A Conserved Basic Patch and Central Kink in the Nipah Virus Phosphoprotein Multimerization Domain Are Essential for Polymerase Function

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Cathepsin cleavage potentiates the Ebola virus glycoprotein to undergo a subsequent fusion-relevant conformational change

Cellular entry of Ebola virus (EBOV), a deadly hemorrhagic fever virus, is mediated by the viral glycoprotein (GP). The receptor-binding subunit of GP must be cleaved (by endosomal cathepsins) in order for entry and infection to proceed. Cleavage appears to proceed through 50-kDa and 20-kDa intermediates, ultimately generating a key 19-kDa core. How 19-kDa GP is subsequently triggered to bind membranes and induce fusion remains a mystery. Here we show that 50-kDa GP cannot be triggered to bind to liposomes in response to elevated temperature but that 20-kDa and 19-kDa GP can. Importantly, 19-kDa GP can be triggered at temperatures ～10°C lower than 20-kDa GP, suggesting that it is the most fusion ready form. Triggering by heat (or urea) occurs only at pH 5, not pH 7.5, and involves the fusion loop, as a fusion loop mutant is defective in liposome binding. We further show that mild reduction (preferentially at low pH) triggers 19-kDa GP to bind to liposomes, with the wild-type protein being triggered to a greater extent than the fusion loop mutant. Moreover, mild reduction inactivates pseudovirion infection, suggesting that reduction can also trigger 19-kDa GP on virus particles. Our results support the hypothesis that priming of EBOV GP, specifically to the 19-kDa core, potentiates GP to undergo subsequent fusion-relevant conformational changes. Our findings also indicate that low pH and an additional endosomal factor (possibly reduction or possibly a process mimicked by reduction) act as fusion triggers.     

Structure of an antibody in complex with its mucin domain linear epitope that is protective against Ebola virus

Antibody 14G7 is protective against lethal Ebola virus challenge and recognizes a distinct linear epitope in the prominent mucin-like domain of the Ebola virus glycoprotein GP. The structure of 14G7 in complex with its linear peptide epitope has now been determined to 2.8 Å. The structure shows that this GP sequence forms a tandem β-hairpin structure that binds deeply into a cleft in the antibody-combining site. A key threonine at the apex of one turn is critical for antibody interaction and is conserved among all Ebola viruses. This work provides further insight into the mechanism of protection by antibodies that target the protruding, highly accessible mucin-like domain of Ebola virus and the structural framework for understanding and characterizing candidate immunotherapeutics.     

Proteomic analysis of human immunodeficiency virus using liquid chromatography/tandem mass spectrometry effectively distinguishes specific incorporated host proteins

A major challenge to studying virus-incorporated host proteins is the fact that they are not encoded by the viral genome. We used Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS) on whole virions to obtain a snapshot of the HIV-1 proteome. We identified known viral and host-cellular proteins and also identified novel components of HIV-1 and confirm these by traditional biochemical methods. Our comparison of wild-type and mutant viruses demonstrates that LC-MS/MS has the specificity to distinguish the presence/absence of a single host protein in intact virions.     

Host cyclophilin A mediates HIV-1 attachment to target cells via heparans

The present study proposes a novel mode of action for cyclophilin A (CypA) in the HIV-1 life cycle. We demonstrate that CypA-deficient viruses do not replicate because they fail to attach to target cells. We show that CypA is exposed at the viral membrane and mediates HIV-1 attachment. We identify heparan as the exclusive cellular binding partner for CypA. Furthermore, CypA binds directly to heparan via a domain rich in basic residues similar to known heparin-binding motifs. This interaction between exposed CypA and cell surface heparans represents the initial step of HIV-1 attachment and is a necessary precursor to gp120-binding to CD4. In conclusion, HIV-1 attachment to target cells is a multi-step process that requires an initial CypA-heparan interaction followed by the gp120-CD4 interaction.     

Contrasting IgG structures reveal extreme asymmetry and flexibility

The crystal structure of IgG1 b12 represents the first visualization of an intact human IgG with a full-length hinge that has all domains ordered and visible. In comparison to intact murine antibodies and hinge-deletant human antibodies, b12 reveals extreme asymmetry, indicative of the extraordinary interdomain flexibility within an antibody. In addition, the structure provides an illustration of the human IgG1 hinge in its entirety and of asymmetry in the composition of the carbohydrate attached to each C(H)2 domain of the Fc. The two separate hinges assume different conformations in order to accommodate the vastly different placements of the two Fab domains relative to the Fc domain. Interestingly, only one of two possible intra-hinge disulfides is formed.     

Structure of a high-affinity "mimotope" peptide bound to HIV-1-neutralizing antibody b12 explains its inability to elicit gp120 cross-reactive antibodies

The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 A resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining complementary experimental approaches in analyzing the antigenic and immunogenic properties of putative molecular mimics.