Mutations in the GEF2 gene of the yeast Saccharomyces cerevisiae have pleiotropic effects. The gef2 mutants display a petite phenotype. These cells grow slowly on several different carbon sources utilized exclusively or primarily by respiration. This phenotype is suppressed by adding large amounts of iron to the growth medium. A defect in mitochondrial function may be the cause of the petite phenotype: the rate of oxygen consumption by intact gef2 cells and by mitochondrial fractions isolated from gef2 mutants was reduced 60%-75% relative to wild type. Cytochrome levels were unaffected in gef2 mutants, indicating that heme accumulation is not significantly altered in these strains. The gef2 mutants were also more sensitive than wild type to growth inhibition by several divalent cations including Cu. We found that the cup5 mutation, causing Cu sensitivity, is allelic to gef2 mutations. The GEF2 gene was isolated, sequenced, and found to be identical to VMA3, the gene encoding the vacuolar H(+)-ATPase proteolipid subunit. These genetic and biochemical analyses demonstrate that the vacuolar H(+)-ATPase plays a previously unknown role in Cu detoxification, mitochondrial function, and iron metabolism.

The X-ray crystal structure of epitope II on the E2 protein of hepatitis C virus, in complex with nonneutralizing antibody mAb#12, has been solved at 2.90-Å resolution. The spatial arrangement of the essential components of epitope II (ie, the C-terminal α-helix and the N-terminal loop) was found to deviate significantly from that observed in those corresponding complexes with neutralizing antibodies. The distinct conformations are mediated largely by the flexibility of a highly conserved glycine residue that connects these components. Thus, it is the particular tertiary structure of epitope II, which is presented in a spatial and temporal manner, that determines the specificity of antibody recognition and, consequently, the outcome of neutralization or nonneutralization.H epatitis C is a major public health problem worldwide. More than 170 million people are infected by the hepatitis C virus (HCV) (1). Approximately 70% of infected people fail to clear the virus during the acute phase of the disease and become chronic carriers (2). Liver cirrhosis, which develops in about 10-20% of chronically infected patients, is linked with a high risk for hepatocellular carcinoma in later life (2, 3). To date, there is neither an effective immune globulin for prophylaxis nor a vaccine for the prevention of hepatitis C. The development of a safe and effective HCV vaccine remains a top priority for the global control of HCV infections.The HCV envelope glycoprotein E2 has long been considered an important immunogenic target in efforts to develop an HCV vaccine candidate. This consideration is largely based on the role of the E2 protein in facilitating the entry of HCV into hepatocytes via interaction with the host entry factors (4-10). Recently, the crystal structure of the E2 core, in complex with a neutralizing antibody, was solved (11). The E2 core study described the interface crucial for host entry factor CD81-mediated entry, thus providing a site of vulnerability that can be exploited in immunogen design. The crystal structure also revealed that nearly 62% of the E2 core amino acid residues are either disordered or in loop structures, the overall effect of which indicates a striking flexibility in the E2 protein structures. Whether the intrinsic structural heterogeneity of the E2 protein is linked to the viral entry process or not is currently unknown.Epitope II resides on the E2 protein between residues 427 and 446, a location that places it in the vicinity of the described E2-CD81 interface in the flexible area of the E2 protein (11-13). Paradoxically, different antibodies are able to bind to a similar set of residues on epitope II; however, their interactions with these residues can lead to either HCV neutralization or nonneutralization, as defined in an in vitro HCV cell culture system (12, 13). In addition, some epitope II-specific nonneutralizing antibodies were shown to interfere with the neutralization by antibodies at epitope I, another epitope on the E2 protein between residues 412 and 426 (12). Furthermore, de...

Antibodies to epitopes in the E2 protein of hepatitis C virus (HCV) reduce the viral infectivity in vivo and in vitro. However, the virus can persist in patients in the presence of neutralizing antibodies. In this study, we generated a panel of monoclonal antibodies that bound specifically to the region between residues 427 and 446 of the E2 protein of HCV genotype 1a, and we examined their capacity to neutralize HCV in a cell culture system. Of the four monoclonal antibodies described here, two were able to neutralize the virus in a genotype 1a-specific manner. The other two failed to neutralize the virus. Moreover, one of the nonneutralizing antibodies could interfere with the neutralizing activity of a chimpanzee polyclonal antibody at E2 residues 412 to 426, as it did with an HCV-specific immune globulin preparation, which was derived from the pooled plasma of chronic hepatitis C patients. Mapping the epitope-paratope contact interfaces revealed that these functionally distinct antibodies shared binding specificity for key amino acid residues, including W 437 , L 438 , L 441 , and F 442 , within the same epitope of the E2 protein. These data suggest that the effectiveness of antibody-mediated neutralization of HCV could be deduced from the interplay between an antibody and a specific set of amino acid residues. Further understanding of the molecular mechanisms of antibody-mediated neutralization and nonneutralization should provide insights for designing a vaccine to control HCV infection in vivo.

Hepatitis C virus (HCV) envelope glycoprotein E2 has been considered as a major target for vaccine design. Epitope II, mapped between residues 427-446 within the E2 protein, elicits antibodies that are either neutralizing or nonneutralizing. The fundamental mechanism of antibody-mediated neutralization at epitope II remains to be defined at the atomic level. Here we report the crystal structure of the epitope II peptide in complex with a monoclonal antibody (mAb#8) capable of neutralizing HCV. The complex structure revealed that this neutralizing antibody engages epitope II via interactions with both the C-terminal α-helix and the N-terminal loop using a bifurcated mode of action. Our structural insights into the key determinants for the antibody-mediated neutralization may contribute to the immune prophylaxis of HCV infection and the development of an effective HCV vaccine.H epatitis C virus (HCV) infection is a major public health problem with an estimated 170 million people infected worldwide (1). HCV is transmitted primarily through direct contact with the blood or other bodily fluids of an infected individual. Although acute hepatitis C is typically mild or even subclinical, the infection becomes chronic in more than 75% of those infected (2, 3). Patients with chronic HCV infection have a high risk of developing cirrhosis and, in some cases, hepatocellular carcinoma (2, 3).Significant advances have been made in the treatment of hepatitis C with the recent introduction of HCV-specific protease and polymerase inhibitors; sustained virologic responses, tantamount to cure, can now be achieved in more than 70% of the most difficult to treat HCV genotype 1-infected patients (4). However, the use of such drugs for treatment is not economically or logistically feasible in most parts of the world; therefore, vaccine development remains an important goal for the global control of HCV infection. Thus far, no HCV vaccine formulation has been able to induce sterilizing immunity, but a recombinant envelope protein vaccine has significantly reduced the rate of chronic HCV infection in a chimpanzee model (5). Thus, designing a vaccine that successfully elicits neutralizing antibodies remains a practical strategy to either prevent primary HCV infection or to reduce the frequency of progression from acute to chronic HCV infection (6).HCV envelope glycoprotein E2 has been studied extensively as a potential candidate for the immune prophylaxis of HCV infection and vaccine development. Several segments of the E2 protein have been identified as key components of conformational or linear epitopes that are critical to antibody-mediated neutralization of HCV in vitro (7-16). Interestingly, naturally evoked antibodies and those produced in vitro that are specifically directed against a short peptide located in the E2 protein between residues 427-446, also known as epitope II, displayed one of three activities: virus neutralization, E2 binding but no neutralization, or interference with virus neutralization (15, 16). To capture the f...

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The identification of a specific immunogenic candidate that will effectively activate the appropriate pathway for neutralizing antibody production is fundamental for vaccine design. By using a monoclonal antibody (1H8) that neutralizes HCV in vitro, we have demonstrated here that 1H8 recognized an epitope mapped between residues A524 and W529 of the E2 protein. We also found that the epitope residues A524, P525, Y527 and W529 were crucial for antibody binding, while the residues T526, Y527 and W529 within the same epitope engaged in the interaction with the host entry factor CD81. Furthermore, we detected “1H8-like” antibodies, defined as those with amino acid-specificity similar to 1H8, in the plasma of patients with chronic HCV infection. The time course study of plasma samples from Patient H, a well-characterized case of post-transfusion hepatitis C, showed that “1H8-like” antibodies could be detected in a sample collected almost two years after the initial infection, thus confirming the immunogenicity of this epitope in vivo. The characterization of this neutralization epitope with a function in host entry factor CD81 interaction should enhance our understanding of antibody-mediated neutralization of HCV infections.

The variations of susceptibility to alloxan induced Diabetes in a total of seventeen rabbits was described. Our study was designed to explore dosage schedules which might improve rabbit responsiveness to and survival after alloxan treatment. A wide range of response to intravenously administered alloxan was observed. Permanent diabetes (blood glucose 350 mg/dl) was found in three rabbits after a single injection (60 mg/kg in one, 100 mg/kg in two). This effect has persisted for eight months. By contrast, two other rabbits injected with a single dose of alloxan (60 mg/kg) developed only transient hyperglycemia. Similarly, four other rabbits either did not respond or had an incomplete response after receiving a total dose of 120 mg/kg. These data suggest that there is extreme variability in individual rabbits susceptibility to the diabetogenic affects of alloxan.

Hepatitis C virus (HCV) glycoprotein E2 is considered a major target for generating neutralizing antibodies against HCV, primarily due to its role of engaging host entry factors, such as CD81, a key cell surface protein associated with HCV entry. Based on a series of biochemical analyses in combination with molecular docking, we present a description of a potential binding interface formed between the E2 protein and CD81. The virus side of this interface includes a hydrophobic helix motif comprised of residues W 437 LAGLF 442 , which encompasses the binding site of a neutralizing monoclonal antibody, mAb41. The helical conformation of this motif provides a structural framework for the positioning of residues F442 and Y443, serving as contact points for the interaction with CD81. The cell side of this interface likewise involves a surface-exposed hydrophobic helix, namely, the D-helix of CD81, which coincides with the binding site of 1D6, a monoclonal anti-CD81 antibody known to block HCV entry. Our illustration of this virus-host interface suggests an important role played by the W 437 LAGLF 442 helix of the E2 protein in the hydrophobic interaction with the D-helix of CD81, thereby facilitating our understanding of the mechanism for antibody-mediated neutralization of HCV. IMPORTANCE Characterization of the interface established between a virus and host cells can provide important information that may be used for the control of virus infections. The interface that enables hepatitis C virus (HCV) to infect human liver cells has not been wellunderstood because of the number of cell surface proteins, factors, and conditions found to be associated with the infection process. Based on a series of biochemical analyses in combination with molecular docking, we present such an interface, consisting of two hydrophobic helical structures, from the HCV E2 surface glycoprotein and the CD81 protein, a major host cell receptor recognized by all HCV strains. Our study reveals the critical role played by hydrophobic interactions in the formation of this virus-host interface, thereby contributing to our understanding of the mechanism for antibody-mediated neutralization of HCV. Hepatitis C virus (HCV) infects more than 170 million people worldwide. Approximately 70% of infected people fail to clear the virus during the acute phase of the disease and become chronic carriers. Liver cirrhosis, which develops in about 10 to 20% of chronically infected patients, is linked with a high risk for hepatocellular carcinoma in later life (1, 2). Although the FDA recently approved a number of highly effective antiviral drugs for treatment of HCV infections, prophylaxis is still an unmet medical need. Disease prevention by use of virus-specific neutralizing antibodies remains the most cost-effective and realistic way to control HCV infection (and reinfection) and significantly reduces the burden of HCV-related diseases (3, 4).Protective immunity against HCV has been difficult to establish in humans, as the antibodies generated during...