Fusion Activity of Transmembrane and Cytoplasmic Domain Chimeras of the Influenza Virus Glycoprotein Hemagglutinin

The role of the sequence of transmembrane and cytoplasmic/intraviral domains of influenza virus hemagglutinin (HA, subtype H7) for HA-mediated membrane fusion was explored. To analyze the influence of the two domains on the fusogenic properties of HA, we designed HA-chimeras in which the cytoplasmic tail and/or transmembrane domain of HA was replaced with the corresponding domains of the fusogenic glycoprotein F of Sendai virus. These chimeras, as well as constructs of HA in which the cytoplasmic tail was replaced by peptides of human neurofibromin type1 (NF1) or c-Raf-1, NF78 (residues 1441 to 1518), and Raf81 (residues 51 to 131), respectively, were expressed in CV-1 cells by using the vaccinia virus-T7 polymerase transient-expression system. Wild-type and chimeric HA were cleaved properly into two subunits and expressed as trimers. Membrane fusion between CV-1 cells and bound human erythrocytes (RBCs) mediated by parental or chimeric HA proteins was studied by a lipid-mixing assay with the lipid-like fluorophore octadecyl rhodamine B chloride (R18). No profound differences in either extent or kinetics could be observed. After the pH was lowered, the above proteins also induced a flow of the aqueous fluorophore calcein from preloaded RBCs into the cytoplasm of the protein-expressing CV-1 cells, indicating that membrane fusion involves both leaflets of the lipid bilayers and leads to formation of an aqueous fusion pore. We conclude that neither HA-specific sequences in the transmembrane and cytoplasmic domains nor their length is crucial for HA-induced membrane fusion activity.     

The Influenza Hemagglutinin Fusion Domain Is an Amphipathic Helical Hairpin That Functions by Inducing Membrane Curvature

The highly conserved N-terminal 23 residues of the hemagglutinin glycoprotein, known as the fusion peptide domain (HAfp23), is vital to the membrane fusion and infection mechanism of the influenza virus. HAfp23 has a helical hairpin structure consisting of two tightly packed amphiphilic helices that rest on the membrane surface. We demonstrate that HAfp23 is a new class of amphipathic helix that functions by leveraging the negative curvature induced by two tightly packed helices on membranes. The helical hairpin structure has an inverted wedge shape characteristic of negative curvature lipids, with a bulky hydrophobic region and a relatively small hydrophilic head region. The F3G mutation reduces this inverted wedge shape by reducing the volume of its hydrophobic base. We show that despite maintaining identical backbone structures and dynamics as the wild type HAfp23, the F3G mutant has an attenuated fusion activity that is correlated to its reduced ability to induce negative membrane curvature. The inverted wedge shape of HAfp23 is likely to play a crucial role in the initial stages of membrane fusion by stabilizing negative curvature in the fusion stalk.     

Lateral Distribution of the Transmembrane Domain of Influenza Virus Hemagglutinin Revealed by Time-resolved Fluorescence Imaging

Influenza virus hemagglutinin (HA) has been suggested to be enriched in liquid-ordered lipid domains named rafts, which represent an important step in virus assembly. We employed Förster resonance energy transfer (FRET) via fluorescence lifetime imaging microscopy to study the interaction of the cytoplasmic and transmembrane domain (TMD) of HA with agly co sylphos pha tidyl ino si tol (GPI)-anchored peptide, an established marker for rafts in the exoplasmic leaflet of living mammalian plasma membranes. Cyan fluorescent protein (CFP) was fused to GPI, whereas the HA sequence was tagged with yellow fluorescent protein (YFP) on its exoplasmic site (TMD-HA-YFP), avoiding any interference of fluorescent proteins with the proposed role of the cytoplasmic domain in lateral organization of HA. Constructs were expressed in Chinese hamster ovary cells (CHO-K1) for which cholesterol-sensitive lipid nanodomains and their dimension in the plasma membrane have been described (Sharma, P., Varma, R., Sarasij, R. C., Ira, Gousset, K., Krishnamoorthy, G., Rao, M., and Mayor, S. (2004) Cell 116, 577–589). Upon transfection in CHO-K1 cells, TMD-HA-YFP is partially expressed as a dimer. Only dimers are targeted to the plasma membrane. Clustering of TMD-HA-YFP with GPI-CFP was observed and shown to be reduced upon cholesterol depletion, a treatment known to disrupt rafts. No indication for association of TMD-HA-YFP with GPI-CFP was found when palmitoylation, an important determinant of raft targeting, was suppressed. Clustering of TMD-HA-YFP and GPI-CFP was also observed in purified plasma membrane suspensions by homoFRET. We concluded that the pal mit oy lated TMD-HA alone is sufficient to recruit HA to cholesterol-sensitive nanodomains. The corresponding construct of the spike protein E2 of Semliki Forest virus did not partition preferentially in such domains.Assembly of enveloped viruses requires the selective recruitment of viral components at distinct sites of the host cell membranes from which viruses bud. One of the most intensely studied enveloped viruses with respect to assembly is the influenza virus, in which budding takes place at the plasma membrane of epithelial cells. Three membrane proteins are embedded in the influenza virus envelope: hemagglutinin (HA),3 which mediates binding of the virus to the host cell and fusion with cell target membrane (1); neuraminidase; and the proton channel M2. The inner viral membrane leaflet is covered by the matrix protein M1, which is supposed to mediate binding of the eight viral RNA-nucleoprotein complexes harboring the genetic information of the virus. Several studies support a role of lipid domains as a platform for enrichment of viral components. HA, the most abundant envelope protein of the influenza virus, has been found to be enriched in detergent-resistant membrane (DRM) fractions (2–4). Typical lipid components of those fractions are saturated phospholipids, glycosphingolipids, and cholesterol, which are known to form liquid-ordered domains (5). This has led to the idea that so-called lipid rafts, which resemble liquid-ordered domains, could function as assembly sites. Support for this hypothesis was given by the observation that the lipid composition of the influenza virus envelope is more similar to that of a raft than to the overall plasma membrane (2, 6).As it has been shown that DRM fractions may not represent the native state of lipid domains, in particular of rafts (7, 8), subsequent efforts have focused on other techniques to assess the lateral organization of HA. Electron microscopy studies using immunogold labeling (4, 9), Förster resonance energy transfer (FRET) measurements between fluorescent HA antibodies in fixed cells (9), and investigations on photoactivatable HA in living cells (10) have revealed cholesterol-sensitive clustering of HA in the plasma membrane of mammalian cells at lengths between 20 and 900 nm.A specific problem encountered in studying the lateral organization of proteins in the plasma membrane is that lipid domains as rafts are typically organized at a submicroscopic level. Indeed, several attempts to image raft domains in biological membranes have suggested that rafts are very small and highly dynamic (11, 12). A guiding study in the characterization of lipid domains in biological membranes has been performed by Mayor and colleagues (13) on the plasma membrane of CHO-K1 cells. Based on homoFRET measurements they have shown that about 20–40% of GFP-tagged glycosylphosphatidylinositol (GPI) (for review see Brown and Rose (14)) and other GPI-anchored proteins are organized with about three to four copies in small cholesterol-sensitive clusters. Mathematical modeling of those experimental data is consistent with a domain diameter of about 5 nm (13).In the present study we investigated the lateral organization of the C terminus of HA, corresponding to the transmembrane domain (TMD), and the cytoplasmic tail (CT) of the protein in the plasma membrane of CHO-K1 cells, taking advantage of the well characterized spatial arrangement of the raft marker GPI in those cells (see above). Lateral organization was studied essentially by fluorescence lifetime imaging microscopy (FLIM)-based FRET between CFP (donor) and YFP (acceptor). For this purpose, we replaced the ectodomain of HA by YFP and studied FRET between this construct and GPI-CFP. As a complementary approach, we performed ensemble measurements in suspensions of plasma membranes purified from cells expressing fluorescent GPI and HA constructs. We measured homoFRET by time-resolved fluorescence anisotropy, providing information on the aggregation/clustering state of the fluorescent constructs, which is important in rationalizing the FLIM-FRET data.     

Comparison of Antiviral Activity between IgA and IgG Specific to Influenza Virus Hemagglutinin: Increased Potential of IgA for Heterosubtypic Immunity

Both IgA and IgG antibodies are known to play important roles in protection against influenza virus infection. While IgG is the major isotype induced systemically, IgA is predominant in mucosal tissues, including the upper respiratory tract. Although IgA antibodies are believed to have unique advantages in mucosal immunity, information on direct comparisons of the in vitro antiviral activities of IgA and IgG antibodies recognizing the same epitope is limited. In this study, we demonstrate differences in antiviral activities between these isotypes using monoclonal IgA and IgG antibodies obtained from hybridomas of the same origin. Polymeric IgA-producing hybridoma cells were successfully subcloned from those originally producing monoclonal antibody S139/1, a hemaggulutinin (HA)-specific IgG that was generated against an influenza A virus strain of the H3 subtype but had cross-neutralizing activities against the H1, H2, H13, and H16 subtypes. These monoclonal S139/1 IgA and IgG antibodies were assumed to recognize the same epitope and thus used to compare their antiviral activities. We found that both S139/1 IgA and IgG antibodies strongly bound to the homologous H3 virus in an enzyme-linked immunosorbent assay, and there were no significant differences in their hemagglutination-inhibiting and neutralizing activities against the H3 virus. In contrast, S139/1 IgA showed remarkably higher cross-binding to and antiviral activities against H1, H2, and H13 viruses than S139/1 IgG. It was also noted that S139/1 IgA, but not IgG, drastically suppressed the extracellular release of the viruses from infected cells. Electron microscopy revealed that S139/1 IgA deposited newly produced viral particles on the cell surface, most likely by tethering the particles. These results suggest that anti-HA IgA has greater potential to prevent influenza A virus infection than IgG antibodies, likely due to increased avidity conferred by its multivalency, and that this advantage may be particularly important for heterosubtypic immunity.     

Functional Study of a Camelid Single Domain Anti-CD22 Antibody

International Journal of Peptide Research and Therapeutics - Variable antigen-binding domain of camelid single chain antibodies (VHH, Nanobody) and its conjugates are considered among very...     

Molecular Dynamics Analysis of Antibody Recognition and Escape by Human H1N1 Influenza Hemagglutinin

The antibody immunoglobulin (Ig) 2D1 is effective against the 1918 hemagglutinin (HA) and also known to cross-neutralize the 2009 pandemic H1N1 influenza HA through a similar epitope. However, the detailed mechanism of neutralization remains unclear. We conducted molecular dynamics (MD) simulations to study the interactions between Ig-2D1 and the HAs from the 1918 pandemic flu (A/South Carolina/1/1918, 18HA), the 2009 pandemic flu (A/California/04/2009, 09HA), a 2009 pandemic flu mutant (A/California/04/2009, 09HA_mut), and the 2006 seasonal flu (A/Solomon Islands/3/2006, 06HA). MM-PBSA analyses suggest the approximate free energy of binding (ΔG) between Ig-2D1 and 18HA is −74.4 kcal/mol. In comparison with 18HA, 09HA and 06HA bind Ig-2D1 ∼6 kcal/mol (ΔΔG) weaker, and the 09HA_mut bind Ig-2D1 only half as strong. We also analyzed the contributions of individual epitope residues using the free-energy decomposition method. Two important salt bridges are found between the HAs and Ig-2D1. In 09HA, a serine-to-asparagine mutation coincided with a salt bridge destabilization, hydrogen bond losses, and a water pocket formation between 09HA and Ig-2D1. In 09HA_mut, a lysine-to-glutamic-acid mutation leads to the loss of both salt bridges and destabilizes interactions with Ig-2D1. Even though 06HA has a similar ΔG to 09HA, it is not recognized by Ig-2D1 in vivo. Because 06HA contains two potential glycosylation sites that could mask the epitope, our results suggest that Ig-2D1 may be active against 06HA only in the absence of glycosylation. Overall, our simulation results are in good agreement with observations from biological experiments and offer novel mechanistic insights, to our knowledge, into the immune escape of the influenza virus.     

抗H5N1亚型禽流感病毒血凝素单克隆抗体的制备及鉴定

目的建立稳定分泌抗H5N1亚型禽流感病毒血凝素单克隆抗体的杂交瘤细胞系,为进一步研究禽流感诊断技术奠定基础。方法以纯化的H5亚型禽流感病毒按常规方法免疫BALBc小鼠,最后一次免疫后第3天取其脾细胞与SP20细胞在聚乙二醇作用下融合,用选择性培养、有限稀释法克隆和血凝抑制试验进行筛选,对获得阳性克隆株用ELISA方法进行亚型鉴定,并用37株H5、H7、H9亚型AIV测定其特异性、覆盖性。结果最后获得了3株分泌特异性抗体的杂交瘤细胞,命名为1E5、4A4、4B1,经长期体外培养和冻存后复苏能稳定地分泌抗体。经鉴定,其亚型均为IgG1、kappa链。腹水HI效价1∶210～1∶216,细胞培养上清HI效价1∶26～1∶28。3株杂交瘤所分泌的单克隆抗体均能与本中心保存的全部20株H5亚型禽流感病毒分离株发生反应,而与15株H9亚型禽流感病毒分离株、2株H7亚型禽流感病毒分离株以及H1H4、H6H15亚型禽流感病毒标准毒株均不反应,与鸡新城疫病毒、鹅新城疫病毒、鹅腺病毒和鸡产蛋下降综合征病毒等均无交叉反应。结论所获3株单克隆抗体可用于禽流感病毒特异性诊断试剂的研制。     

Phage Lysin LysK Can Be Truncated to Its CHAP Domain and Retain Lytic Activity against Live Antibiotic-Resistant Staphylococci

A truncated derivative of the phage endolysin LysK containing only the CHAP (cysteine- and histidine-dependent amidohydrolase/peptidase) domain exhibited lytic activity against live clinical staphylococcal isolates, including methicillin-resistant Staphylococcus aureus. This is the first known report of a truncated phage lysin which retains high lytic activity against live staphylococcal cells.     

Guiding the Immune Response against Influenza Virus Hemagglutinin toward the Conserved Stalk Domain by Hyperglycosylation of the Globular Head Domain

Influenza virus hemagglutinin consists of a highly variable and immunodominant head domain and a more conserved but immunosubdominant stalk domain. We introduced seven N-linked glycosylation sites in the hemagglutinin head domain to shield the immunodominant antigenic sites. The hyperglycosylated hemagglutinin enhanced stalk-directed seroreactivity while dampening the head response in immunized mice. Upon influenza virus challenge, mice vaccinated with the hyperglycosylated hemagglutinin were better protected against morbidity and mortality than mice receiving the wild-type hemagglutinin.     

Structure of a Classical Broadly Neutralizing Stem Antibody in Complex with a Pandemic H2 Influenza Virus Hemagglutinin

We report the structural characterization of the first antibody identified to cross-neutralize multiple subtypes of influenza A viruses. The crystal structure of mouse antibody C179 bound to the pandemic 1957 H2N2 hemagglutinin (HA) reveals that it targets an epitope on the HA stem similar to those targeted by the recently identified human broadly neutralizing antibodies. C179 also inhibits the low-pH conformational change of the HA but uses a different angle of approach and both heavy and light chains.     

黑色素瘤细胞单域抗体文库的构建及鉴定

黑色素瘤作为一种多发于皮肤部位的恶性肿瘤,严重危害着动物和人们的健康。与传统抗体比较,单域抗体具有结构简单、分子量小、免疫原性弱等特点,使其在疾病的诊断及治疗方面具有广阔的应用空间。本研究以B16-F10细胞蛋白质为研究对象,通过反复冻融与超声破碎相结合的方式获得B16-F10蛋白质作为抗原,免疫成年雄性羊驼。采用噬菌体单域抗体展示技术,构建了质量优良的B16-F10细胞蛋白质单域抗体免疫文库,其库容为5.76 × 10¹⁰,VHH重组率为96%,文库丰度为3.00 × 10¹⁰个/mL。该结果为研究黑色素瘤的生物学特性提供了新思路,同时也为后续筛选B16-F10单域抗体奠定了基础。     

黑色素瘤细胞单域抗体文库的构建及鉴定

黑色素瘤作为一种多发于皮肤部位的恶性肿瘤,严重危害着动物和人们的健康。与传统抗体比较,单域抗体具有结构简单、分子量小、免疫原性弱等特点,使其在疾病的诊断及治疗方面具有广阔的应用空间。本研究以B16-F10细胞蛋白质为研究对象,通过反复冻融与超声破碎相结合的方式获得B16-F10蛋白质作为抗原,免疫成年雄性羊驼。采用噬菌体单域抗体展示技术,构建了质量优良的B16-F10细胞蛋白质单域抗体免疫文库,其库容为5.76 × 10¹⁰,VHH重组率为96%,文库丰度为3.00 × 10¹⁰个/mL。该结果为研究黑色素瘤的生物学特性提供了新思路,同时也为后续筛选B16-F10单域抗体奠定了基础。     

Identification and Characterization of a Novel Single Domain Antibody Against Ebola Virus

Virologica Sinica - Ebola virus (EBOV) belongs to the Filoviridae family and causes severe illnesses such as hemorrhagic fever with a high mortality rate up to 90%. Now two antibody drugs termed...     

The Chloride Conductance of Tight Junctions of Rat Ileum Can Be Increased by cAMP But Not by Carbachol

It is well known, that in mammalian small intestine, cAMP increases Cl⁻ permeability of the apical membrane of enterocytes as part of its secretory action. Paradoxically, this is usually accompanied by an increase of the transepithelial resistance. In the present study we report that in the presence of bumetanide (to block basolateral Cl⁻ uptake) cAMP always decreased the transepithelial resistance. We examined whether this decrease in resistance was due to a cAMP-dependent increase of the paracellular electrolyte permeability in addition to the increase of the Cl⁻ permeability of the apical cell membrane. We used diffusion potentials induced by serosal replacement of NaCl, and transepithelial current passage to evoke transport number effects. The results revealed that cAMP (but not carbachol) could increase the Cl⁻ permeability of the tight junctions in rat ileum. Moreover, we observed a variation in transepithelial resistance of individual tissue preparations, inversely related to the cation selectivity of the tissue, suggesting that Na⁺ permeability of the tight junctions can vary between preparations. Received: 7 September 1996/Revised: 5 November 1996     

Mutation Frequencies at Defined Single Codon Sites in Vesicular Stomatitis Virus and Poliovirus Can Be Increased Only Slightly by Chemical Mutagenesis

Mutagenesis by a variety of chemical mutagens conferred only 1.1- to 2.8-fold increases in mutation frequencies at defined single base sites in vesicular stomatitis virus and poliovirus.     

Evaluation of Disulfide Bond Position to Enhance the Thermal Stability of a Highly Stable Single Domain Antibody

Single domain antibodies are the small recombinant variable domains derived from camelid heavy-chain-only antibodies. They are renowned for their stability, in large part due to their ability to refold following thermal or chemical denaturation. In addition to refolding after heat denaturation, A3, a high affinity anti-Staphylococcal Enterotoxin B single domain antibody, possesses a melting temperature of ∼84°C, among the highest reported for a single domain antibody. In this work we utilized the recently described crystal structure of A3 to select locations for the insertion of a second disulfide bond and evaluated the impact that the addition of this second bond had on the melting temperature. Four double-disulfide versions of A3 were constructed and each was found to improve the melting temperature relative to the native structure without reducing affinity. Placement of the disulfide bond at a previously published position between framework regions 2 and 3 yielded the largest improvement (>6°C), suggesting this location is optimal, and seemingly provides a universal route to raise the melting temperature of single domain antibodies. This study further demonstrates that even single domain antibodies with extremely high melting points can be further stabilized by addition of disulfide bonds.     

Intracellular Expression of a Single Domain Antibody Reduces Cytotoxicity of 15-Acetyldeoxynivalenol in Yeast

15-Acetyldeoxynivalenol (15-AcDON) is a low molecular weight sesquiterpenoid trichothecene mycotoxin associated with Fusarium ear rot of maize and Fusarium head blight of small grain cereals. The accumulation of mycotoxins such as deoxynivalenol (DON) and 15-AcDON within harvested grain is subject to stringent regulation as both toxins pose dietary health risks to humans and animals. These toxins inhibit peptidyltransferase activity, which in turn limits eukaryotic protein synthesis. To assess the ability of intracellular antibodies (intrabodies) to modulate mycotoxin-specific cytotoxocity, a gene encoding a camelid single domain antibody fragment (V_HH) with specificity and affinity for 15-AcDON was expressed in the methylotropic yeast Pichia pastoris. Cytotoxicity and V_HH immunomodulation were assessed by continuous measurement of cellular growth. At equivalent doses, 15-AcDON was significantly more toxic to wild-type P. pastoris than was DON. In turn, DON was orders of magnitude more toxic than 3-acetyldeoxynivalenol. Intracellular expression of a mycotoxin-specific V_HH within P. pastoris conveyed significant (p = 0.01) resistance to 15-AcDON cytotoxicity at doses ranging from 20 to 100 μg·ml⁻¹. We also documented a biochemical transformation of DON to 15-AcDON to account for the attenuation of DON cytotoxicity at 100 and 200 μg·ml⁻¹. The proof of concept established within this eukaryotic system suggests that in planta V_HH expression may lead to enhanced tolerance to mycotoxins and thereby limit Fusarium infection of commercial agricultural crops.     

In vitro Antiviral Activity of Recombinant Antibodies of IgG and IgA Isotypes to Hemagglutinin of the Influenza A Virus

Seasonal and highly infectious strains of the influenza A and influenza B viruses cause millions of cases of severe complications in elderly people, children, and patients with immune diseases each year. Immunoglobulin A (IgA), which is an active component of humoral immunity, can prevent the spread of the virus in the upper respiratory tract. The preparation and study of the properties of recombinant virus-specific IgA could be an important approach to finding new means of preventing and treating influenza. Based on CHO DG44 cells, we developed stable monoclonal cell lines that produce monomeric and dimeric antibodies FI6-IgA1 and FI6-IgA2m1 to hemagglutinin (HA) of the influenza A virus. When studying the productivity, growth, and stability of the obtained clones, we found that the dimeric form of antibodies of IgA1 isotype is superior to other forms. The dimeric form of IgA antibodies plays a key role in mucosal immunity. Recognizing the prospects of using dimeric IgA as prophylactic and therapeutic mucosal drugs for viral infections, we studied their virus-neutralizing and antiviral activities on MDCK cell culture and compared them with the antibodies of the IgG1 isotype. This study presents the data on antiviral and virus-neutralizing activities of the FI6-IgA1 dimers to seasonal and highly infectious strains of influenza A virus.