Glycosylation Of Viral Proteins Research Articles

N-glycosylation of the SARS-CoV-2 spike protein at Asn331 and Asn343 is involved in spike-ACE2 binding, virus entry, and regulation of IL-6.

The coronavirus disease 2019 (COVID-19) pandemic is an ongoing global public health crisis. The causative agent, the SARS-CoV-2 virus, enters host cells via molecular interactions between the viral spike protein and the host cell ACE2 surface protein. The SARS-CoV-2 spike protein is extensively decorated with up to 66 N-linked glycans. Glycosylation of viral proteins is known to function in immune evasion strategies but may also function in the molecular events of viral entry into host cells. Here, we show that N-glycosylation at Asn331 and Asn343 of SARS-CoV-2 spike protein is required for it to bind to ACE2 and for the entry of pseudovirus harboring the SARS-CoV-2 spike protein into cells. Interestingly, high-content glycan binding screening data have shown that N-glycosylation of Asn331 and Asn343 of the RBD is important for binding to the specific glycan molecule G4GN (Galβ-1,4 GlcNAc), which is critical for spike-RBD-ACE2 binding. Furthermore, IL-6 was identified through antibody array analysis of conditioned media of the corresponding pseudovirus assay. Mutation of N-glycosylation of Asn331 and Asn343 sites of the spike receptor-binding domain (RBD) significantly reduced the transcriptional upregulation of pro-inflammatory signaling molecule IL-6. In addition, IL-6 levels correlated with spike protein levels in COVID-19 patients'serum. These findings establish the importance of RBD glycosylation in SARS-CoV-2 pathogenesis, which can be exploited for the development of novel therapeutics for COVID-19.

Validation of Candidate Host Cell Entry Factors for Bovine Herpes Virus Type-1 Based on a Genome-Wide CRISPR Knockout Screen.

To identify host factors that affect Bovine Herpes Virus Type 1 (BoHV-1) infection we previously applied a genome wide CRISPR knockout screen targeting all bovine protein coding genes. By doing so we compiled a list of both pro-viral and anti-viral proteins involved in BoHV-1 replication. Here we provide further analysis of those that are potentially involved in viral entry into the host cell. We first generated single cell knockout clones deficient in some of the candidate genes for validation. We provide evidence that Polio Virus Receptor-related protein (PVRL2) serves as a receptor for BoHV-1, mediating more efficient entry than the previously identified Polio Virus Receptor (PVR). By knocking out two enzymes that catalyze HSPG chain elongation, HST2ST1 and GLCE, we further demonstrate the significance of HSPG in BoHV-1 entry. Another intriguing cluster of candidate genes, COG1, COG2 and COG4-7 encode six subunits of the Conserved Oligomeric Golgi (COG) complex. MDBK cells lacking COG6 produced fewer but bigger plaques compared to control cells, suggesting more efficient release of newly produced virions from these COG6 knockout cells, due to impaired HSPG biosynthesis. We further observed that viruses produced by the COG6 knockout cells consist of protein(s) with reduced N-glycosylation, potentially explaining their lower infectivity. To facilitate candidate validation, we also detailed a one-step multiplex CRISPR interference (CRISPRi) system, an orthogonal method to KO that enables quick and simultaneous deployment of three CRISPRs for efficient gene inactivation. Using CRISPR3i, we verified eight candidates that have been implicated in the synthesis of surface heparan sulfate proteoglycans (HSPGs). In summary, our experiments confirmed the two receptors PVR and PVRL2 for BoHV-1 entry into the host cell and other factors that affect this process, likely through the direct or indirect roles they play during HSPG synthesis and glycosylation of viral proteins.

Site-specific glycosylation of SARS-CoV-2: Big challenges in mass spectrometry analysis.

Glycosylation of viral proteins is required for the progeny formation and infectivity of virtually all viruses. It is increasingly clear that distinct glycans also play pivotal roles in the virus's ability to shield and evade the host's immune system. Recently, there has been a great advancement in structural identification and quantitation of viral glycosylation, especially spike proteins. Given the ongoing pandemic and the high demand for structure analysis of SARS‐CoV‐2 densely glycosylated spike protein, mass spectrometry methodologies have been employed to accurately determine glycosylation patterns. There are still many challenges in the determination of site‐specific glycosylation of SARS‐CoV‐2 viral spike protein. This is compounded by some conflicting results regarding glycan site occupancy and glycan structural characterization. These are probably due to differences in the expression systems, form of expressed spike glycoprotein, MS methodologies, and analysis software. In this review, we recap the glycosylation of spike protein and compare among various studies. Also, we describe the most recent advancements in glycosylation analysis in greater detail and we explain some misinterpretation of previously observed data in recent publications. Our study provides a comprehensive view of the spike protein glycosylation and highlights the importance of consistent glycosylation determination.

Protein and peptide research applied to Covid-19 and SARS-Cov-2

The emergence of SARS-Cov-2 remains a challenge for the global scientific community. Many efforts have been made to rapidly develop vaccines against SARS-Cov-2. Currently available vaccines, based on the spike glycoprotein (S), are effective in generating neutralising antibodies to prevent severe acute forms of Covid-19. However, the emergence and spread of SARS-Cov-2 variants with mutations in the S protein raises questions about the need to adapt the vaccine strategy to the evolution of the virus. Proteome analysis of many SARS-Cov-2 clinical isolates suggests the development of vaccines based on peptides corresponding to epitopes located in the constant regions of viral proteins that could be effective despite the appearance of variants or the emergence of new strains of beta coronavirus. Other therapeutic innovations against Covid-19 could come from the development of peptides that inhibit structural and non-structural viral proteins. Beyond the therapeutic aspects, research on Covid-19-related proteins and peptides is also directed towards the development of diagnostic tools. The study of the glycosylation of viral proteins and the ACE-2 receptor is important for the development of therapies and diagnostics.

Cyclopentenone Prostaglandins: Biologically Active Lipid Mediators Targeting Inflammation.

Cyclopentenone prostaglandins (cyPGs) are biologically active lipid mediators, including PGA2, PGA1, PGJ2, and its metabolites. cyPGs are essential regulators of inflammation, cell proliferation, apoptosis, angiogenesis, cell migration, and stem cell activity. cyPGs biologically act on multiple cellular targets, including transcription factors and signal transduction pathways. cyPGs regulate the inflammatory response by interfering with NF-κB, AP-1, MAPK, and JAK/STAT signaling pathways via both a group of nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-γ) dependent and PPAR-γ independent mechanisms. cyPGs promote the resolution of chronic inflammation associated with cancers and pathogen (bacterial, viral, and parasitic) infection. cyPGs exhibit potent effects on viral infections by repressing viral protein synthesis, altering viral protein glycosylation, inhibiting virus transmission, and reducing virus-induced inflammation. We summarize their anti-proliferative, pro-apoptotic, cytoprotective, antioxidant, anti-angiogenic, anti-inflammatory, pro-resolution, and anti-metastatic potential. These properties render them unique therapeutic value, especially in resolving inflammation and could be used in adjunct with other existing therapies. We also discuss other α, β -unsaturated carbonyl lipids and cyPGs like isoprostanes (IsoPs) compounds.

Abstract 713: Inhibition of pseudo SARS-CoV-2 binding activity of a anti-cancer polypharmacological agent analogs

Abstract Severe acute respiratory syndrome coronavirus (SARS-CoV-2), a single-stranded RNA virus with largest genomes (∼ 29,800 bases) of all viruses, causes the infectious disease, COVID-19. SARS-CoV-2 is a zoonotic betacoronavirus transmitted through airborne and fecal-oral routes infecting nasal, lung, and intestinal tissues causing over 26 million confirmed coronavirus disease 2019 (COVID-19) cases and nearly 900,000 associated deaths worldwide as of September 2020. The surface of SARS-CoV-2 is covered with copies of spike glycoprotein (SGP) that plays a pivotal role in virus entry in host cells utilizing angiotensin-converting enzyme 2 (ACE2) as an attachment and entry receptor. We have successfully made a high titer replication-incompetent third-generation lentiviral vector pseudotyped with SARS-2-CoV SGP (pLV-S). Upon successful attachment and entry, the lentiviral vector causes enhanced green fluorescent protein (eGFP) to be expressed in the infected cell. Here, we show how the polypharmacological agent, 2-Deoxy-D-glucose (2-DG), a glucose mimic, and well characterized anti-cancer drug, and its analogs inhibit spike protein binding to the ACE2 receptors. This is as suggested from the 2DG and two analogs inhibition, with a low micromolar affinity, the pLV-S attachment and entry resulting in reduced cell fluorescence of infected HEK293 cells. In addition, we describe 2DG and analogs has the potential to deliver polypharmacological effects on the COVID-through inhibition of glycolysis (at energy status), modulation of inflammatory responses (cytokine storm) and alterations in glycosylation of viral proteins. A unique class of adjuvants and mitigants in the anti-SARS-CoV-2 activity is presented here. Citation Format: Rao V. Papineni, Amitava Adhikary, Ritesh Tandon, Dipanwita Mitra. Inhibition of pseudo SARS-CoV-2 binding activity of a anti-cancer polypharmacological agent analogs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 713.

Glycans of SARS-CoV-2 Spike Protein in Virus Infection and Antibody Production.

Viral protein glycosylation represents a successful strategy employed by the parasite to take advantage of host–cell machinery for modification of its own proteins. The resulting glycans have unneglectable roles in viral infection and immune response. The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which presents on the surface of matured virion and mediates viral entry into the host, also undergoes extensive glycosylation to shield it from the human defense system. It is believed that the ongoing COVID-19 pandemic with more than 90,000,000 infections and 1,900,000 deaths is partly due to its successful glycosylation strategy. On the other hand, while glycan patches on S protein have been reported to shield the host immune response by masking “nonself” viral peptides with “self-glycans,” the epitopes are also important in eliciting neutralizing antibodies. In this review, we will summarize the roles of S protein glycans in mediating virus–receptor interactions, and in antibody production, as well as indications for vaccine development.

Glycan Cluster Shielding and Antibody Epitopes on Lassa Virus Envelop Protein.

An understanding of how an antiviral monoclonal antibody recognizes its target is vital for the development of neutralizing antibodies and vaccines. The extensive glycosylation of viral proteins almost certainly affects the antibody response, but the investigation of such effects is hampered by the huge range of structures and interactions of surface glycans through their inherent complexity and flexibility. Here, we built an atomistic model of a fully glycosylated envelope protein complex of the Lassa virus and performed molecular dynamics simulations to characterize the impact of surface glycans on the antibody response. The simulations attested to the variety of conformations and interactions of surface glycans. The results show that glycosylation nonuniformly shields the surface of the complex and only marginally affects protein dynamics. The glycans gather in distinct clusters through interaction with protein residues, and only a few regions are left accessible by an antibody. We successfully recovered known protein epitopes by integrating the simulation results with existing sequence- and structure-based epitope prediction methods. The results emphasize the rich structural environment of glycans and demonstrate that shielding is not merely envelopment by a uniform blanket of sugars. This work provides a molecular basis for integrating otherwise elusive structural properties of glycans into vaccine and neutralizing antibody developments.

Does Antigen Glycosylation Impact the HIV-Specific T Cell Immunity?

It is largely unknown how post-translational protein modifications, including glycosylation, impacts recognition of self and non-self T cell epitopes presented by HLA molecules. Data in the literature indicate that O- and N-linked glycosylation can survive epitope processing and influence antigen presentation and T cell recognition. In this perspective, we hypothesize that glycosylation of viral proteins and processed epitopes contribute to the T cell response to HIV. Although there is some evidence for T cell responses to glycosylated epitopes (glyco-epitopes) during viral infections in the literature, this aspect has been largely neglected for HIV. To explore the role of glyco-epitope specific T cell responses in HIV infection we conducted in silico and ex vivo immune studies in individuals with chronic HIV infection. We found that in silico viral protein segments with potentially glycosylable epitopes were less frequently targeted by T cells. Ex vivo synthetically added glycosylation moieties generally masked T cell recognition of HIV derived peptides. Nonetheless, in some cases, addition of simple glycosylation moieties produced neo-epitopes that were recognized by T cells from HIV infected individuals. Herein, we discuss the potential importance of these observations and compare limitations of the employed technology with new methodologies that may have the potential to provide a more accurate assessment of glyco-epitope specific T cell immunity. Overall, this perspective is aimed to support future research on T cells recognizing glycosylated epitopes in order to expand our understanding on how glycosylation of viral proteins could alter host T cell immunity against viral infections.

A Path toward SARS-CoV-2 Attenuation: Metabolic Pressure on CTP Synthesis Rules the Virus Evolution.

In the context of the COVID-19 pandemic, we describe here the singular metabolic background that constrains enveloped RNA viruses to evolve towards likely attenuation in the long term, possibly after a step of increased pathogenicity. Cytidine triphosphate (CTP) is at the crossroad of the processes allowing SARS-CoV-2 to multiply, because CTP is in demand for four essential metabolic steps. It is a building block of the virus genome, it is required for synthesis of the cytosine-based liponucleotide precursors of the viral envelope, it is a critical building block of the host transfer RNAs synthesis and it is required for synthesis of dolichol-phosphate, a precursor of viral protein glycosylation. The CCA 3’-end of all the transfer RNAs required to translate the RNA genome and further transcripts into the proteins used to build active virus copies is not coded in the human genome. It must be synthesized de novo from CTP and ATP. Furthermore, intermediary metabolism is built on compulsory steps of synthesis and salvage of cytosine-based metabolites via uridine triphosphate (UTP) that keep limiting CTP availability. As a consequence, accidental replication errors tend to replace cytosine by uracil in the genome, unless recombination events allow the sequence to return to its ancestral sequences. We document some of the consequences of this situation in the function of viral proteins. This unique metabolic setup allowed us to highlight and provide a raison d’être to viperin, an enzyme of innate antiviral immunity, which synthesizes 3ʹ-deoxy-3′,4ʹ-didehydro-CTP (ddhCTP) as an extremely efficient antiviral nucleotide.

Intact Glycopeptide Analysis of Influenza A/H1N1/09 Neuraminidase Revealing the Effects of Host and Glycosite Location on Site-Specific Glycan Structures.

Influenza H1N1 virus has posed a serious threat to human health. The glycosylation of neuraminidase (NA) could affect the infectivity and virulence of the influenza virus, but detailed site-specific glycosylation information of NA is still missing. In this study, intact glycopeptide analysis is performed on an influenza NA (A/H1N1/California/2009) that is expressed in human 293T and insect Hi-5 cells. The data indicate that three of four potential N-linked glycosylation sites are glycosylated, including one partial glycosylation site from both cell lines. The NA expressed in human cells has more complex glycans than that of insect cells, suggesting the importance of selecting an appropriate expression system for the production of functional glycoproteins. Different types of glycans are identified from different glycosites of NA expressed in human cells, which implies the site-dependence of glycosylation on NA. This study provides valuable information for the research of influenza virus as well as the functions of viral protein glycosylation.

Influenza Virus Hemagglutinin Glycoproteins with Different N-Glycan Patterns Activate Dendritic Cells In Vitro.

Influenza virus hemagglutinin (HA) N-glycans play important regulatory roles in the control of virus virulence, antigenicity, receptor-binding specificity, and viral escape from the immune response. Considered essential for controlling innate and adaptive immune responses against influenza virus infections, dendritic cells (DCs) trigger proinflammatory and adaptive immune responses in hosts. In this study, we engineered Chinese hamster ovary (CHO) cell lines expressing recombinant HA from pandemic H1, H5, and H7 influenza viruses. rH1HA, rH5HA, and rH7HA were obtained as wild-type proteins or in the presence of kifunensine (KIF) or further with endo-β-N-acetylglucosaminidase-treated KIF (KIF+E) to generate single-N-acetylglucosamine (GlcNAc) N-glycans consisting of (i) terminally sialylated complex-type N-glycans, (ii) high-mannose-type N-glycans, and (iii) single-GlcNAc-type N-glycans. Our results show that high-mannose-type and single-GlcNAc-type N-glycans, but not complex-type N-glycans, are capable of inducing more active hIL12 p40, hIL12 p70, and hIL-10 production in human DCs. Significantly higher HLA-DR, CD40, CD83, and CD86 expression levels, as well reduced endocytotic capacity in human DCs, were noted in the high-mannose-type rH1HA and single-GlcNAc-type rH1HA groups than in the complex-type N-glycan rH1HA group. Our data indicate that native avian rHA proteins (H5N1 and H7N9) are more immunostimulatory than human rHA protein (pH1N1). The high-mannose-type or single-GlcNAc-type N-glycans of both avian and human HA types are more stimulatory than the complex-type N-glycans. HA-stimulated DC activation was accomplished partially through a mannose receptor(s). These results provide more understanding of the contribution of glycosylation of viral proteins to the immune responses and may have implications for vaccine development. Influenza viruses trigger seasonal epidemics or pandemics with mild-to-severe consequences for human and poultry populations. DCs are the most potent professional antigen-presenting cells, which play a crucial role in the link between innate and adaptive immunity. In this study, we obtained stable-expression CHO cells to produce rH1HA, rH5HA, and rH7HA proteins containing distinct N-glycan patterns. These rHA proteins, each with a distinct N-glycan pattern, were used to investigate interactions with mouse and human DCs. Our data indicate that native avian rHA proteins (H5N1 and H7N9) are more immunostimulatory than human rHA protein (pH1N1). High-mannose-type and single-GlcNAc-type N-glycans were more effective than complex-type N-glycans in triggering mouse and human DC activation and maturation. We believe these results provide some useful information for influenza vaccine development regarding how influenza virus HA proteins with different types of N-glycans activate DCs.

Recent advances in synthetic carbohydrate-based human immunodeficiency virus vaccines.

An effective vaccine for human immunodeficiency virus (HIV) is urgently needed to prevent HIV infection and progression to acquired immune deficiency syndrome (AIDS). As glycosylation of viral proteins becomes better understood, carbohydrate-based antiviral vaccines against special viruses have attracted much attention. Significant efforts in carbohydrate synthesis and immunogenicity research have resulted in the development of multiple carbohydrate-based HIV vaccines. This review summarizes recent advances in synthetic carbohydrate-based vaccines design strategies and the applications of these vaccines in the prevention of HIV.

Cytomegalovirus-mediated activation of pyrimidine biosynthesis drives UDP-sugar synthesis to support viral protein glycosylation.

Human cytomegalovirus (HCMV) induces numerous changes to the host metabolic network that are critical for high-titer viral replication. We find that HCMV infection substantially induces de novo pyrimidine biosynthetic flux. This activation is important for HCMV replication because inhibition of pyrimidine biosynthetic enzymes substantially decreases the production of infectious virus, which can be rescued through medium supplementation with pyrimidine biosynthetic intermediates. Metabolomic analysis revealed that pyrimidine biosynthetic inhibition considerably reduces the levels of various UDP-sugar metabolites in HCMV-infected, but not mock-infected, cells. Further, UDP-sugar biosynthesis, which provides the sugar substrates required for glycosylation reactions, was found to be induced during HCMV infection. Pyrimidine biosynthetic inhibition also attenuated the glycosylation of the envelope glycoprotein B (gB). Both glycosylation of gB and viral growth were restored by medium supplementation with either UDP-sugar metabolites or pyrimidine precursors. These results indicate that HCMV drives de novo-synthesized pyrimidines to UDP-sugar biosynthesis to support virion protein glycosylation. The importance of this link between pyrimidine biosynthesis and UDP-sugars appears to be partially shared among diverse virus families, because UDP-sugar metabolites rescued the growth attenuation associated with pyrimidine biosynthetic inhibition during influenza A and vesicular stomatitis virus infection, but not murine hepatitis virus infection. In total, our results indicate that viruses can specifically modulate pyrimidine metabolic flux to provide the glycosyl subunits required for protein glycosylation and production of high titers of infectious progeny.

CpG-E1E2 glycosylation-mutants of Hepatitis C Virus enhance specific cellular immune response and neutralizing antibodes (113.14)

Abstract Hepatitis C virus (HCV) infects 170 million people world wide, and about 80% of the infected individuals develop chronic hepatitis with a risk of progression to cirrhosis and hepatocellular carcinoma. In contrast to hepatitis A and B virus, there is no vaccine for HCV. Thus, the development of an idea vaccine against hepatitis C remains a high prority goal. N-linked glycosylation of viral proteins have been implicated in immunicity. HCV neucleocapsids consist of two highly glycosylated envelop protein E1 and E2, in which the E1 contains 5 N-linked glycans and the E2 has 11 N-glycosylation sites. These oligose chains are essential for correct folding, assemble, and antigenic variation of virus and important for inducing immune response. In this study, the effects of the N-linked glycosylation of HCV E1-E2 fusion protein, a naturally poor immunogen, on the induction of specific immune response were examined. Different E1E2 glycosylation mutants were constructed into pVAX -1 and were used as DNA vaccine. We found that some fusion E1E2 mutants and CpG-E1E2 mutants could enhance much higher specific cellular and humoral reesponses than the wild type of E1E2 in the immunized BALB/c mice. Specific mutants were determined and could be as a HCV candidcate vaccine for its higher induction of specific cellular and neutralizing antibody immune responses.

Abstract 2725: 2-Deoxyglucose inhibits KSHV replication under normoxic conditions

Abstract BACKGROUND: Cancer cells are more glycolytic than normal cells and thereby take up more glucose, as demonstrated by the positron emission tomography (PET) scan. This can be exploited for cancer therapy using glycolytic inhibitors such as 2-deoxy-D-glucose (2DG). Due to its similarity to mannose, 2DG also interferes with N-linked glycosylation resulting in an unfolded protein response (UPR), which slows growth of tumors. Additionally, 2DG inhibits productive infection of enveloped viruses, e.g. herpesviruses, due to its interference with glycosylation of viral proteins. Kaposi's sarcoma (KS) is a cancer that is caused by infection of a gammaherpesvirus; KS might be particularly vulnerable to 2DG, given the chemical's properties as both an anti-cancer and an anti-viral agent. We are investigating the mechanisms and their resulting effects by which cell culture models for KS are sensitive to 2DG treatment in the context of viral replication. DESIGN: Two models of herpesvirus infection were used for 2DG treatment in vitro: KS-associated herpesvirus (KSHV) and murine herpesvirus-68 (MHV-68). Human embryonic kidney cells latently infected with a recombinant KSHV genome (HEK293rKSHV.219) were induced to lytic reactivation upon addition of sodium butyrate. Murine embryonic fibroblasts were infected de novo with MHV-68 to cause lytic replication. In each model, cells were treated with nontoxic doses of 2DG. RNA and protein levels were measured at 24 hours treatment; cell proliferation and viral copy number were measured at 72 hours. RESULTS: In two different models of gammaherpesvirus infection we find that 2DG inhibits expression of early and late viral lytic genes, as well as interferes with the ability of the host to produce infectious virions. While 2DG is used at nontoxic concentrations, the compound inhibits cell proliferation. Treatment with inhibitors of glycolysis–but not glycosylation–does not significantly decrease viral copy number or infectious particles released from infected cells. Furthermore, 2DG is correlated with UPR induction by the phosphorylation of the alpha subunit of eukaryotic translation initiation factor-2 as well as up-regulation of the endoplasmic reticulum (ER) chaperone glucose-regulated protein-78 at both the RNA and protein levels. CONCLUSION: Overall, these results indicate that a major mechanism by which 2DG inhibits viral replication might be through ER stress/UPR. Furthermore, 2DG action takes place early in the viral lytic cycle, preventing the induction of viral genes, which correlates to the inability to produce mature viral particles. These data suggest that 2DG may be clinically beneficial in the treatment of herpesvirus-induced cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2725. doi:10.1158/1538-7445.AM2011-2725

Effect of tunicamycin on the biogenesis of hepatitis C virus glycoproteins.

Hepatitis C virus (HCV) infects humans, with a prevalence around 3% of population, causing acute and chronic hepatitis and hepatocellular carcinoma. We studied the effect of inhibition of glycosylation on the assembly of the HCV particle. HCV possesses two envelope glycoproteins E1 and E2 that are highly modified by N-glycans. These glycan residues are crucial for viral entry and maturation of the progeny. Here, we examined the influence of inhibition of N-glycosylation on expression of E1 and E2. Since the propagation of HCV in cell culture is limited, we used a recombinant baculovirus producing viral-like particles in insect cells. Our data showed that blocking of N-glycan transfer to the nascent polypeptide chain with the antibiotic tunicamycin resulted in the loss of E1 and E2. We also found that a dose of tunicamycin that did not influence the cell viability significantly reduced the E2 level in infected cells. The results indicate that blocking of glycosylation at an early step efficiently reduces the assembly of HCV virions. Thus, we suggest that derivatives of tunicamycin that preferentially block glycosylation of viral proteins may become potential therapeutic agents against HCV.

Development of a proteomic platform for EBV and KSHV serological screening

In the context of HIV infection, there is an increased susceptibility to EBV- and KSHV-associated malignancies. Evaluating a possible role for specific humoral responses to individual viral proteins in the control of viral load and prevention of EBV or KSHV related disease development in this population has been hampered by the limited availability of suitable screening reagents. We have developed a protein array platform that can be utilized for EBV and KSHV serological screening. The array consists of EBV and KSHV proteins printed onto glass slides. All eighty-four EBV open reading frames and eighty-six KSHV open reading frames were cloned into bacterial vectors and the inserts validated by DNA sequencing in both directions. Authenticated inserts were transferred to a yeast vector that expresses proteins as N-terminal GST-fusions. Seventy-nine EBV proteins and eighty-one KSHV proteins were successfully purified from yeast. First-generation EBV/KSHV protein arrays have been printed with these proteins plus a variety of control proteins that include GST, and human IgG, IgM, and IgA. Glycosylation of viral proteins may affect immunogenicity. To evaluate this aspect, EBV and KSHV virion glycoproteins are being expressed in insect cells modified with the human glycosylation machinery. Once purified, these proteins will be added to the EBV/KSHV protein arrays. The ability to compare serological responses to the complete repertoire of individual EBV and KSHV encoded proteins should provide new insight into B cell mediated immune regulation of these viruses.

Spike protein VP4 assembly with maturing rotavirus requires a postendoplasmic reticulum event in polarized caco-2 cells.

Rotavirus assembly is a multistep process that requires the successive association of four major structural proteins in three concentric layers. It has been assumed until now that VP4, the most external viral protein that forms the spikes of mature virions, associates with double-layer particles within the endoplasmic reticulum (ER) in conjunction with VP7 and with the help of a nonstructural protein, NSP4. VP7 and NSP4 are two glycosylated proteins. However, we recently described a strong association of VP4 with raft-type membrane microdomains, a result that makes the ER a highly questionable site for the final assembly of rotavirus, since rafts are thought to be absent from this compartment. In this study, we used tunicamycin (TM), a drug known to block the first step of protein N glycosylation, as a tool to dissect rotavirus assembly. We show that, as expected, TM blocks viral protein glycosylation and also decreases virus infectivity. In the meantime, viral particles were blocked as enveloped particles in the ER. Interestingly, TM does not prevent the targeting of VP4 to the cell surface nor its association with raft membranes, whereas the infectivity associated with the raft fractions strongly decreased. VP4 does not colocalize with the ER marker protein disulfide-isomerase even when viral particles were blocked by TM in this compartment. These results strongly support a primary role for raft membranes in rotavirus final assembly and the fact that VP4 assembly with the rest of the particle is an extrareticular event.

Identification of a glycosylation site in the woodchuck hepatitis virus preS2 protein and its role in protein trafficking.

The middle surface antigen (M-sAg) of hepadnaviruses is one of three envelope proteins that share a common C-terminal S domain. M-sAg contains the preS2 domain in addition to the S region. The preS2 region of woodchuck hepatitis virus (WHV) contains a potential glycosylation site Asn-Gln-Thr at amino acid (aa) positions 3-5. In this study, we mutated this site by site-directed mutagenesis and confirmed that glycosylation occurs here. In in vitro translation assays, the mutation Thr to Asn at aa 5 significantly impaired glycosylation of M-sAg. The mutated M-sAg formed abnormal clustered structures in transfected cells as determined by immunofluorescent staining. Confocal microscopic analysis showed that an enrichment of this glycosylation-deficient protein in the Golgi apparatus occurred, which is not typical for the wild-type protein. These results are consistent with earlier findings that incorrect glycosylation of viral proteins may interfere with virus assembly.