibit the virus entry [10] . Studies have shown that blocking of N-glycan biosynthesis with small-molecule inhibitors inhibits entry of the SARS-CoV-2. Besides, blocking the processing of O-glycans was shown to partially block the virus entry. Analysis of the crystal structure showed that the key glycans regulating this process could be located at Asn-90 or Asn-322 position of the Spike protein [72] . However, the detailed mechanisms for this site-specific glycosylation regulating the entry of SARS-CoV-2 remain unclear.

Some coronaviruses have E1 and E2 envelope proteins. Previous data showed that E1 protein is an O-linked glycoprotein while E2 is N-linked glycosylated [73] [74] [75] [76] . The M protein of coronavirus is the most abundant protein, and plays a central role in virus assembly. SARS-CoV M protein contains an N-glycosylation site at N4 [76, 77] . Although the glycosylation of the coronavirus M protein is a highly conserved feature, this glycosylation is not important for viral assembly or replication [78] .

Ebola virus (EBOV) belongs to the filovirus family, and its surface glycoprotein (GP) is composed of trimers of GP1/GP2 heterodimers. The GP1 subunit is important for receptor binding, while the transmembrane-related GP2 subunit is necessary for membrane fusion. EBOV GP1 contains 15 N-glycosylation sites. A previous study showed that loss of any of N-glycosylation sites does not affect the expression of GP but enhance pseudovirion transduction [13] . Removal of GP1 N-glycans does not affect the binding of pseudoviral particles to the cell surface, but increases protease sensitivity of the cathepsin B [13] . In antibody neutralization assays, elimination of N-linked glycans in GP1 can significantly increase the sensitivity of antibody neutralization, while introducing N618D to the GP1 subunit (7Gm8 G), which could further enhance neutralization sensitivity of the virus particles [13, 79] . The GP2 subunit of filovirus contains two highly conserved N-linked glycosylation sites at N563 and N618 positions [79] . Deletion of the glycosylation site at N563 could lead to enhanced virus entry, possibly through partial destruction of the stability of the GP. Removal of a single glycan at N563 or N618 could not increase the sensitivity to neutralizing antibodies. Besides, antibody sensitivity is increased in viruses lacking all the N-linked glycans in the GP1 or the glycan at N618 in the GP2, compared with viruses with GP1 single mutation [79] .

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-transmitted bunyavirus with a segmented, negative-strand RNA genome, which includes large (L), medium (M), and small (S) segments. The M segment encodes a membrane protein precursor that matures into two glycoproteins, Gn and Gc. Recent studies have shown that SFTSV-specific neutralizing antibodies, mainly induced by Gn and Gc, play a vital role in the protective immune response [80] . Gn has two N-linked glycosylation sites, at Asn 33 and Asn 63 [81] . SFTSV Gc is a type I transmembrane protein, whose extracellular domain is stabilized by 13 disulfide bonds and can form putative trimmers at acidic pH [82] . Analysis of the electron density of Gc has shown that there are three N-linked glycosylation sites in the extracellular domain of SFTSV Gc: Asn 853, Asn 914, and Asn 936 82 . DC-SIGN and Non-muscular myosin heavy chain IIA (NMMHC-IIA) have been reported to be the initial binding receptor for SFTSV [83, 84] . Whether the N-linked glycans in Gn and Gc are critical for virus binding and entry remains to be defined.

Hantaan virus (HTNV) also encodes two glycoproteins, Gn and Gc. Gn contains five potential N-linked glycosylation sites at N134, N235, N347, N399, and N609 while Gc contains a potential N-linked glycosylation site at N928 [85] . All glycosylation sites on the extracellular domain of Gn (N134, N235, N347, and N399) and Gc (N928) are glycosylated, and the fifth Gn site (N609) which faces the cytosol remains unutilized [86] . N134 is most critical for guiding the correct folding of Gn. In the absence of this site, Gn is stagnated in the ER and cannot be recognized by anti-Gn MAb [86] . In addition, glycosylation at the third Gn site (N347) significantly affects its folding, while the lack of N235 and N399 sites in Gn and N928 site in Gc is insufficient to affect folding and targeting [86] . Besides, mutations of the N134, N347, and N399 N-glycosylation sites of Gn reduce the immunoreactivity of Gc protein. Conversely, lack of N-glycans on Gc has been shown to reduce immunoreactivity of the Gn protein [86] .

Rift Valley Fever is a disease caused by Rift Vally Fever virus (RVFV) and is transmitted by mosquitoes. The RNA genome of the RVFV consists of large (L), medium (M), and small (S) segments. The M segment encodes the Gn and Gc glycoproteins. RVFV has five putative N-glycosylation sites, located at N438 of the Gn protein, and N794, N829, N1035, and N1077 of Gc protein, respectively [87] . Glycosylation at N438 of RVFV Gn or N1077 of the Gc plays an important role in mediating virus infection through DC-SIGN [87] . A previous study showed that in Jurkat-DC-SIGN cells, there was suppression of the infectivity of N438Q or N1035Q mutants, while N438Q/N1035Q double mutation had little effect on the viral infection of these cells [87] . Besides, RVFV Gn and Gc glycoproteins are crucial in inducing neutralizing antibodies [88, 89] .

Nipah virus (NiV) is an emerging paramyxovirus with deadly consequences. NiV infects the respiratory and nervous systems and leads to fatal encephalitis, the main cause of human death [90, 91] . The fusion (F) and attachment (G) envelope glycoproteins of the NiV mediate virus entry, cell-cell fusion, and syncytium formation. The NiV-F protein has five potential N-linked glycosylation sites, four of which are highly glycosylated [92] . Removal of N-glycans on F protein has little effect on protein processing and expression, but F3 and F5 N-glycan mutants have a higher fusion level compared to the wild-type NiV [92] . Removal of N-glycans in the NiV F protein results in significantly enhanced virus entry. Meanwhile, the N-glycans on NiV-F could protect the virus from neutralization by antibodies [92] . There are seven potential N-linked glycosylation sites in NiV-G, of which six are highly glycosylated [93] . The function of NiV-G N-glycan in membrane fusion is highly dependent on its environment, and some NiV-G N-glycans even reduce the fusion efficiency [93] . Like NiV, another paramyxovirus, Hendra virus (HeV), also requires two different membrane-anchored glycoproteins G and F for membrane fusion and viral entry [94] . The HeV-G head domain may have five N-linked glycosylation sites, which are partially occupied by carbohydrates [94] . To date, data on the N-linked glycosylation of these two HeV glycoproteins remain scant.

Human Respiratory Syncytial Virus (HRSV) belongs to the Pneumovirus genus, Paramyxoviridae. The virus envelope has two main glycoproteins, G and F, which are responsible for virus attachment to cells and membrane fusion [95] . The glycosylation sites in the G protein are highly variable, while those in the F protein are relatively conserved. The RSV F has five N-glycosylation sites, which are N27 and N70 located in the F2 subunit, N116 and N126 in the p27 peptide, as well as N500 in the F1 subunit. Every single N-glycosylation site in the RSV F protein is not dispensible for virus replication, but they have synergistic contribution to the efficiency of viral infection [96] . The virus can not replicate if all the N-glycans in the RSV F protein are removed. Data have demonstrated that the N-glycan at the N500 position is essential for syncytium formation in RSV-infected Hep-2 cells [96] . In addition, the frequency of syncytia formation in RSV F N116Q-infected cells was also significantly reduced, indicating that N-glycosylation at N116 affects syncytia formation. Another study showed that removal of the N-glycosylation site at position N116 can enhance the antigenicity of F protein [97] . The G protein contains potential N-glycosylation and O-glycosylation sites. Variations in the O-glycosylation are the main reasons for the high variation between virus strains and may contribute to immune evasion [96] .

Lymphocytic choroid meningitis virus (LCMV) belongs to the arenavirus family. There are three main structural proteins: one nucleocapsid protein (NP) and two glycoproteins (GP1 and GP2) [98] . AS previous study predicted nine potential N-linked glycosylation sites on the LCMV glycoprotein: 6 on GP1 and 3 on GP2 [8] . The three N-glycosylation sites on GP2 are conserved in all the members of this virus. In contrast, the number and location of N-glycosylation sites in GP1 are highly diverse. In addition, folding of the glycoprotein complex (GPC) precursor requires N-linked glycosylation in GP1 [99] . N-linked glycosylation site mutations of GP1 at positions 87, 97 and 104 impair the expression and processing of GPC [8] . Removal of glycans at position 234 or addition of glycans at position 379/381 in GP2 have been shown to impair GP-mediated cell fusion without altering expression or processing [8] . N-glycosylation mutations may affect VLP infectivity during viral fusion events. Mutations in N-glycosylation, at some sites reduce, the VLP infectivity, while the mutant S373A increases the infectivity. Among them, the N-glycan attached to the Asp173 mediates masking the neutralizing GP-1D epitope on Arm-5 antibody clone to prevent neutralization [8] . All three N-glycosylation mutants (T234A, S398A, and T403A) showed reduced adaptability in mouse macrophages. The rLCMV produced by N-glycosylation deletion mutant in GP1 (G104N) or GP2 (E379N/A381T) significantly reduced viral fitness in neurons [100] . These data indicated that LCMV glycosylation regulates viral fitness and cell tropism, and affects the viral growth.

Lassa virus (LASV), a member of the arenavirus family, causes Lassa fever [101] . LASV GP1 is responsible for attachment of the virus to the host cell receptor, while GP2 mediates the fusion of the virus and the endosomal membrane [102, 103] . There are 11 N-glycosylation motifs in GPC, including 7 N-glycans on GP1 and 4 N-glycans on GP2 [9] . The glycosylation of GP1/GP2 is necessary for their transport, antigenicity, and infectivity [8, 104, 105] . Recent studies have shown that 25-hydroxycholesterol (25HC) could decrease the maturation of N-glycan of LASV glycoproteins, thus reducing production of infectious virus [106] . N-glycosylation of LASV glycoprotein requires STT3B, which is essential for viral infectivity [107] . In pseudotyped LASV, the N-linked glycosylation site mutants at N89 and N365 were shown to completely inhibit infectivity, while removal of N-linked glycans at positions N109 and N119 only partially inhibited the infectivity [9] . Besides, the specific glycan chains affect functions of CD4+ T cells and CD8+ T cells in splenic lymphocytes and help LASV escape the immune response by reducing the host's recognition of GPC and preventing induction of immune response [9] . The role of N-glycosylation at 3 rd (N99), 5 th (N119), 6 th (N167), 8 th (N365) and 9 th (N373) sites can mask key epitopes in the GPC and escape humoral immune response [9] . These GPC sites may be targets for the development of effective therapeutic or preventive antibodies against Lassa fever.

Hepatitis B virus (HBV) is a human DNA virus that encodes three envelope glycoproteins, small (S), medium (M), and large (L). These three proteins share a potential N-glycosylation site at Asn-146 (N146) of their S domain [108, 109] . Early N-glycan modification on the ER has a major impact on the production of infectious virus particles [108, 110] . The hyper-glycosylated antigens induce earlier and longer-lasting humoral immune responses compared to the wild-type. N-glycosylation may also be a predictive marker for increased risk for cancer in patients with chronic HBV infection, which deserves more evaluation [108] . Although many features of the HBV life cycle are associated with viral glycosylation, the role of N-glycosylation in the pathogenesis of the virus remains unknown.

Herpes virus is a large enveloped double-stranded DNA virus, which include many important human and animal pathogens. Herpes simplex virus 1 (HSV-1) is a contagious neurotropic herpes virus, which encode 12 envelope glycoproteins and play important roles in the viral replication [111] [112] [113] . Glycoproteins gB, gC, and gD have been implicated in the attachment of virus to receptors, while gB, gD, and heterodimer gH/gL are essential for HSV attachment and penetration [111, [114] [115] [116] [117] [118] [119] . HSV-1 gH contains 838 amino acids and has 7 consensus sites for N-linked oligosaccharides as well as 11 sites for O-linked glycosylation [120] [121] [122] . gL contains 224 amino acids and has one consensus site for N-linked oligosaccharides and 3 potential sites for O-linked glycosylation [120, 121, 123] . HSV-1 gC-1 is heavily glycosylated which contains nine consensus sites for N-linked glycosylation and numerous clustered O-linked glycans in a peptide stretch delimited by amino acids 30 and 124 [124] . All the three potential N-glycosylation sites in gD protein are actually glycosylated [125] . Moreover, both gD1 and gD2 are O-glycosylated, and gD2 undergoes greater O-glycosylation compared to gD1 [125] . Two conserved N-linked glycosylation (N48 and N58) sites for gK are critical for virusinduced cell fusion and replication.

Retinoic acid was shown to suppress viral yield in Vero cells and altered N-glycosylation of viral envelope proteins [126] . The use of tunicamycin to inhibit N-glycosylation of HSV-1 envelope glycoproteins was associated with the production of noninfectious viral particles [126, 127] . Seventy-four O-linked glycosylation sites have been identified on 8 of the 12 HSV-1 envelope proteins [128] . Of the 74 O-glycosylation sites, 34 are localized on the four HSV-1 membrane proteins (gB, gD, gH and gL), which are essential for viral infectivity in vitro [128] . Twenty-one glycosylation sites were identified in gB, which is essential for fusion with host cell membranes. The remaining 40 O-glycosites were distributed among four HSV-1 glycoproteins (gC, gE, gI and gG), which are all critical in virus-host interactions and host immune response modulation [128] .

Varicella-zoster virus (VZV), a human alphaherpesviruses, encodes various structural glycoproteins that are important for its assembly and trafficking [129, 130] . At least seven of its proteins are glycosylated -glycoproteins B, C, E, H, I, K, and L (gB, gC, gE, gH, gI, gK, and gL) [131] . Among them, gE is the most abundant VZV glycoprotein, and its loss leads to impaired viral reproduction [132, 133] . Like HSV-1, multiple G proteins have N and O-glycosylation sites, but there are few reports on the specific biological functions of these glycosylation sites.

Other viruses, such as Rabies virus [134] , Human Papillomavirus [135] , and Metapneumovirus [136] have N-linked glycans, which play important roles in infectivity, protein folding, tropism, proteolytic processing, as well as immune escape.

Nonstructural viral proteins can also be glycosylated. The flavivirus genome encodes seven NS proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 [137] [138] [139] . DENV NS1 has two glycosylation sites at Asn-130 and Asn-207 [21, 140, 141] (Figure 1 ), which are conserved in the family flaviviridae. By destroying the endothelial glycocalyx-like layer (EGL), DENV NS1 has been shown to induce vascular leakage [142] . Mutations of the NS1 protein at the N207 glycosylation site could not lead to EGL degradation or internalization by endothelial cells. Just like DENV, the N207 glycosylation sites of WNV and ZIKV NS1 are essential for internalization. N207 is important in clathrin-mediated endocytosis, a common pathway involved in flavivirus NS1-mediated pathogenesis, where it is important for NS1 endosomal transport. Mutations of Asn-207 slightly delay viral replication [143] .

Glycosylation at N130 plays an important role in viral proliferation, NS1 protein secretion, and cytopathic effects. Mutations at the N130 glycosylation site of DENV1 and DENV2 NS1 have been reported to prevent DENV from producing live viruses in mammalian and mosquito cells [144, 145] . In DENV4, N130 mutations suppressed viral replication in mammalian and C6/36 cells. The absence of N130 in NS1 inhibited viral neurovirulence in mice models [145] [146] [147] .

Like DENV, JEV, and ZIKV NS1 have two N-linked glycosylation sites at N130 and N207 [148] . Yellow fever (YF) NS1 has glycosylation sites at positions 130 and 208 [149] . In addition, some viruses, such as WNV, St. Louis encephalitis (SLE) and Murray valley encephalitis virus (MVEV) have a third glycosylation site at the 175 th amino acid position [150] . Entebbe bat virus (ENTV) has four potential N-linked glycosylation sites in NS1, located at Asn-106, Asn-130, Asn-207, and Asn-326 [151] while the tick-borne encephalitis virus (TBEV) NS1 has three putative N-linked glycosylation sites at residues 85, 207, and 223 [152] . In brain endothelial cells, WNV and JEV NS1 only interact with the glycocalyx, while YFV NS1 exerts the strongest effects on liver endothelial cells [148] . Multiple amino acid changes in the first glycosylation motif of WNV NS1 protein promoted neuroinvasion in mice models [153] . These findings imply that elucidation of the mechanisms involved in flavivirus NS1 glycosylation is essential for the development of antiviral therapies and NS1-based vaccine approaches.

The nsp3 and nsp4 nonstructural proteins of coronavirus undergo N-linked glycosylation in ER. For instance, mouse hepatitis virus MHV nsp3 has a glycosylation site at N1525. The glycosylation site of nsp4 in gamma-coronavirus bronchitis virus (IBV) is at residue N48. For the nsp4 of MHV, two glycosylation sites have been predicted at residues N176 and N237 [76, 154] . These two nonstructural proteins play vital roles in the life cycles of coronaviruses; however, the functions of their glycosylation have not been elucidated. Other nonstructural proteins of coronaviruses, such as ORF3a, 8ab, and 3b are also glycosylated, but the significance of these modifications are yet to be determined [155] .

In addition, some other viral nonstructural proteins, such as Rotavirus NSP4 [156] and HCV NS4B [157, 158] , may also be glycosylated, but not discussed in detail here for the limited space of the review.

Glycomics is an important study field in cell biology. Viral-related N-glycosylation modifications, which confers various advantages on viral survival and virulence, have become a research hotspot. Targeting glycans may be a promising approach for inhibition of viral infections. Not only is glycosylation important for pathogens, it is also vital for hosts. Enveloped viruses hijack host glycosylation mechanisms and take advantage of host-derived glycans to modify their own glycoproteins, which benefits many aspects of viral pathogenesis, including entry into host cells, membrane fusion, and infectivity. Moreover, N-linked glycosylation of envelope proteins play important roles in neutralizing antibody sensitivity and immune escape. These glycoproteins are the only antigens expressed on viral surfaces; therefore, they are essential for vaccine development. For some viruses, glycans in nonstructural proteins are vital to their life cycles. Even though the involved mechanisms are yet to be established, their roles should not be underestimated. Elucidating the mechanisms of viral protein glycosylation during viral infection and replication will inform the development of specific antiviral therapies and vaccines.

Global trends in emerging infectious diseases

Global aspects of viral glycosylation

Iminosugars: a host-targeted approach to combat Flaviviridae infections

Deletion of the highly conserved N-glycan at Asn260 of HIV-1 gp120 affects folding and lysosomal degradation of gp120, and results in loss of viral infectivity

A single N-linked glycosylation site in the Japanese encephalitis virus prM protein is critical for cell type-specific prM protein biogenesis, virus particle release, and pathogenicity in mice

N-Linked glycosylation of west Nile virus envelope proteins influences particle assembly and infectivity

Influenza virus N-linked glycosylation and innate immunity

Glycosylation modulates arenavirus glycoprotein expression and function

Effects of N-linked glycan on Lassa virus envelope glycoprotein cleavage, infectivity, and immune response

Inhibition of SARS-CoV-2 viral entry upon blocking N-and O-glycan elaboration

Essential role of Dengue virus envelope protein N glycosylation at asparagine-67 during viral propagation

N-glycosylation in the pre-membrane protein is essential for the Zika virus life cycle

Comprehensive functional analysis of N-linked glycans on Ebola virus GP1

Virus glycosylation: role in virulence and immune interactions

Dengue/dengue haemorrhagic fever: history and current status

Observations related to pathogenesis of dengue hemorrhagic fever. VI. Hypotheses and discussion

Dengue virus life cycle: viral and host factors modulating infectivity

Solution structure of Dengue virus capsid protein reveals another fold

Flavivirus capsid is a dimeric alpha-helical protein

The heterogeneous nuclear ribonucleoprotein K (hnRNP K) interacts with Dengue virus core protein

Dengue virus glycosylation: what do we know? Front Microbiol

Crucial role of the N-glycans on the viral E-envelope glycoprotein in DC-SIGN-mediated Dengue virus infection

Cryo-EM reconstruction of Dengue virus in complex with the carbohydrate recognition domain of DC-SIGN

Alpha-Glucosidase inhibitors reduce Dengue virus production by affecting the initial steps of virion morphogenesis in the endoplasmic reticulum

The emergence of Zika virus and its new clinical syndromes

Zika virus infection complicated by Guillain-Barre syndrome-case report

Neurological implications of Zika virus infection in adults

Envelope protein glycosylation mediates Zika virus pathogenesis

N-glycosylation of viral E protein is the determinant for vector midgut invasion by flaviviruses. mBio

Functional analysis of glycosylation of Zika virus envelope protein

The ablation of envelope protein glycosylation enhances the neurovirulence of ZIKV and cell apoptosis in newborn mice

N-Linked glycosylation of the West Nile virus envelope protein is not a requisite for avian virulence or vector competence

Genetic and phenotypic variation of West Nile virus

Glycosylation and antigenic variation among Kunjin virus isolates

Extensive nucleotide changes and deletions within the envelope glycoprotein gene of Euro-African West Nile viruses

DC-SIGN enhances infection of cells with glycosylated West Nile virus in vitro and virus replication in human dendritic cells induces production of IFN-alpha and TNF-alpha

Requirement of glycosylation of West Nile virus envelope protein for infection of, but not spread within, Culex quinquefasciatus mosquito vectors

West Nile virus envelope protein glycosylation is required for efficient viral transmission by Culex vectors

Viral envelope protein glycosylation is a molecular determinant of the neuroinvasiveness of the New York strain of West Nile virus

Mutation of putative N-linked glycosylation sites in Japanese encephalitis virus premembrane and envelope proteins enhances humoral immunity in BALB/C mice after DNA vaccination

DC-SIGN binding contributed by an extra N-linked glycosylation on Japanese Encephalitis Virus envelope protein reduces the ability of viral brain invasion

Role of N-linked glycans in the functions of hepatitis C virus envelope proteins incorporated into infectious virions

Role of N-linked glycans in the functions of hepatitis C virus envelope glycoproteins

Hepatitis C virus envelope glycoprotein E2 glycans modulate entry, CD81 binding, and neutralization

The neutralizing activity of anti-hepatitis C virus antibodies is modulated by specific glycans on the E2 envelope protein

Envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage 1 West Nile virus strains

Glycosylation of the dengue 2 virus E protein at N67 is critical for virus growth in vitro but not for growth in intrathoracically inoculated Aedes aegypti mosquitoes

Selection and partial characterization of dengue 2 virus mutants that induce fusion at elevated pH

Genetic determinants of dengue type 4 virus neurovirulence for mice

Both E protein glycans adversely affect dengue virus infectivity but are beneficial for virion release

Role of N-glycosylation on Zika virus E protein secretion, viral assembly and infectivity

Glycan deletions in the HIV-1 gp120 V1/V2 domain compromise viral infectivity, sensitize the mutant virus strains to carbohydrate-binding agents and represent a specific target for therapeutic intervention

Several N-glycans on the HIV envelope glycoprotein gp120 preferentially locate near disulphide bridges and are required for efficient infectivity and virus transmission

The highly conserved glycan at asparagine 260 of HIV-1 gp120 is indispensable for viral entry

Role of N-linked glycans in a human immunodeficiency virus envelope glycoprotein: effects on protein function and the neutralizing antibody response

Effects of glycosylation on the properties and functions of influenza virus hemagglutinin

Role of conserved glycosylation sites in maturation and transport of influenza a virus hemagglutinin

Oligosaccharides in the stem region maintain the influenza virus hemagglutinin in the metastable form required for fusion activity

Role of overlapping glycosylation sequons in antigenic properties, intracellular transport and biological activities of influenza A/H2N2 virus haemagglutinin

Effects of host-dependent glycosylation of hemagglutinin on receptor-binding properties on H1N1 human influenza a virus grown in MDCK cells and in embryonated eggs

Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety

Glycosylation changes in the globular head of H3N2 influenza hemagglutinin modulate receptor binding without affecting virus virulence

Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence

Mutations at the cleavage site of the hemagglutinin after the pathogenicity of influenza virus A/chick/Penn/83 (H5N2)

Loss of a single N-linked glycan from the hemagglutinin of influenza virus is associated with resistance to collectins and increased virulence in mice

Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics

Glycosylation of neuraminidase determines the neurovirulence of influenza A/WSN/33 virus

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding

Function, and antigenicity of the SARS-CoV-2 spike glycoprotein

Cryo-EM structure of the 2019-nCov spike in the prefusion conformation

Comprehensive characterization of N-and O-glycosylation of SARS-CoV-2 human receptor angiotensin converting enzyme 2

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

Site of addition of N-acetyl-galactosamine to the E1 glycoprotein of mouse hepatitis virus-A59

Tunicamycin resistant glycosylation of coronavirus glycoprotein: demonstration of a novel type of viral glycoprotein

Proteolytic activation of the spike protein at a novel RRRR/S motif is implicated in furin-dependent entry, syncytium formation, and infectivity of coronavirus infectious bronchitis virus in cultured cells

Inhibition of N-linked glycosylation by tunicamycin may contribute to the treatment of SARS-CoV-2

Assembly of coronavirus spike protein into trimers and its role in epitope expression

Studies on membrane topology, N-glycosylation and functionality of SARS-CoV membrane protein

The role of conserved N-linked glycans on Ebola Virus glycoprotein 2

Development of a SFTSV DNA vaccine that confers complete protection against lethal infection in ferrets

Structures of phlebovirus glycoprotein Gn and identification of a neutralizing antibody epitope

Structure of a phleboviral envelope glycoprotein reveals a consolidated model of membrane fusion

Severe fever with thrombocytopenia virus glycoproteins are targeted by neutralizing antibodies and can use DC-SIGN as a receptor for pH-dependent entry into human and animal cell lines

Nonmuscle myosin heavy chain IIA is a critical factor contributing to the efficiency of early infection of severe fever with thrombocytopenia syndrome virus

Hantaan virus M RNA: coding strategy, nucleotide sequence, and gene order

Analysis of N-linked glycosylation of hantaan virus glycoproteins and the role of oligosaccharide side chains in protein folding and intracellular trafficking

N-glycans on the Rift Valley fever virus envelope glycoproteins Gn and Gc redundantly support viral infection via DC-SIGN

An equine herpesvirus type 1 (EHV-1) vector expressing Rift Valley fever virus (RVFV) Gn and Gc induces neutralizing antibodies in sheep

Naturally acquired Rift Valley fever virus neutralizing antibodies predominantly target the Gn glycoprotein

Nipah virus-a potential agent of bioterrorism?

Nipah encephalitis outbreak in Malaysia

N-Glycans on Nipah virus fusion protein protect against neutralization but reduce membrane fusion and viral entry

N-Glycans on the Nipah virus attachment glycoprotein modulate fusion and viral entry as they protect against antibody neutralization

Henipavirus mediated membrane fusion, virus entry and targeted therapeutics

Binding of human respiratory syncytial virus to cells: implication of sulfated cell surface proteoglycans

Characterization of the role of N-glycosylation sites in the respiratory syncytial virus fusion protein in virus replication, syncytium formation and antigenicity

Removal of the N-glycosylation sequon at position N116 located in p27 of the respiratory Syncytial virus fusion protein elicits enhanced antibody responses after DNA immunization

Post-Translational processing of the glycoproteins of lymphocytic choriomeningitis virus

Neutralizing epitopes of lymphocytic choriomeningitis virus are conformational and require both glycosylation and disulfide bonds for expression

LCMV glycosylation modulates viral fitness and cell tropism

Characterization of the Lassa virus GP1 ectodomain shedding: implications for improved diagnostic platforms

Envelope glycoprotein of arenaviruses

Genetic diversity among Lassa virus strains

The Lassa virus glycoprotein precursor GP-C is proteolytically processed by subtilase SKI-1/S1P

Targeting the proteolytic processing of the viral glycoprotein precursor is a promising novel antiviral strategy against arenaviruses

25-hydroxycholesterol inhibition of Lassa virus infection through aberrant GP1 glycosylation

Comprehensive interactome analysis reveals that STT3B is required for N-glycosylation of Lassa virus glycoprotein

N-glycosylation and N-glycan processing in HBV biology and pathogenesis

Functions of the internal pre-S domain of the large surface protein in hepatitis B virus particle morphogenesis

Treatment of hepatitis B virus-infected cells with alpha-glucosidase inhibitors results in production of virions with altered molecular composition and infectivity

Different receptors binding to distinct interfaces on herpes simplex virus gD can trigger events leading to cell fusion and viral entry

A single-aminoacid substitution in herpes simplex virus 1 envelope glycoprotein B at a site required for binding to the paired immunoglobulin-like type 2 receptor alpha (PILRalpha) abrogates PILRalpha-dependent viral entry and reduces pathogenesis

Targeting STT3A-oligosaccharyltransferase with NGI-1 causes herpes simplex virus 1 dysfunction

Glycoprotein targeted therapeutics: a new era of anti-herpes simplex virus-1 therapeutics

Herpes virus fusion and entry: a story with many characters

Viral entry mechanisms: cellular and viral mediators of herpes simplex virus entry

Glycoprotein C-independent binding of herpes simplex virus to cells requires cell surface heparan sulphate and glycoprotein B

Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity

Cell fusion induced by herpes simplex virus glycoproteins gB, gD, and gH-gL requires a gD receptor but not necessarily heparan sulfate

Prediction of O-glycosylation of mammalian proteins: specificity patterns of UDP-GalNac:polypeptide N-acetylgalactosaminyltransferase

Structural and antigenic analysis of a truncated form of the herpes simplex virus glycoprotein gH-gL complex

Assembly of asparagine-linked oligosaccharides

A novel herpes simplex virus glycoprotein, gL, forms a complex with glycoprotein H (gH) and affects normal folding and surface expression of gH

O-Linked glycosylation of the mucin domain of the herpes simplex virus type 1-specific glycoprotein gC-1 is temporally regulated in a seed-and-spread manner

Comparative study on O-linked oligosaccharides of glycoprotein D of herpes simplex virus types 1 and 2

Retinoic acid reduces the yield of herpes simplex virus in Vero cells and alters the N-glycosylation of viral envelope proteins

Processing of herpes simplex virus-1 glycans in cells defective in glycosyl transferases of the Golgi system: relationship to cell fusion and virion egress

A strategy for O-glycoproteomics of enveloped viruses-the O-glycoproteome of herpes simplex virus type 1

Varicella-Zoster virus glycoprotein gpI/gpiv receptor: expression, complex formation, and antigenicity within the vaccinia virus-T7 RNA polymerase transfection system

Characterization of interaction of gH and gL glycoproteins of varicella-zoster virus: their processing and trafficking

Varicella-Zoster virus glycoprotein M homolog is glycosylated, is expressed on the viral envelope, and functions in virus cell-to-cell spread

The requirement of varicella zoster virus glycoprotein E (gE) for viral replication and effects of glycoprotein I on gE in melanoma cells

Structural analysis of the varicella-zoster virus gp98-gp62 complex: posttranslational addition of N-linked and O-linked oligosaccharide moieties

Efficient N-glycosylation at position 37, but not at position 146, in the street rabies virus glycoprotein reduces pathogenicity

Glycosylation of human papillomavirus type 16 L1 protein

Characterization of human metapneumovirus F protein-promoted membrane fusion: critical roles for proteolytic processing and low pH

Coupling of replication and assembly in flaviviruses

The IMPORTance of the nucleus during flavivirus replication

Specific requirements for elements of the 5' and 3' terminal regions in flavivirus RNA synthesis and viral replication

Functional and antigenic domains of the dengue-2 virus nonstructural glycoprotein NS-1

Newly synthesized dengue-2 virus nonstructural protein NS1 is a soluble protein but becomes partially hydrophobic and membrane-associated after dimerization

Endocytosis of flavivirus NS1 is required for NS1-mediated endothelial hyperpermeability and is abolished by a single N-glycosylation site mutation

Critical role of Dengue Virus NS1 protein in viral replication

Characterization of Asn130-to-Ala mutant of dengue type 1 virus NS1 protein

Growth restriction of dengue virus type 2 by site-specific mutagenesis of virus-encoded glycoproteins

Chimeric tick-borne encephalitis and dengue type 4 viruses: effects of mutations on neurovirulence in mice

Deglycosylation of the NS1 protein of dengue 2 virus, strain 16681: construction and characterization of mutant viruses

Flavivirus NS1 triggers tissue-specific vascular endothelial dysfunction reflecting disease tropism

Mutagenesis of the N-linked glycosylation sites of the yellow fever virus NS1 protein: effects on virus replication and mouse neurovirulence

Development and characterization of non-glycosylated E and NS1 mutant viruses as a potential candidate vaccine for West Nile virus

Characterization of Sepik and Entebbe bat viruses closely related to yellow fever virus

Genome sequence of tick-borne encephalitis virus (Western subtype) and comparative analysis of nonstructural proteins with other flaviviruses

Multiple amino acid changes at the first glycosylation motif in NS1 protein of West Nile virus are necessary for complete attenuation for mouse neuroinvasiveness

Murine hepatitis virus nonstructural protein 4 regulates virus-induced membrane modifications and replication complex function

Post-Translational modifications of coronavirus proteins: roles and function

Expression of nonstructural rotavirus protein NSP4 mimics Ca2+ homeostasis changes induced by rotavirus infection in cultured cells

Identification of a novel determinant for membrane association in hepatitis C virus nonstructural protein 4B

An amphipathic alpha-helix at the C terminus of hepatitis C virus nonstructural protein 4B mediates membrane association

We thank Ms. Qianqian Zhang, Fujian Agriculture and Forestry University, for the critical reading of the manuscript and valuable discussions.

No potential conflict of interest was reported by the author(s).

Data sharing not applicable to this article as no data sets were generated or analyzed during the current study. Data cited in this review are published and available online or upon request from the authors of the respective publications.