Viral Surface Proteins Research Articles

Progress on chicken T cell immunity to viruses.

Avian virus infection remains one of the most important threats to the poultry industry. Pathogens such as avian influenza virus (AIV), avian infectious bronchitis virus (IBV), and infectious bursal disease virus (IBDV) are normally controlled by antibodies specific for surface proteins and cellular immune responses. However, standard vaccines aimed at inducing neutralizing antibodies must be administered annually and can be rendered ineffective because immune-selective pressure results in the continuous mutation of viral surface proteins of different strains circulating from year to year. Chicken T cells have been shown to play a crucial role in fighting virus infection, offering lasting and cross-strain protection, and offer the potential for developing universal vaccines. This review provides an overview of our current knowledge of chicken T cell immunity to viruses. More importantly, we point out the limitations and barriers of current research and a potential direction for future studies.

Influenza A virus surface proteins are organized to help penetrate host mucus.

Influenza A virus (IAV) enters cells by binding to sialic acid on the cell surface. To accomplish this while avoiding immobilization by sialic acid in host mucus, viruses rely on a balance between the receptor-binding protein hemagglutinin (HA) and the receptor-cleaving protein neuraminidase (NA). Although genetic aspects of this balance are well-characterized, little is known about how the spatial organization of these proteins in the viral envelope may contribute. Using site-specific fluorescent labeling and super-resolution microscopy, we show that HA and NA are asymmetrically distributed on the surface of filamentous viruses, creating a spatial organization of binding and cleaving activities that causes viruses to step consistently away from their NA-rich pole. This Brownian ratchet-like diffusion produces persistent directional mobility that resolves the virus's conflicting needs to both penetrate mucus and stably attach to the underlying cells, potentially contributing to the prevalence of the filamentous phenotype in clinical isolates of IAV.

Polyvalent Diazonium Polymers Provide Efficient Protection of Oncolytic Adenovirus Enadenotucirev from Neutralizing Antibodies while Maintaining Biological Activity In Vitro and In Vivo.

Oncolytic viruses offer many advantages for cancer therapy when administered directly to confined solid tumors. However, the systemic delivery of these viruses is problematic because of the host immune response, undesired interactions with blood components, and inherent targeting to the liver. Efficacy of systemically administered viruses has been improved by masking viral surface proteins with polymeric materials resulting in modulation of viral pharmacokinetic profile and accumulation in tumors in vivo. Here we describe a new class of polyvalent reactive polymer based on poly( N-(2-hydroxypropyl)methacrylamide) (polyHPMA) with diazonium reactive groups and their application in the modification of the chimeric group B oncolytic virus enadenotucirev (EnAd). A series of six copolymers with different chain lengths and density of reactive groups was synthesized and used to coat EnAd. Polymer coating was found to be extremely efficient with concentrations as low as 1 mg/mL resulting in complete (>99%) ablation of neutralizing antibody binding. Coating efficiency was found to be dependent on both chain length and reactive group density. Coated viruses were found to have reduced transfection activity both in vitro and in vivo, with greater protection against neutralizing antibodies resulting in lower transgene production. However, in the presence of neutralizing antibodies, some in vivo transgene expression was maintained for coated virus compared to the uncoated control. The decrease in transgene expression was found not to be solely due to lower cellular uptake but due to reduced unpackaging of the virus within the cells and reduced replication, indicating that the polymer coating does not cause permanent inactivation of the virus. These data suggest that virus activity may be modulated by the appropriate design of coating polymers while retaining protection against neutralizing antibodies.

Towards a thermodynamic mechanistic model for the effect of temperature on arthropod vector competence for transmission of arboviruses

Arboviruses such as West Nile virus (WNV), bluetongue virus (BTV), dengue virus (DENV) and chikungunya virus (CHIKV) infect their arthropod vectors over a range of average temperatures depending on the ambient temperature. How the transmission efficiency of an arbovirus (i.e. vector competence) varies with temperature influences not only the short term risk of arbovirus outbreaks in humans and livestock but also the long term impact of climate change on the geographical range of the virus. The strength of the interaction between viral surface (glyco)protein (GP) and the host cell receptor (Cr) on binding of virus to host cell is defined by the thermodynamic dissociation constant Kd_receptor which is assumed to equal 10−3 M (at 37 °C) for binding of a sialic acid (SA) on the arthropod midgut epithelial cell surface to a SA-binding site on the surface of BTV, for example. Here virus binding affinity is modelled with increasing number of GP/Cr contacts at temperatures from 10 °C to 35 °C taking into account the change in entropy on immobilization of the whole virus on binding (ΔSa_immob). Based on published data, three thermodynamic GP/Cr binding scenarios, namely enthalpy-driven, entropy-assisted and entropy-driven, are shown to affect the temperature sensitivity of virus binding in different ways. Thus for enthalpy-driven GP/Cr binding, viruses bind host cells much more strongly at 10 °C than 35 °C. A mechanistic model is developed for the number of arthropod midgut cells with bound virus and by building in a kinetic component for the rate of arbovirus replication and subsequent spread to the arthropod salivary glands, a model for the effect of temperature on vector competence is developed. The model separates the opposing effects of temperature on midgut cell binding affinity from the kinetic component of virogenesis. It successfully accommodates both increases in vector competence with temperature as for DENV and WNV in mosquitoes and decreases as for the CHIKV 2010–1909 strain in various populations of Aedes albopictus mosquitoes. Enhanced cell binding at lower temperatures through enthalpy-driven GP/Cr binding compensates for the lower replication rate to some degree such that some transmission can still occur at lower temperatures. In contrast, the strength of entropy-driven GP/Cr binding diminishes at low temperatures although there is no minimum temperature threshold for transmission efficiency. The magnitude of ΔSa_immob is an important data gap. It is concluded that thermodynamic and kinetic data obtained at the molecular level will prove important in modelling vector competence with temperature.

Egg-based influenza split virus vaccine with monoglycosylation induces cross-strain protection against influenza virus infections

Each year influenza virus infections cause hundreds of thousands of deaths worldwide and a significant level of morbidity with major economic burden. At the present time, vaccination with inactivated virus vaccine produced from embryonated chicken eggs is the most prevalent method to prevent the infections. However, current influenza vaccines are only effective against closely matched circulating strains and must be updated and administered yearly. Therefore, generating a vaccine that can provide broad protection is greatly needed for influenza vaccine development. We have previously shown that vaccination of the major surface glycoprotein hemagglutinin (HA) of influenza virus with a single N-acetylglucosamine at each of the N-glycosylation sites [monoglycosylated HA (HAmg)] can elicit better cross-protection compared with the fully glycosylated HA (HAfg). In the current study, we produced monoglycosylated inactivated split H1N1 virus vaccine from chicken eggs by the N-glycosylation process inhibitor kifunensine and the endoglycosidase Endo H, and intramuscularly immunized mice to examine its efficacy. Compared with vaccination of the traditional influenza vaccine with complex glycosylations from eggs, the monoglycosylated split virus vaccine provided better cross-strain protection against a lethal dose of virus challenge in mice. The enhanced antibody responses induced by the monoglycosylated vaccine immunization include higher neutralization activity, higher hemagglutination inhibition, and more HA stem selectivity, as well as, interestingly, higher antibody-dependent cellular cytotoxicity. This study provides a simple and practical procedure to enhance the cross-strain protection of influenza vaccine by removing the outer part of glycans from the virus surface through modifications of the current egg-based process.

Glycosylation of HIV Env Impacts IgG Subtype Responses to Vaccination.

The envelope protein (Env) is the only surface protein of the human immunodeficiency virus (HIV) and as such the exclusive target for protective antibody responses. Experimental evidences from mouse models suggest a modulating property of Env to steer antibody class switching towards the less effective antibody subclass IgG1 accompanied with strong TH2 helper responses. By simple physical linkage we were able to imprint this bias, exemplified by a low IgG2a/IgG1 ratio of antigen-specific antibodies, onto an unrelated antigen, namely the HIV capsid protein p24. Here, our results indicate the glycan moiety of Env as the responsible immune modulating activity. Firstly, in Card9−/− mice lacking specific C-Type lectin responsiveness, DNA immunization significantly increased the IgG2a/IgG1 ratio for the Env-specific antibodies while the antibody response against the F-protein of the respiratory syncytial virus (RSV) serving as control antigen remained unchanged. Secondly, sequential shortening of the Env encoding sequence revealed the C2V3 domain as responsible for the strong IgG1 responses and TH2 cytokine production. Removing all potential N-glycosylation sites from the C2V3 domain by site-specific mutagenesis reversed the vaccine-induced immune response towards a Th1-dominated T-cell response and a balanced IgG2a/IgG1 ratio. Accordingly, the stretch of oligomannose glycans in the C2V3 domain of Env might mediate a specific uptake and/or signaling modus in antigen presenting cells by involving interaction with an as yet unknown C-type lectin receptor. Our results contribute to a deeper understanding of the impact of Env glycosylation on HIV antigen-specific immune responses, which will further support HIV vaccine development.

Structure-Based Vaccine Antigen Design.

Enabled by new approaches for rapid identification and selection of human monoclonal antibodies, atomic-level structural information for viral surface proteins, and capacity for precision engineering of protein immunogens and self-assembling nanoparticles, a new era of antigen design and display options has evolved. While HIV-1 vaccine development has been a driving force behind these technologies and concepts, clinical proof-of-concept for structure-based vaccine design may first be achieved for respiratory syncytial virus (RSV), where conformation-dependent access to neutralization-sensitive epitopes on the fusion glycoprotein determines the capacity to induce potent neutralizing activity. Success with RSV has motivated structure-based stabilization of other class I viral fusion proteins for use as immunogens and demonstrated the importance of structural information for developing vaccines against other viral pathogens, particularly difficult targets that have resisted prior vaccine development efforts. Solving viral surface protein structures also supports rapid vaccine antigen design and application of platform manufacturing approaches for emerging pathogens.

Mutations in the H7 HA and PB1 genes of avian influenza a viruses increase viral pathogenicity and contact transmission in guinea pigs

ABSTRACT Avian influenza A viruses (AIV) of the H7 subtype continue to evolve posing a pandemic threat. However, molecular markers of H7N7 AIV pathogenicity and transmission in mammals remain poorly understood. In this study, we performed a systematic in vitro and in vivo analysis by comparing an H7N7 highly pathogenic AIV and its ferret adapted variant. Passaging an H7N7 AIV in ferrets led to six mutations in genes encoding the viral polymerase complex and the viral surface proteins. Here, we show that mutations in the H7 hemagglutinin gene cause increased pathogenicity in mice. Contact transmission between guinea pigs required additional mutations in the gene encoding the polymerase subunit PB1. Thus, particular vigilance is required with respect to HA and PB1 mutations as predictive molecular markers to assess the pandemic risk posed by emerging H7 avian influenza viruses.

Enhancement of antigen-specific humoral immune responses and protein solubility through conjugation of bacterial flagellin, Vibrio vulnificus FlaB, to the N-terminus of porcine epidemic diarrhea virus surface protein antigen S0.

Porcine epidemic diarrhea (PED) is a highly contagious enteric swine disease. The large economic impact of PED on the swine industry worldwide has made the development of an effective PED vaccine a necessity. S0, a truncated region of the porcine epidemic diarrhea virus (PEDV) spike protein, has been suggested as a candidate antigen for PED subunit vaccines; however, poor solubility problems when the protein is expressed in Escherichia coli, and the inherent problems of subunit vaccines, such as low immunogenicity, remain. Flagellin has been widely used as a fusion partner to enhance the immunogenicity and solubility of many difficult-to-express proteins; however, the conjugation effect of flagellin varies depending on the target antigen or the position of the fusion placement. Here, we conjugated flagellin, Vibrio vulnificus FlaB, to the N- and C-termini of S0 and evaluated the ability of the fusion to enhance the solubility and immunogenicity of S0. Flagellin conjugation in the presence of the trigger factor chaperone tig greatly improved the solubility of the fusion protein (up to 99%) regardless of its conjugation position. Of importance, flagellin conjugated to the N-terminus of S0 significantly enhanced S0-specific humoral immune responses compared to other recombinant antigens in Balb/c mice. The mechanism of this phenomenon was investigated through in vitro and in vivo studies. These findings provide important information for the development of a novel PED vaccine and flagellin-based immunotherapeutics.

An E. coli-produced single-chain variable fragment (scFv) targeting hepatitis B virus surface protein potently inhibited virion secretion

Hepatitis B virus (HBV) envelopes as well as empty subviral particles carry in their lipid membranes the small (S), middle (M), and large (L) surface proteins, collectively known as hepatitis B surface antigen (HBsAg). Due to their common S domain all three proteins share a surface-exposed hydrophilic antigenic loop (AGL) with a complex disulfide bridge-dependent structure. The AGL is critical for HBV infectivity and virion secretion, and thus represents a major target for neutralizing antibodies. Previously, a human monoclonal antibody (mAb) targeting a conformational epitope in the AGL, IgG12, exhibited 1000-fold higher neutralizing activity than hepatitis B immune globulin (HBIG). Here we designed a single-chain variable fragment (scFv) homolog of IgG12, G12-scFv, which could be efficiently produced in soluble form in the cytoplasm of E. coli SHuffle cells. Independent in vitro assays verified specific binding of G12-scFv to a conformational S epitope shared with IgG12. Despite 20-fold lower affinity, G12-scFv but not an irrelevant scFv potently neutralized HBV infection of susceptible hepatoma cells (IC50 = 1.8 nM). Strikingly, low concentrations of G12-scFv blocked virion secretion from HBV producing cells (IC50 = 1.25 nM) without disturbing intracellular viral replication, whereas extracellular HBsAg was reduced only at >100-fold higher though still nontoxic concentration. The inhibitory effects correlated with S binding specificity and presumably also G12-scFv internalization into cells. Together these data suggest G12-scFv as a highly specific yet easily accessible novel tool for basic, diagnostic, and possibly future therapeutic applications.

Identification of Diaryl-Quinoline Compounds as Entry Inhibitors of Ebola Virus.

Ebola virus is the causative agent of Ebola virus disease in humans. The lethality of Ebola virus infection is about 50%, supporting the urgent need to develop anti-Ebola drugs. Glycoprotein (GP) is the only surface protein of the Ebola virus, which is functionally critical for the virus to attach and enter the host cells, and is a promising target for anti-Ebola virus drug development. In this study, using the recombinant HIV-1/Ebola pseudovirus platform we previously established, we evaluated a small molecule library containing various quinoline compounds for anti-Ebola virus entry inhibitors. Some of the quinoline compounds specifically inhibited the entry of the Ebola virus. Among them, compound SYL1712 was the most potent Ebola virus entry inhibitor with an IC50 of ~1 μM. The binding of SYL1712 to the vial glycoprotein was computationally modeled and was predicted to interact with specific residues of GP. We used the time of the addition assay to show that compound SYL1712 blocks Ebola GP-mediated entry. Finally, consistent with being an Ebola virus entry inhibitor, compound SYL1712 inhibited infectious Ebola virus replication in tissue culture under biosafety level 4 containment, with an IC50 of 2 μM. In conclusion, we identified several related molecules with a diaryl-quinoline scaffold as potential anti-EBOV entry inhibitors, which can be further optimized for anti-Ebola drug development.

The Hepatitis B Surface Antigen Binding Protein: An Immunoglobulin G Constant Region-Like Protein That Interacts With HBV Envelop Proteins and Mediates HBV Entry.

Hepatitis B virus (HBV) infection is a leading cause of liver cirrhosis, liver cancer, and liver failure, affecting 350 million people worldwide. Currently available anti-HBV drugs include (PEGylated-) interferon-α and nucleos(t)ide analogs, which can cause significant side effects and drug-resistance in many cases of long-term treatment. The lack of a reliable and robust in vitro infection system is a major barrier for understanding the HBV life cycle and discovering novel therapeutic targets. In the present study, we demonstrate that overexpression of the hepatitis B surface antigen binding protein (SBP) in HepG2 cells (HepG2-SBP) resulted in their susceptibility to HBV infection. HepG2-SBP cells supported the uptake of the viral surface protein (HBsAg-preS), HBV-pseudotyped virus, and live HBV in patient sera. Moreover, SBP-mediated HBsAg-preS uptake, and HBV pseudotyped virus infections were efficiently blocked by preS1- and SBP-specific antibodies. These observations suggest that SBP is involved in HBV entry and that HepG2-SBP cells can serve as a cellular model to study the post-binding steps of HBV infection.

Differences in antigenic sites and other functional regions between genotype A and G mumps virus surface proteins

The surface proteins of the mumps virus, the fusion protein (F) and haemagglutinin-neuraminidase (HN), are key factors in mumps pathogenesis and are important targets for the immune response during mumps virus infection. We compared the predicted amino acid sequences of the F and HN genes from Dutch mumps virus samples from the pre-vaccine era (1957–1982) with mumps virus genotype G strains (from 2004 onwards). Genotype G is the most frequently detected mumps genotype in recent outbreaks in vaccinated communities, especially in Western Europe, the USA and Japan. Amino acid differences between the Jeryl Lynn vaccine strains (genotype A) and genotype G strains were predominantly located in known B-cell epitopes and in N-linked glycosylation sites on the HN protein. There were eight variable amino acid positions specific to genotype A or genotype G sequences in five known B-cell epitopes of the HN protein. These differences may account for the reported antigenic differences between Jeryl Lynn and genotype G strains. We also found amino acid differences in and near sites on the HN protein that have been reported to play a role in mumps virus pathogenesis. These differences may contribute to the occurrence of genotype G outbreaks in vaccinated communities.

Human cytomegalovirus-infected cells release extracellular vesicles that carry viral surface proteins

Extracellular vesicles (EVs) released by virus-infected cells typically incorporate host and viral components inside the vesicles (cargo molecules). Here, we investigated if human cytomegalovirus (HCMV) proteins are incorporated in EV outer membrane released by HCMV-infected cells. We separated EVs from HCMV using an iodixanol step-gradient and found that the separated vesicles carried EV markers such as the tetraspanin CD63 and Rab27A. Flow analysis of individual EVs demonstrated that on average, 15 ± 3.7% of EVs were positive for gB, 5.3 ± 2.3% were positive for gH and 3.74 ± 1.5% were positive for both gB and gH. In light of previous findings demonstrating HIV envelope proteins in EV membranes, the presence of viral protein at the surface of EVs released by HCMV-infected cells indicated that viral membrane proteins incorporated in EVs released by virus-infected cells may be a general phenomenon.

Common and Distinct Capsid and Surface Protein Requirements for Secretion of Complete and Genome-Free Hepatitis B Virions.

During the morphogenesis of hepatitis B virus (HBV), an enveloped virus, two types of virions are secreted: (i) a minor population of complete virions containing a mature nucleocapsid with the characteristic, partially double-stranded, relaxed circular DNA genome and (ii) a major population containing an empty capsid with no DNA or RNA (empty virions). Secretion of both types of virions requires interactions between the HBV capsid or core protein (HBc) and the viral surface or envelope proteins. We have studied the requirements from both HBc and envelope proteins for empty virion secretion in comparison with those for secretion of complete virions. Substitutions within the N-terminal domain of HBc that block secretion of DNA-containing virions reduced but did not prevent secretion of empty virions. The HBc C-terminal domain was not essential for empty virion secretion. Among the three viral envelope proteins, the smallest, S, alone was sufficient for empty virion secretion at a basal level. The largest protein, L, essential for complete virion secretion, was not required but could stimulate empty virion secretion. Also, substitutions in L that eliminated secretion of complete virions reduced but did not eliminate empty virion secretion. S mutations that blocked secretion of the hepatitis D virus (HDV), an HBV satellite, did not block secretion of either empty or complete HBV virions. Together, these results indicate that both common and distinct signals on empty capsids and mature nucleocapsids interact with the S and L proteins during the formation of complete and empty virions.IMPORTANCE Hepatitis B virus (HBV) is a major cause of severe liver diseases, including cirrhosis and cancer. In addition to the complete infectious virion particle, which contains an outer envelope layer and an interior capsid that, in turn, encloses a DNA genome, HBV-infected cells also secrete noninfectious, incomplete viral particles in large excess over the number of complete virions. In particular, the empty (or genome-free) virion shares with the complete virion the outer envelope and interior capsid but contains no genome. We have carried out a comparative study on the capsid and envelope requirements for the secretion of these two types of virion particles and uncovered both shared and distinct determinants on the capsid and envelope for their secretion. These results provide new information on HBV morphogenesis and have implications for efforts to develop empty HBV virions as novel biomarkers and a new generation of HBV vaccine.

Late therapeutic intervention with a respiratory syncytial virus L-protein polymerase inhibitor, PC786, on respiratory syncytial virus infection in human airway epithelium.

Background and PurposeEffective anti‐respiratory syncytial virus (RSV) agents are still not available for clinical use. Current major targets are virus surface proteins, such as a fusion protein involved in viral entry, but agents effective after RSV infection is established are required. Here we have investigated the effects of late therapeutic intervention with a novel inhaled RSV polymerase inhibitor, PC786, on RSV infection in human airway epithelium.Experimental ApproachAir liquid interface‐cultured bronchial or small airway epithelium was infected with RSVA2. PC786 was applied apically or basolaterally once daily following peak virus load on Day 3 post inoculation. Apical wash was collected daily for determination of viral burden by PCR and plaque assay (primary endpoints) and biomarker analyses. The effects were compared with those of ALS‐8112, an anti‐RSV nucleoside analogue, and GS‐5806, a fusion‐protein inhibitor, which were treated basolaterally.Key ResultsLate intervention with GS‐5806 did not show significant anti‐viral effects, but PC786 produced potent, concentration‐dependent inhibition of viral replication with viral load falling below detectable limits 3 days after treatment commenced in airway epithelium. These effects were superior to those of ALS‐8112. PC786 showed inhibitory activities against RSV‐induced increases of CCL5, IL‐6, double‐strand DNA and mucin. The effects of PC786 were also confirmed in small airway epithelium.Conclusion and ImplicationsLate therapeutic intervention with the RSV polymerase inhibitor, PC786, reduced the viral burden quickly in human airway epithelium. Thus, PC786 demonstrates the potential to be an effective therapeutic agent to treat active RSV infection.

Inhibiting EBV: The Key to Carefree Smooching

There are no vaccines or therapies to treat Epstein-Barr virus (EBV). Snijder etal. (2018) isolated a potent human antibody against EBV that blocks infection of both B cells and epithelial cells. Structural analysis of the antibody complexed with a viral surface protein complex identified a site that may be useful in vaccine development.

Screening of neuraminidase inhibitory activities of some medicinal plants traditionally used in Lingnan Chinese medicines

BackgroundNeuraminidase (NA) is one of the key surface protein of the influenza virus, and has been established as a primary drug target for anti-influenza therapies. This study aimed to screen bioactive herbal extracts from some medicinal plants traditionally used in Lingnan Chinese Medicines by NA activity high-throughput screening assay.MethodsOne hundred ninety herbal extracts from 95 medicinal plants collected in Guangzhou were screened for their potential inhibitory activities against A (H1N1) influenza neuraminidase, and the most active extracts were further evaluated for their anti-influenza virus activities using virus-induced cytopathic effect (CPE).ResultsAmong the tested 190 herbal extracts, 14 extracts inhibited significantly NA activity (IC50 < 40 μg/mL), and the extracts 1–5, which were obtained from Amomurn villosum Lour, Melaphis chinensis (Bell) Baker, Sanguisorba officinalis and Flos Caryophylli, showed potent inhibitory activity against NA with IC50 values ranging from 4.1 to 9.6 μg/mL. Moreover, the most bioactive extracts 1–5 were found to protect MDCK cells from A (H1N1) influenza virus infection with very low cytotoxicity to the host cells (EC50 values ranged from 1.8 to 14.1 μg/mL, CC50 values ranged from 97.0 to 779.2 μg/mL, SI values ranged from 14 to 438). In addition, quantitative RT-PCR analysis showed that the extracts 1–5 inhibited viral RNA synthesis in a dose-dependent manner.ConclusionWe performed in vitro screening of anti-neuraminidase activities of herbal extracts from medicinal plants used in Lingnan Chinese Medicines, and the results indicate that some bioactive extracts are worth further studies to identify the bioactive components responsible for anti-influenza virus activities, to elucidate their modes of action and finally determine their clinical potentials.

A Sulfonozanamivir Analogue Has Potent Anti-influenza Virus Activity.

Influenza virus infection continues to cause significant, often severe, respiratory illness worldwide. A validated target for the development of anti-influenza agents is the virus surface protein sialidase. In the current study, we have discovered a highly potent inhibitor of influenza virus sialidase, based on a novel sialosyl sulfonate template. The synthesised 3-guanidino sialosyl α-sulfonate, a sulfonozanamivir analogue, inhibits viral replication in vitro at the nanomolar level, comparable to that of the anti-influenza drug zanamivir. Using protein X-ray crystallography we show that the sialosyl α-sulfonate template binds within the sialidase active site in a 1 C4 chair conformation. The C1-sulfonate moiety forms crucial and strong-binding interactions with the active site's triarginyl cluster, while the 3-guanidino moiety interacts significantly with conserved active site residues. This sulfonozanamivir analogue provides a new direction in anti-influenza virus drug development.

Divergent preS Sequences in Virion-Associated Hepatitis B Virus Genomes and Subviral HBV Surface Antigen Particles From HBV e Antigen-Negative Patients.

Hepatitis B virus (HBV) surface proteins (HBsAg) coat the viral particle and form subviral particles (SVPs). Loss of HBsAg represents a functional cure and is an important treatment goal. We analyzed the impact of the HBV genotypes A-E and pre-S mutations on SVP expression in hepatitis B virus e antigen (HBeAg)-negative chronic HBV-infected patients. A HBV genome harboring a preS1-deletion was analyzed in hepatoma cells. We observed a genotype-specific ratio of the 3 surface proteins (SHBs/MHBs/LHBs), reflecting differences in the morphology and composition of SVPs. Deletions/mutations in the preS1/preS2 domain, detected in released viral genomes, did not affect the molecular weight of MHBs and LHBs in these patients. In contrast, LHB molecular weight was altered in vitro using an HBV genome harboring a preS1-deletion derived from one of these patients. Differences in composition of SVPs may result in genotype-specific immunogenicity and pathogenesis. In the patients with preS-mutations, secreted HBsAg and released viral genomes cannot be derived from the same genetic source. As viral genomes are derived from covalently closed circular DNA (cccDNA), HBsAg is presumably derived from integrated DNA. This important HBsAg source should be considered for novel antiviral strategies in HBeAg-negative chronic HBV-infected patients.