Single N-linked Glycosylation Site Research Articles

N-glycosylation in the Pre-Membrane Protein Is Essential for the Zika Virus Life Cycle.

Asparagine (N)-linked protein glycosylation plays an important role in protein synthesis and modification. Two Zika virus (ZIKV) structural proteins, the pre-membrane (prM) and envelope (E) protein are N-glycosylated. The prM protein of all ZIKV strains contains a single N-linked glycosylation site, while not all strains contain an N-linked site in the E protein. Our aim was to examine the impact of prM and E N-linked glycosylation on ZIKV infectivity and cell trafficking. Using a ZIKV infectious clone, we found that when the N-glycan sites were removed, the prM- and the prM/E-double mutants did not produce an infectious virus in the supernatant. Further, by using ZIKV prME constructs, we found that N-glycosylation was necessary for effective secretion of ZIKV virions. The absence of the N-glycan on prM or E caused protein aggregation in the rough endoplasmatic reticulum (ER) compartment. The aggregation was more pronounced for the prM-mutation, and the mutant virus lost the ER-Golgi intermediate compartment (ERGIC) localization. In addition, lack of the N-glycan on prM induced nuclear translocation of CCAAT-enhancer-binding protein homologous protein (CHOP), an ER stress marker. To conclude, we show that the prM N-glycan is essential for the ZIKV infectious cycle, and plays an important role in viral protein trafficking, protein folding, and virion assembly.

Molecular characterization of MHC class II DRA cDNA in Deoni cattle

The present study was undertaken to clone and characterize DRA gene in Deoni cattle. The cDNA for the DRA gene was amplified by using specific primers designed based on available cattle sequences and purified products were cloned in competent E.coli (DH5á) strain. The full length 1013bp product of cDNA of DRA contained a single ORF of 762 nucleotides that coded for 253 amino acids translated product. Twenty four amino acids formed signal peptide while 229 constituted mature peptide. The deduced amino acid sequences resembled those of class II molecules of other species for all the conserved residues having critical functional role. But a single N-linked glycosylation site in á1 was observed in cattle and buffalo when compared to human and swine which contain a second site in á2 domain. The signal peptide was found more variable among the species compared. Comparison of nucleotide and amino acid sequences among related species and dendrogram constructed revealed that the cattle sequences are more similar to buffalo sequences.

The Zika virus envelope protein glycan loop regulates virion antigenicity

Because antibodies are an important component of flavivirus immunity, understanding the antigenic structure of flaviviruses is critical. Compared to dengue virus (DENV), the loop containing the single N-linked glycosylation site on Zika virus (ZIKV) envelope (E) proteins extends further towards the DII fusion loop (DII-FL) on neighboring E proteins within E dimers on mature viruses. Although ZIKV is poorly neutralized by DII-FL antibodies, we demonstrated significantly increased neutralization sensitivity of ZIKV particles incorporating the DENV glycan loop. Increased neutralization sensitivity was independent of E protein glycosylation: ZIKV lacking E protein glycans remained poorly neutralized, whereas ZIKV loop chimeras with or without an E protein glycan were potently neutralized. ZIKV particles lacking the E protein glycan were capable of infecting Raji cells expressing the lectin DC-SIGNR, suggesting the prM glycan of partially mature particles can facilitate entry. Our study provides insight into the determinants of ZIKV E protein function and antigenicity.

Engineering Aglycosylated IgG Variants with Wild-Type or Improved Binding Affinity to Human Fc Gamma RIIA and Fc Gamma RIIIAs

The binding of human IgG1 to human Fc gamma receptors (hFcγRs) is highly sensitive to the presence of a single N-linked glycosylation site at asparagine 297 of the Fc, with deglycosylation resulting in a complete loss of hFcγR binding. Previously, we demonstrated that aglycosylated human IgG1 Fc variants can engage the human FcγRII class of the low-affinity hFcγRs, demonstrating that N-linked glycosylation of the Fc is not a strict requirement for hFcγR engagement. In the present study, we demonstrate that aglycosylated IgG variants can be engineered to productively engage with FcγRIIIA, as well as the human Fc gamma RII subset. We also assess the biophysical properties and serum half-life of the aglycosylated IgG variants to measure stability. Aglycosylated constructs N297D/S298T (DTT)–K326I/A327Y/L328G (IYG) and N297D/S298A–IYG optimally drove tumor cell phagocytosis. A mathematical model of phagocytosis suggests that hFcγRI and hFcγRIIIA dimers were the main drivers of phagocytosis. In vivo tumor control of B16F10 lung metastases further confirmed the variant DTT–IYG to be the best at restoring wild-type-like properties in prevention of lung metastases. While deuterium incorporation was similar across most of the protein, several peptides within the CH2 domain of DTT–IYG showed differential deuterium uptake in the peptide region of the FG loop as compared to the aglycosylated N297Q. Thus, in this study, we have found an aglycosylated variant that may effectively substitute for wild-type Fc. These aglycosylated variants have the potential to allow therapeutic antibodies to be produced in virtually any expression system and still maintain effector function.

N-linked glycosylation of N48 is required for equilibrative nucleoside transporter 1 (ENT1) function.

Human equilibrative nucleoside transporter 1 (hENT1) transports nucleosides and nucleoside analogue drugs across cellular membranes and is necessary for the uptake of many anti-cancer, anti-parasitic and anti-viral drugs. Previous work, and in silico prediction, suggest that hENT1 is glycosylated at Asn(48) in the first extracellular loop of the protein and that glycosylation plays a role in correct localization and function of hENT1. Site-directed mutagenesis of wild-type (wt) hENT1 removed potential glycosylation sites. Constructs (wt 3xFLAG-hENT1, N48Q-3xFLAG-hENT1 or N288Q-3xFLAG-hENT2) were transiently transfected into HEK293 cells and cell lysates were treated with or without peptide-N-glycosidase F (PNGase-F), followed by immunoblotting analysis. Substitution of N48 prevents hENT1 glycosylation, confirming a single N-linked glycosylation site. N48Q-hENT1 protein is found at the plasma membrane in HEK293 cells but at lower levels compared with wt hENT1 based on S-(4-nitrobenzyl)-6-thioinosine (NBTI) binding analysis (wt 3xFLAG-ENT1 Bmax, 41.5±2.9pmol/mg protein; N48Q-3xFLAG-ENT1 Bmax, 13.5±0.45pmol/mg protein) and immunofluorescence microscopy. Although present at the membrane, chloroadenosine transport assays suggest that N48Q-hENT1 is non-functional (wt 3xFLAG-ENT1, 170.80±44.01pmol/mg protein; N48Q-3xFLAG-ENT1, 57.91±17.06pmol/mg protein; mock-transfected 74.31±19.65pmol/mg protein). Co-immunoprecipitation analyses suggest that N48Q ENT1 is unable to interact with self or with wt hENT1. Based on these data we propose that glycosylation at N48 is critical for the localization, function and oligomerization of hENT1.

Identification, cDNA Cloning, and Characterization of Luteinizing Hormone Beta Subunit (lhb) Gene in Catla catla

ABSTRACTReproductive hormones play a significant role in the gonadal development and gametogenesis process of animals. In the present study luteinizing hormone beta, (lhb) subunit gene was cloned and characterized from the brain of Catla catla. The lhb full-length of cDNA sequence is 629 bp which consists of 43bp 5'-UTR (untranslated region) 447bp, ORF(open reading frame) and 139 bp of 3'-UTR respectively. The coding region of lhb gene encoded a peptide of 148 amino acids. The coding sequence of lhb gene consist of a single N-linked glycosylation site (NET) and 12 cysteine knot residues. Phylogenetic analysis of C. catla Lhβ deduced amino acid sequence showed high similarity with Carassius auratus followed by Gobiocypris rarus. 3D structure Lhβ protein comprises of five β-sheets and six coils/loops. The qPCR results revealed lhb mRNA is mainly expressed in the pituitary, ovary while moderate expression was observed in brain and testis. To best our knowledge, this is the first report on the identification, molecular characterization and structural information regarding luteinizing hormone in Indian major carp.

Molecular cloning and characterization of the gonadotropin subunits GPα, FSHβ, and LHβ genes in the stinging catfish Heteropneustes fossilis: phylogeny, seasonal expression and pituitary localization.

Gonadotropins are heterodimeric glycoproteins secreted by the pituitary, and consist of a common glycoprotein hormone alpha (GPα) and the function-specific follicle-stimulating hormone beta subunit (FSHβ) or luteinizing hormone beta subunit (LHβ). In the present study, the subunit protein genes were cloned and characterized from the pituitary of the catfish Heteropneustes fossilis. Full-length cDNAs of GPα, FSHβ, and LHβ are 511 base pairs (bp), 659 bp and 660 bp long, and encode 92, 108, and 112 aminoacids long mature proteins, respectively. GPα has 10 cysteines with 2 N-linked glycosylation sites while LHβ contains 12 cysteines with a single N-linked glycosylation site. In contrast, FSHβ has 13 cysteines, 1 additional over the conserved 12 cysteines of other vertebrates, and a single glycosylation site between Cys 3 and Cys 4. Phylogenetic analyses of the deduced proteins confirm their homology and relationships with the respective gonadotropin subunit proteins of gnathostome vertebrates. Tissue expression analysis by semi-quantitative RT-PCR shows that GPα mRNA is expressed only in the pituitary while both FSHβ and LHβ mRNA are expressed in extra-pituitary sites. The subunit mRNAs show both seasonal and sex dimorphic variations especially in the expression of FSHβ and LHβ transcripts. In the sexually quiescent phase, the transcript expression is low while in the recrudescent phase, the expressions are differential, high, and varied with regard to sex and reproductive phase. In situ hybridization of the mRNAs gave positive signals in gonadotropes in the pars distalis of the pituitary, which exhibited seasonal variation in staining intensity and numbers.

Molecular cloning, characterization and mRNA expression of duck interleukin-17F

Interleukin-17F (IL-17F) is a proinflammatory cytokine that plays an important role in gut homeostasis. A full-length duck IL-17F (duIL-17F) cDNA with a 510-bp coding region was identified in ConA-activated splenic lymphocytes. duIL-17F is predicted to encode 166 amino acids, including a 26-amino acid signal peptide, a single N-linked glycosylation site, and six cysteine residues that are conserved in mammalian IL-17. duIL-17F shares 77.5% amino acid sequence identity with chicken IL-17F (chIL-17F), 37–46% with corresponding mammalian homologues, and 53.5% with the previously described duck IL-17A (duIL-17A). The duIL-17F transcripts were expressed in a wide range of untreated tissues; levels were highest in the liver and moderate in the thymus, bursa, kidney, and intestinal tissues. Expression levels of duIL-17F transcript were slightly up-regulated in ConA- and LPS-activated splenic lymphocytes but not in poly I:C stimulated cells. duIL-17F forms heterodimers with duIL-17A. Recombinant duIL-17F, like duIL-17A, induced IL-1β, IL-6, and IL-8 expression in duck embryonic fibroblasts (DEFs). duIL-17A, but not duIL-17F expression, was significantly up-regulated in the liver and spleen of Salmonella Typhimurium-infected ducks. Further analysis of the contributions of IL-17F to different Salmonella spp. or other disease models will be required to expand our understanding of its biological functions.

Cloning and characterization of goose interleukin-17A cDNA

Interleukin-17 (IL-17 or IL-17A) is a proinflammatory cytokine produced by activated T cells. IL-17A plays important roles in inflammation and host defense. In this study, the cDNA of the goose IL-17A (GoIL-17A) gene was cloned from thymocytes. Recombinant GoIL-17A (rGoIL-17A) was expressed using a baculovirus expression system and then biologically characterized. The complete open reading frame (ORF) of GoIL-17A contains 510 base pairs that encode 169 amino acid residues, including a 29-amino acid signal peptide and a single potential N-linked glycosylation site. This protein has a molecular weight of 18.9kDa. The amino acid sequence showed 95.9%, 84.6%, 45.0% and 38.4% similarity with the corresponding duck, chicken, rat, and human IL-17A sequences, respectively. The six conserved cysteine residues were also observed in GoIL-17A. A recombinant, mature form of GoIL-17A was produced and its biological activities in goose embryonic fibroblasts were investigated. RT-PCR analysis revealed a marked up-regulation of IL-6 and IL-8 mRNA expression in goose embryonic fibroblasts treated with 1–50μg of rGoIL-17A for 12h. The GoIL-17A gene sequence and the biologically active recombinant protein may be useful for understanding the role of IL-17A in immune regulation.

Reporters for the analysis of N-glycosylation in Candida albicans

A large proportion of Candida albicans cell surface proteins are decorated post-translationally by glycosylation. Indeed N-glycosylation is critical for cell wall biogenesis in this major fungal pathogen and for its interactions with host cells. A detailed understanding of N-glycosylation will yield deeper insights into host-pathogen interactions. However, the analysis of N-glycosylation is extremely challenging because of the complexity and heterogeneity of these structures. Therefore, in an attempt to reduce this complexity and facilitate the analysis of N-glycosylation, we have developed new synthetic C. albicans reporters that carry a single N-linked glycosylation site derived from Saccharomyces cerevisiae Suc2. These glycosylation reporters, which carry C.albicans Hex1 or Sap2 signal sequences plus carboxy-terminal FLAG3 and His6 tags, were expressed in C.albicans from the ACT1 promoter. The reporter proteins were successfully secreted and hyperglycosylated by C.albicans cells, and their outer chain glycosylation was dependent on Och1 and Pmr1, which are required for N-mannan synthesis, but not on Mnt1 and Mnt2 which are only required for O-mannosylation. These reporters are useful tools for the experimental dissection of N-glycosylation and other related processes in C.albicans, such as secretion.

Requirement of Glycosylation of West Nile Virus Envelope Protein for Infection of, but Not Spread within, Culex quinquefasciatus Mosquito Vectors

Most of sequenced West Nile virus (WNV) genomes encode a single N-linked glycosylation site on their envelope (E) proteins. We previously found that WNV lacking the E protein glycan was severely inhibited in its ability to replicate and spread within two important mosquito vector species, Culex pipiens and Cx. tarsalis. However, recent work with a closely related species, Cx. pipiens pallens, found no association between E protein glycosylation and either replication or dissemination. To examine this finding further, we expanded upon our previous studies to include an additional Culex species, Cx. quinquefasciatus. The non-glycosylated WNV-N154I virus replicated less efficiently in mosquito tissues after intrathoracic inoculation, but there was little difference in replication efficiency in the midgut after peroral infection. Interestingly, although infectivity was inhibited when WNV lacked the E protein glycan, there was little difference in viral spread throughout the mosquito. These data indicate that E protein glycosylation affects WNV-vector interactions in a species-specific manner.

Crystal Structure of a Bony Fish β2-Microglobulin: INSIGHTS INTO THE EVOLUTIONARY ORIGIN OF IMMUNOGLOBULIN SUPERFAMILY CONSTANT MOLECULES

Crystal Structure of a Bony Fish β2-Microglobulin: INSIGHTS INTO THE EVOLUTIONARY ORIGIN OF IMMUNOGLOBULIN SUPERFAMILY CONSTANT MOLECULES

A Single Asparagine-Linked Glycosylation Site of the Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Facilitates Inhibition by Mannose-Binding Lectin through Multiple Mechanisms

Mannose-binding lectin (MBL) is a serum protein that plays an important role in host defenses as an opsonin and through activation of the complement system. The objective of this study was to assess the interactions between MBL and severe acute respiratory syndrome-coronavirus (SARS-CoV) spike (S) glycoprotein (SARS-S). MBL was found to selectively bind to retroviral particles pseudotyped with SARS-S. Unlike several other viral envelopes to which MBL can bind, both recombinant and plasma-derived human MBL directly inhibited SARS-S-mediated viral infection. Moreover, the interaction between MBL and SARS-S blocked viral binding to the C-type lectin, DC-SIGN. Mutagenesis indicated that a single N-linked glycosylation site, N330, was critical for the specific interactions between MBL and SARS-S. Despite the proximity of N330 to the receptor-binding motif of SARS-S, MBL did not affect interactions with the ACE2 receptor or cathepsin L-mediated activation of SARS-S-driven membrane fusion. Thus, binding of MBL to SARS-S may interfere with other early pre- or postreceptor-binding events necessary for efficient viral entry.

Escape from Autologous Neutralizing Antibodies in Acute/Early Subtype C HIV-1 Infection Requires Multiple Pathways

One aim for an HIV vaccine is to elicit neutralizing antibodies (Nab) that can limit replication of genetically diverse viruses and prevent establishment of a new infection. Thus, identifying the strengths and weaknesses of Nab during the early stages of natural infection could prove useful in achieving this goal. Here we demonstrate that viral escape readily occurred despite the development of high titer autologous Nab in two subjects with acute/early subtype C infection. To provide a detailed portrayal of the escape pathways, Nab resistant variants identified at multiple time points were used to create a series of envelope (Env) glycoprotein chimeras and mutants within the background of a corresponding newly transmitted Env. In one subject, Nab escape was driven predominantly by changes in the region of gp120 that extends from the beginning of the V3 domain to the end of the V5 domain (V3V5). However, Nab escape pathways in this subject oscillated and at times required cooperation between V1V2 and the gp41 ectodomain. In the second subject, escape was driven by changes in V1V2. This V1V2-dependent escape pathway was retained over time, and its utility was reflected in the virus's ability to escape from two distinct monoclonal antibodies (Mabs) derived from this same patient via introduction of a single potential N-linked glycosylation site in V2. Spatial representation of the sequence changes in gp120 suggested that selective pressure acted upon the same regions of Env in these two subjects, even though the Env domains that drove escape were different. Together the findings argue that a single mutational pathway is not sufficient to confer escape in early subtype C HIV-1 infection, and support a model in which multiple strategies, including potential glycan shifts, direct alteration of an epitope sequence, and cooperative Env domain conformational masking, are used to evade neutralization.

Molecular characterization of duck interleukin-17

Interleukin-17 (IL17), belonging to the Th17 family, is a proinflammatory cytokine produced by activated T cells. A 1034 bp cDNA encoding duck IL17 ( duIL17) was cloned from Con A-activated splenic lymphocytes of ducks. The encoded protein, which is predicted to consist of 169 amino acids, has a molecular weight of 18.8 kDa and includes a 29 residue NH 2-terminal signal peptide, a single potential N-linked glycosylation site, and six cysteine residues that are conserved in mammalian IL17. The duIL17 shared 84% amino acid sequence identity with the previously described chicken IL17 (chIL17), 36–47% to mammalian homologues, and open reading frame 13 of Herpesvirus saimiri (HVS13). The genomic structure of duIL17 was quite similar to its chicken and mammalian counterparts. The duIL17 mRNA expression was detected only in Con A-activated splenic lymphocytes by RT-PCR, although its expression was undetectable in a variety of normal tissues. Two mAbs against chIL17 showed cross-reactivity with duIL17 as detected by indirect ELISA and Western blot analysis. These findings indicate that the structure of IL17 is highly conserved among poultry, and two mAbs detecting common epitopes of IL17 are available for molecular and immunological studies of IL17 in birds.

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

Many, but not all, strains of West Nile virus (WNV) contain a single N-linked glycosylation site on their envelope (E) proteins. Previous studies have shown that E-glycosylated strains are more neuroinvasive in mice than non-glycosylated strains. E protein glycosylation also appears to play a role in attachment and entry of WNV into host cells in vitro; however, studies examining how E protein glycosylation affects the interactions of WNV with its mosquito vectors in vivo have not yet been performed. We mutated the E protein glycosylation site from NYS to IYS in a previously described full-length clone of the NY99 genotype of WNV (WT), resulting in a virus that lacked the glycan at aa154. WNV-N154I replicated less efficiently than WNV-WT in Culex mosquito tissues, although the extent of the decrease was greater in Cx. pipiens than in Cx. tarsalis. Following peroral infection, mosquitoes infected with WNV-N154I were less likely to transmit virus than those infected with WNV-WT. Interestingly, all but one of the mosquitoes infected with WNV-N154I transmitted a revertant virus, suggesting that there is strong selective pressure toward E protein glycosylation. Together these data suggest that loss of the glycan at aa154 on the WNV E protein can severely restrict viral spread in the mosquito vector.

Regions of Melanocortin 2 (MC2) Receptor Accessory Protein Necessary for Dual Topology and MC2 Receptor Trafficking and Signaling

MRAP, melanocortin 2 (MC2) receptor accessory protein, is required for trafficking by the MC2 (ACTH) receptor. MRAP is a single transmembrane protein that forms highly unusual antiparallel homodimers. We used molecular complementation to ask where MRAP achieves dual topology. Fragments of yellow fluorescent protein (YFP) were fused to the NH2 or COOH terminus of MRAP such that YFP fluorescence could occur only in antiparallel homodimers; fluorescence was present in the endoplasmic reticulum. MRAP retained dual topology after deletion of most of the amino terminus. In contrast, deletion of residues 31-37, just NH2-terminal to the transmembrane domain, forced MRAP into a single Nexo/Ccyt orientation and blocked its ability to promote MC2 receptor trafficking and homodimerize. When the transmembrane domain of MRAP was replaced with the corresponding region from RAMP3, dual topology was retained but MRAP was inactive. Insertion of MRAP residues 29-37 conferred dual topology to RAMP3, normally in an Nexo/Ccyt orientation. When expressed with MRAPDelta1-30, MRAPDelta10-20, or MRAPDelta21-30, MC2 receptor was localized on the plasma membrane but unable to respond to ACTH. Residues 18-21 of MRAP were critical; MC2 receptor expressed with MRAP(18-21A) localized to the plasma membrane but did not bind 125I-ACTH or increase cAMP in response to ACTH. A newly identified MRAP homolog, MRAP2, lacks amino acids 18LDYI21 of MRAP and, like MRAP(18-21A), allows MC2 receptor trafficking but not signaling. MRAP2 with an LDYI insertion functions like MRAP. These results demonstrate that MRAP not only facilitates MC2 receptor trafficking but also allows properly localized receptor to bind ACTH and consequently signal.

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

The prM protein of Japanese encephalitis virus (JEV) contains a single potential N-linked glycosylation site, N(15)-X(16)-T(17), which is highly conserved among JEV strains and closely related flaviviruses. To investigate the role of this site in JEV replication and pathogenesis, we manipulated the RNA genome by using infectious JEV cDNA to generate three prM mutants (N15A, T17A, and N15A/T17A) with alanine substituting for N(15) and/or T(17) and one mutant with silent point mutations introduced into the nucleotide sequences corresponding to all three residues in the glycosylation site. An analysis of these mutants in the presence or absence of endoglycosidases confirmed the addition of oligosaccharides to this potential glycosylation site. The loss of prM N glycosylation, without significantly altering the intracellular levels of viral RNA and proteins, led to an approximately 20-fold reduction in the production of extracellular virions, which had protein compositions and infectivities nearly identical to those of wild-type virions; this reduction occurred at the stage of virus release, rather than assembly. This release defect was correlated with small-plaque morphology and an N-glycosylation-dependent delay in viral growth. A more conservative mutation, N15Q, had the same effect as N15A. One of the four prM mutants, N15A/T17A, showed an additional defect in virus growth in mosquito C6/36 cells but not human neuroblastoma SH-SY5Y or hamster BHK-21 cells. This cell type dependence was attributed to abnormal N-glycosylation-independent biogenesis of prM. In mice, the elimination of prM N glycosylation resulted in a drastic decrease in virulence after peripheral inoculation. Overall, our findings indicate that this highly conserved N-glycosylation motif in prM is crucial for multiple stages of JEV biology: prM biogenesis, virus release, and pathogenesis.

Characterization of the cell membrane-associated products of the Neuregulin 4 gene

The NRG4 gene is a member of a family of four genes that encode a class of epidermal growth factors. This gene has been reported to express a protein designated here as NRG4A1. We describe here a novel splice variant of the NRG4 gene, NRG4A2, which encodes a C-terminal region containing a predicted type I PDZ-binding peptide. Both NRG4A1 and NRG4A2 were shown to be expressed on the cell surface, as expected by the presence of a predicted transmembrane sequence, and were modified at a single N-linked glycosylation site in the extracellular domain. Significant stabilization of expression of both proteins was seen in the presence of the proteosome inhibitor MG-132 suggesting that they are normally degraded by this system. N-terminal cleavage was inhibited in both isotypes by the broad-spectrum matrix metalloproteinase inhibitor, galardin (GM 6001). A glycosylated, secreted form of NRG4A1 was detected in the cell medium which showed biological activity in two assays, phosphorylation of the HER4 receptor and stimulation of neurite formation in PC-12 cells stably expressing HER4. Transfection and expression of green fluorescent protein-tagged proteins and immunofluorescent staining with specific anti-peptide antibodies showed that NRG4A1 is localized to membrane ruffles, while NRG4A2 has a more punctate membrane distribution.

Influence of porcine reproductive and respiratory syndrome virus GP5 glycoprotein N-linked glycans on immune responses in mice

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically significant viral diseases in the swine industry. Infection with PRRSV following vaccination is common, since protection is incomplete. Persistent infection may be one of the biggest obstacles to control of the disease. "Glycan shielding" was postulated to be a primary mechanism to explain evasion from neutralizing immune response, ensuring in vivo persistence of virus, such as HIV, SIV, and HBV. The objective of this study was to construct recombinant adenoviruses expressing single or multiple N-linked glycosylation site (NGS) mutant GP5 of PRRSV, and evaluate the expression in cell culture, and potential to induce immune responses in BALB/c mice. Six recombinant adenoviruses were constructed each expressing wild-type GP5 and 1-4 NGS mutants: N44S, N44/51S, N30/44/51S, N30/33/44/51S and N30/33S. Inoculation of BALB/c mice with all five recombinants expressing NGS mutant GP5 resulted in a significant neutralizing antibody responses which were significantly higher than that of recombinant adenovirus expressing wild-type GP5. But there were no significant difference in lymphocyte proliferation responses induced by wild type and NGS mutant GP5. It indicated that glycosylations of GP5 at residues N30, N33, N44 and N51 are critical for induction of neutralizing antibodies. These NGS mutant PRRSV GP5 will be useful to characterize the effects of glycosylation on immunogenicity in the natural host, and may lead to a new approach for the generation of PRRSV vaccines.