Protective neutralizing antibodies from human survivors of Crimean-Congo hemorrhagic fever.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a World Health Organization priority pathogen. CCHFV infections cause a highly lethal hemorrhagic fever for which specific treatments and vaccines are urgently needed. Here, we characterize the human immune response to natural CCHFV infection to identify potent neutralizing monoclonal antibodies (nAbs) targeting the viral glycoprotein. Competition experiments showed that these nAbs bind six distinct antigenic sites in the Gc subunit. These sites were further delineated through mutagenesis and mapped onto a prefusion model of Gc. Pairwise screening identified combinations of non-competing nAbs that afford synergistic neutralization. Further enhancements in neutralization breadth and potency were attained by physically linking variable domains of synergistic nAb pairs through bispecific antibody (bsAb) engineering. Although multiple nAbs protected mice from lethal CCHFV challenge in pre- or post-exposure prophylactic settings, only a single bsAb, DVD-121-801, afforded therapeutic protection. DVD-121-801 is a promising candidate suitable for clinical development as a CCHFV therapeutic.

Pan-protective anti-alphavirus human antibodies target a conserved E1 protein epitope.

Alphaviruses are emerging, mosquito-transmitted pathogens that cause musculoskeletal and neurological disease in humans. Although neutralizing antibodies that inhibit individual alphaviruses have been described, broadly reactive antibodies that protect against both arthritogenic and encephalitic alphaviruses have not been reported. Here, we identify DC2.112 and DC2.315, two pan-protective yet poorly neutralizing human monoclonal antibodies (mAbs) that avidly bind to viral antigen on the surface of cells infected with arthritogenic and encephalitic alphaviruses. These mAbs engage a conserved epitope in domain II of the E1 protein proximal to and within the fusion peptide. Treatment with DC2.112 or DC2.315 protects mice against infection by both arthritogenic (chikungunya and Mayaro) and encephalitic (Venezuelan, Eastern, and Western equine encephalitis) alphaviruses through multiple mechanisms, including inhibition of viral egress and monocyte-dependent Fc effector functions. These findings define a conserved epitope recognized by weakly neutralizing yet protective antibodies that could be targeted for pan-alphavirus immunotherapy and vaccine design.

Systematic Analysis of Monoclonal Antibodies against Ebola Virus GP Defines Features that Contribute to Protection.

Antibodies are promising post-exposure therapies against emerging viruses, but which antibody features and in vitro assays best forecast protection are unclear. Our international consortium systematically evaluated antibodies against Ebola virus (EBOV) using multidisciplinary assays. For each antibody, we evaluated epitopes recognized on the viral surface glycoprotein (GP) and secreted glycoprotein (sGP), readouts of multiple neutralization assays, fraction of virions left un-neutralized, glycan structures, phagocytic and natural killer cell functions elicited, and in vivo protection in a mouse challenge model. Neutralization and induction of multiple immune effector functions (IEFs) correlated most strongly with protection. Neutralization predominantly occurred via epitopes maintained on endosomally cleaved GP, whereas maximal IEF mapped to epitopes farthest from the viral membrane. Unexpectedly, sGP cross-reactivity did not significantly influence in vivo protection. This comprehensive dataset provides a rubric to evaluate novel antibodies and vaccine responses and a roadmap for therapeutic development for EBOV and related viruses.

Antibodies from a Human Survivor Define Sites of Vulnerability for Broad Protection against Ebolaviruses.

Experimental monoclonal antibody (mAb) therapies have shown promise for treatment of lethal Ebola virus (EBOV) infections, but their species-specific recognition of the viral glycoprotein (GP) has limited their use against other divergent ebolaviruses associated with human disease. Here, we mined the human immune response to natural EBOV infection and identified mAbs with exceptionally potent pan-ebolavirus neutralizing activity and protective efficacy against three virulent ebolaviruses. These mAbs recognize an inter-protomer epitope in the GP fusion loop, a critical and conserved element of the viral membrane fusion machinery, and neutralize viral entry by targeting a proteolytically primed, fusion-competent GP intermediate (GPCL) generated in host cell endosomes. Only a few somatic hypermutations are required for broad antiviral activity, and germline-approximating variants display enhanced GPCL recognition, suggesting that such antibodies could be elicited more efficiently with suitably optimized GP immunogens. Our findings inform the development of both broadly effective immunotherapeutics and vaccines against filoviruses.

Design and characterization of protective pan-ebolavirus and pan-filovirus bispecific antibodies.

Monoclonal antibodies (mAbs) are an important class of antiviral therapeutics. MAbs are highly selective, well tolerated, and have long in vivo half-life as well as the capacity to induce immune-mediated virus clearance. Their activities can be further enhanced by integration of their variable fragments (Fvs) into bispecific antibodies (bsAbs), affording simultaneous targeting of multiple epitopes to improve potency and breadth and/or to mitigate against viral escape by a single mutation. Here, we explore a bsAb strategy for generation of pan-ebolavirus and pan-filovirus immunotherapeutics. Filoviruses, including Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV), cause severe hemorrhagic fever. Although there are two FDA-approved mAb therapies for EBOV infection, these do not extend to other filoviruses. Here, we combine Fvs from broad ebolavirus mAbs to generate novel pan-ebolavirus bsAbs that are potently neutralizing, confer protection in mice, and are resistant to viral escape. Moreover, we combine Fvs from pan-ebolavirus mAbs with those of protective MARV mAbs to generate pan-filovirus protective bsAbs. These results provide guidelines for broad antiviral bsAb design and generate new immunotherapeutic candidates.

Probing differences among Aß oligomers with two triangular trimers derived from Aß.

The assembly of the ß-amyloid peptide (Aß) to form oligomers and fibrils is closely associated with the pathogenesis and progression of Alzheimer's disease. Aß is a shape-shifting peptide capable of adopting many conformations and folds within the multitude of oligomers and fibrils the peptide forms. These properties have precluded detailed structural elucidation and biological characterization of homogeneous, well-defined Aß oligomers. In this paper, we compare the structural, biophysical, and biological characteristics of two different covalently stabilized isomorphic trimers derived from the central and C-terminal regions Aß. X-ray crystallography reveals the structures of the trimers and shows that each trimer forms a ball-shaped dodecamer. Solution-phase and cell-based studies demonstrate that the two trimers exhibit markedly different assembly and biological properties. One trimer forms small soluble oligomers that enter cells through endocytosis and activate capase-3/7-mediated apoptosis, while the other trimer forms large insoluble aggregates that accumulate on the outer plasma membrane and elicit cellular toxicity through an apoptosis-independent mechanism. The two trimers also exhibit different effects on the aggregation, toxicity, and cellular interaction of full-length Aß, with one trimer showing a greater propensity to interact with Aß than the other. The studies described in this paper indicate that the two trimers share structural, biophysical, and biological characteristics with oligomers of full-length Aß. The varying structural, assembly, and biological characteristics of the two trimers provide a working model for how different Aß trimers can assemble and lead to different biological effects, which may help shed light on the differences among Aß oligomers.

A Powassan virus domain III nanoparticle immunogen elicits neutralizing and protective antibodies in mice.

Powassan virus (POWV) is an emerging tick borne flavivirus (TBFV) that causes severe neuroinvasive disease. Currently, there are no approved treatments or vaccines to combat POWV infection. Here, we generated and characterized a nanoparticle immunogen displaying domain III (EDIII) of the POWV E glycoprotein. Immunization with POWV EDIII presented on nanoparticles resulted in significantly higher serum neutralizing titers against POWV than immunization with monomeric POWV EDIII. Furthermore, passive transfer of EDIII-reactive sera protected against POWV challenge in vivo. We isolated and characterized a panel of EDIII-specific monoclonal antibodies (mAbs) and identified several that potently inhibit POWV infection and engage distinct epitopes within the lateral ridge and C-C' loop of the EDIII. By creating a subunit-based nanoparticle immunogen with vaccine potential that elicits antibodies with protective activity against POWV infection, our findings enhance our understanding of the molecular determinants of antibody-mediated neutralization of TBFVs.

Protocadherin-1 is essential for cell entry by New World hantaviruses.

The zoonotic transmission of hantaviruses from their rodent hosts to humans in North and South America is associated with a severe and frequently fatal respiratory disease, hantavirus pulmonary syndrome (HPS)1,2. No specific antiviral treatments for HPS are available, and no molecular determinants of in vivo susceptibility to hantavirus infection and HPS are known. Here we identify the human asthma-associated gene protocadherin-1 (PCDH1)3-6 as an essential determinant of entry and infection in pulmonary endothelial cells by two hantaviruses that cause HPS, Andes virus (ANDV) and Sin Nombre virus (SNV). In vitro, we show that the surface glycoproteins of ANDV and SNV directly recognize the outermost extracellular repeat domain of PCDH1-a member of the cadherin superfamily7,8-to exploit PCDH1 for entry. In vivo, genetic ablation of PCDH1 renders Syrian golden hamsters highly resistant to a usually lethal ANDV challenge. Targeting PCDH1 could provide strategies to reduce infection and disease caused by New World hantaviruses.

Near-germline human monoclonal antibodies neutralize and protect against multiple arthritogenic alphaviruses.

Arthritogenic alphaviruses are globally distributed, mosquito-transmitted viruses that cause rheumatological disease in humans and include Chikungunya virus (CHIKV), Mayaro virus (MAYV), and others. Although serological evidence suggests that some antibody-mediated heterologous immunity may be afforded by alphavirus infection, the extent to which broadly neutralizing antibodies that protect against multiple arthritogenic alphaviruses are elicited during natural infection remains unknown. Here, we describe the isolation and characterization of MAYV-reactive alphavirus monoclonal antibodies (mAbs) from a CHIKV-convalescent donor. We characterized 33 human mAbs that cross-reacted with CHIKV and MAYV and engaged multiple epitopes on the E1 and E2 glycoproteins. We identified five mAbs that target distinct regions of the B domain of E2 and potently neutralize multiple alphaviruses with differential breadth of inhibition. These broadly neutralizing mAbs (bNAbs) contain few somatic mutations and inferred germline-revertants retained neutralizing capacity. Two bNAbs, DC2.M16 and DC2.M357, protected against both CHIKV- and MAYV-induced musculoskeletal disease in mice. These findings enhance our understanding of the cross-reactive and cross-protective antibody response to human alphavirus infections.

Longitudinally monitored immune biomarkers predict the timing of COVID-19 outcomes.

The clinical outcome of SARS-CoV-2 infection varies widely between individuals. Machine learning models can support decision making in healthcare by assessing fatality risk in patients that do not yet show severe signs of COVID-19. Most predictive models rely on static demographic features and clinical values obtained upon hospitalization. However, time-dependent biomarkers associated with COVID-19 severity, such as antibody titers, can substantially contribute to the development of more accurate outcome models. Here we show that models trained on immune biomarkers, longitudinally monitored throughout hospitalization, predicted mortality and were more accurate than models based on demographic and clinical data upon hospital admission. Our best-performing predictive models were based on the temporal analysis of anti-SARS-CoV-2 Spike IgG titers, white blood cell (WBC), neutrophil and lymphocyte counts. These biomarkers, together with C-reactive protein and blood urea nitrogen levels, were found to correlate with severity of disease and mortality in a time-dependent manner. Shapley additive explanations of our model revealed the higher predictive value of day post-symptom onset (PSO) as hospitalization progresses and showed how immune biomarkers contribute to predict mortality. In sum, we demonstrate that the kinetics of immune biomarkers can inform clinical models to serve as a powerful monitoring tool for predicting fatality risk in hospitalized COVID-19 patients, underscoring the importance of contextualizing clinical parameters according to their time post-symptom onset.

Peptide-Based Vaccines: Current Progress and Future Challenges.

Vaccines have had a profound impact on the management and prevention of infectious disease. In addition, the development of vaccines against chronic diseases has attracted considerable interest as an approach to prevent, rather than treat, conditions such as cancer, Alzheimer's disease, and others. Subunit vaccines consist of nongenetic components of the infectious agent or disease-related epitope. In this Review, we discuss peptide-based vaccines and their potential in three therapeutic areas: infectious disease, Alzheimer's disease, and cancer. We discuss factors that contribute to vaccine efficacy and how these parameters may potentially be modulated by design. We examine both clinically tested vaccines as well as nascent approaches and explore current challenges and potential remedies. While peptide vaccines hold substantial promise in the prevention of human disease, many obstacles remain that have hampered their clinical use; thus, continued research efforts to address these challenges are warranted.

Human monoclonal antibodies against chikungunya virus target multiple distinct epitopes in the E1 and E2 glycoproteins.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes persistent arthritis in a subset of human patients. We report the isolation and functional characterization of monoclonal antibodies (mAbs) from two patients infected with CHIKV in the Dominican Republic. Single B cell sorting yielded a panel of 46 human mAbs of diverse germline lineages that targeted epitopes within the E1 or E2 glycoproteins. MAbs that recognized either E1 or E2 proteins exhibited neutralizing activity. Viral escape mutations localized the binding epitopes for two E1 mAbs to sites within domain I or the linker between domains I and III; and for two E2 mAbs between the ß-connector region and the B-domain. Two of the E2-specific mAbs conferred protection in vivo in a stringent lethal challenge mouse model of CHIKV infection, whereas the E1 mAbs did not. These results provide insight into human antibody response to CHIKV and identify candidate mAbs for therapeutic intervention.

Design and evaluation of bi- and trispecific antibodies targeting multiple filovirus glycoproteins.

Filoviruses (family Filoviridae) include five ebolaviruses and Marburg virus. These pathogens cause a rapidly progressing and severe viral disease with high mortality rates (generally 30-90%). Outbreaks of filovirus disease are sporadic and, until recently, were limited to less than 500 cases. However, the 2013-2016 epidemic in western Africa, caused by Ebola virus (EBOV), illustrated the potential of filovirus outbreaks to escalate to a much larger scale (over 28,000 suspected cases). mAbs against the envelope glycoprotein represent a promising therapeutic platform for managing filovirus infections. However, mAbs that exhibit neutralization or protective properties against multiple filoviruses are rare. Here we examined a panel of engineered bi- and trispecific antibodies, in which variable domains of mAbs that target epitopes from multiple filoviruses were combined, for their capacity to neutralize viral infection across filovirus species. We found that bispecific combinations targeting EBOV and Sudan virus (another ebolavirus), provide potent cross-neutralization and protection in mice. Furthermore, trispecific combinations, targeting EBOV, Sudan virus, and Marburg virus, exhibited strong neutralization potential against all three viruses. These results provide important insights into multispecific antibody engineering against filoviruses and will inform future immunotherapeutic discoveries.

Engineered Dengue Virus Domain III Proteins Elicit Cross-Neutralizing Antibody Responses in Mice.

Dengue virus is the most globally prevalent mosquito-transmitted virus. Primary infection with one of four cocirculating serotypes (DENV-1 to -4) causes a febrile illness, but secondary infection with a heterologous serotype can result in severe disease, due in part to antibody-dependent enhancement of infection (ADE). In ADE, cross-reactive but nonneutralizing antibodies, or subprotective levels of neutralizing antibodies, promote uptake of antibody-opsonized virus in Fc-γ receptor-positive cells. Thus, elicitation of broadly neutralizing antibodies (bNAbs), but not nonneutralizing antibodies, is desirable for dengue vaccine development. Domain III of the envelope glycoprotein (EDIII) is targeted by bNAbs and thus is an attractive immunogen. However, immunization with EDIII results in sera with limited neutralization breadth. We developed "resurfaced" EDIII immunogens (rsDIIIs) in which the A/G strand epitope that is targeted by bNAb 4E11 is maintained but less desirable epitopes are masked. RsDIIIs bound 4E11, but not serotype-specific or nonneutralizing antibodies. One rsDIII and, unexpectedly, wild-type (WT) DENV-2 EDIII elicited cross-neutralizing antibody responses against DENV-1 to -3 in mice. While these sera were cross-neutralizing, they were not sufficiently potent to protect AG129 immunocompromised mice at a dose of 200 µl (50% focus reduction neutralization titer [FRNT50], ∼1:60 to 1:130) against mouse-adapted DENV-2. Our results provide insight into immunogen design strategies based on EDIII.IMPORTANCE Dengue virus causes approximately 390 million infections per year. Primary infection by one serotype causes a self-limiting febrile illness, but secondary infection by a heterologous serotype can result in severe dengue syndrome, which is characterized by hemorrhagic fever and shock syndrome. This severe disease is thought to arise because of cross-reactive, non- or poorly neutralizing antibodies from the primary infection that are present in serum at the time of secondary infection. These cross-reactive antibodies enhance the infection rather than controlling it. Therefore, induction of a broadly and potently neutralizing antibody response is desirable for dengue vaccine development. Here, we explore a novel strategy for developing immunogens based on domain III of the E glycoprotein, where undesirable epitopes (nonneutralizing or nonconserved) are masked by mutation. This work provides fundamental insight into the immune response to domain III that can be leveraged for future immunogen design.

Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site.

The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic Bcl-2-associated X protein (BAX) is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how this may be inhibited is not yet completely understood. Here we describe synthetic antibody fragments (Fabs) as structural and biochemical probes to investigate the potential mechanisms of BAX regulation. These synthetic Fabs bind with high affinity to BAX and inhibit its activation by the BH3-only protein tBID (truncated Bcl2 interacting protein) in assays using liposomal membranes. Inhibition of BAX by a representative Fab, 3G11, prevented mitochondrial translocation of BAX and BAX-mediated cytochrome c release. Using NMR and hydrogen-deuterium exchange mass spectrometry, we showed that 3G11 forms a stoichiometric and stable complex without inducing a significant conformational change on monomeric and inactive BAX. We identified that the Fab-binding site on BAX involves residues of helices α1/α6 and the α1-α2 loop. Therefore, the inhibitory binding surface of 3G11 overlaps with the N-terminal activation site of BAX, suggesting a novel mechanism of BAX inhibition through direct binding to the BAX N-terminal activation site. The synthetic Fabs reported here reveal, as probes, novel mechanistic insights into BAX inhibition and provide a blueprint for developing inhibitors of BAX activation.

Pan-ebolavirus and Pan-filovirus Mouse Monoclonal Antibodies: Protection against Ebola and Sudan Viruses.

UNLABELLED: The unprecedented 2014-2015 Ebola virus disease (EVD) outbreak in West Africa has highlighted the need for effective therapeutics against filoviruses. Monoclonal antibody (MAb) cocktails have shown great potential as EVD therapeutics; however, the existing protective MAbs are virus species specific. Here we report the development of pan-ebolavirus and pan-filovirus antibodies generated by repeated immunization of mice with filovirus glycoproteins engineered to drive the B cell responses toward conserved epitopes. Multiple pan-ebolavirus antibodies were identified that react to the Ebola, Sudan, Bundibugyo, and Reston viruses. A pan-filovirus antibody that was reactive to the receptor binding regions of all filovirus glycoproteins was also identified. Significant postexposure efficacy of several MAbs, including a novel antibody cocktail, was demonstrated. For the first time, we report cross-neutralization and in vivo protection against two highly divergent filovirus species, i.e., Ebola virus and Sudan virus, with a single antibody. Competition studies indicate that this antibody targets a previously unrecognized conserved neutralizing epitope that involves the glycan cap. Mechanistic studies indicated that, besides neutralization, innate immune cell effector functions may play a role in the antiviral activity of the antibodies. Our findings further suggest critical novel epitopes that can be utilized to design effective cocktails for broad protection against multiple filovirus species. IMPORTANCE: Filoviruses represent a major public health threat in Africa and an emerging global concern. Largely driven by the U.S. biodefense funding programs and reinforced by the 2014 outbreaks, current immunotherapeutics are primarily focused on a single filovirus species called Ebola virus (EBOV) (formerly Zaire Ebola virus). However, other filoviruses including Sudan, Bundibugyo, and Marburg viruses have caused human outbreaks with mortality rates as high as 90%. Thus, cross-protective immunotherapeutics are urgently needed. Here, we describe monoclonal antibodies with cross-reactivity to several filoviruses, including the first report of a cross-neutralizing antibody that exhibits protection against Ebola virus and Sudan virus in mice. Our results further describe a novel combination of antibodies with enhanced protective efficacy. These results form a basis for further development of effective immunotherapeutics against filoviruses for human use. Understanding the cross-protective epitopes are also important for rational design of pan-ebolavirus and pan-filovirus vaccines.

Interrogation of side chain biases for oligomannose recognition by antibody 2G12 via structure-guided phage display libraries.

Monoclonal antibodies (mAbs) are essential reagents for deciphering gene or protein function and have been a fruitful source of therapeutic and diagnostic agents. However, developing anticarbohydrate antibodies to target glycans for those purposes has been less successful because the molecular basis for glycan-mAb interactions is poorly understood relative to protein- or peptide-binding mAbs. Here, we report our investigation on glycan-mAb interactions by using the unique architectural scaffold of 2G12, an antibody that targets oligomannoses on the HIV-1 glycoprotein gp120, as the template for engineering highly specific mAbs to target glycans. We first analyzed 24 different X-ray structures of antiglycan mAbs from the Protein Data Bank to determine side chain amino acid distributions in of glycan-mAb interactions. We identified Tyr, Arg, Asn, Ser, Asp, and His as the six most prevalent residues in the glycan-mAb contacts. We then utilized this information to construct two phage display libraries ("Lib1" and "Lib2") in which positions on the heavy chain variable domains of 2G12 were allowed to vary in restricted manner among Tyr, Asp, Ser, His, Asn, Thr, Ala and Pro to interrogate the minimal physicochemical requirements for oligomannose recognition. We analyzed the sequences of 39 variants from Lib1 and 14 variants from Lib2 following selection against gp120, the results showed that there is a high degree of malleability within the 2G12 for glycan recognitions. We further characterized five unique phage clones from both libraries that exhibited a gp120-specific binding profile. Expression of two of these variants as soluble mAbs indicated that, while specificity of gp120-binding was retained, the affinity of these mutants was significantly reduced relative to WT 2G12. Nonetheless, the results indicate these is some malleability in the identity of contact residues and provide a novel insight into the nature of glycan-antibody interactions and how they may differ from protein-antibody binding interactions.

Structural and Functional Studies on the Marburg Virus GP2 Fusion Loop.

Marburg virus (MARV) and the ebolaviruses belong to the family Filoviridae (the members of which are filoviruses) that cause severe hemorrhagic fever. Infection requires fusion of the host and viral membranes, a process that occurs in the host cell endosomal compartment and is facilitated by the envelope glycoprotein fusion subunit, GP2. The N-terminal fusion loop (FL) of GP2 is a hydrophobic disulfide-bonded loop that is postulated to insert and disrupt the host endosomal membrane during fusion. Here, we describe the first structural and functional studies of a protein corresponding to the MARV GP2 FL. We found that this protein undergoes a pH-dependent conformational change, as monitored by circular dichroism and nuclear magnetic resonance. Furthermore, we report that, under low pH conditions, the MARV GP2 FL can induce content leakage from liposomes. The general aspects of this pH-dependent structure and lipid-perturbing behavior are consistent with previous reports on Ebola virus GP2 FL. However, nuclear magnetic resonance studies in lipid bicelles and mutational analysis indicate differences in structure exist between MARV and Ebola virus GP2 FL. These results provide new insight into the mechanism of MARV GP2-mediated cell entry.

Conditional trimerization and lytic activity of HIV-1 gp41 variants containing the membrane-associated segments.

Fusion of host and viral membranes is a critical step during infection by membrane-bound viruses. The HIV-1 glycoproteins gp120 (surface subunit) and gp41 (fusion subunit) represent the prototypic system for studying this process; in the prevailing model, the gp41 ectodomain forms a trimeric six-helix bundle that constitutes a critical intermediate and provides the energetic driving force for overcoming barriers associated with membrane fusion. However, most structural studies of gp41 variants have been performed either on ectodomain constructs lacking one or more of the membrane-associated segments (the fusion peptide, FP, the membrane-proximal external region, MPER, and the transmembrane domain, TM) or on variants consisting of these isolated segments alone without the ectodomain. Several recent reports have suggested that the HIV-1 ectodomain, as well as larger construct containing the membrane-bound segments, dissociates from a trimer to a monomer in detergent micelles. Here we compare the properties of a series of gp41 variants to delineate the roles of the ectodomain, FP, and MPER and TM, all in membrane-mimicking environments. We find that these proteins are prone to formation of a monomer in detergent micelles. In one case, we observed exclusive monomer formation at pH 4 but conditional trimerization at pH 7 even at low micromolar (∼5 µM) protein concentrations. Liposome release assays demonstrate that these gp41-related proteins have the capacity to induce content leakage but that this activity is also strongly modulated by pH with much higher activity at pH 4. Circular dichroism, nuclear magnetic resonance, and binding assays with antibodies specific to the MPER provide insight into the structural and functional roles of the FP, MPER, and TM and their effect on structure within the larger context of the fusion subunit.

A switch from parallel to antiparallel strand orientation in a coiled-coil X-ray structure via two core hydrophobic mutations.

The coiled-coil is one of the most ubiquitous and well studied protein structural motifs. Significant effort has been devoted to dissecting subtle variations of the typical heptad repeat sequence pattern that can designate larger topological features such as relative α-helical orientation and oligomer size. Here we report the X-ray structure of a model coiled-coil peptide, HA2-Del-L2seM, which forms an unanticipated core antiparallel dimer with potential sites for discrete higher-order multimerization (trimer or tetramer). In the X-ray structure, a third, partially-ordered α-helix is weakly associated with the antiparallel dimer and analytical ultracentrifugation experiments indicate the peptide forms a well-defined tetramer in solution. The HA2-Del-L2seM sequence is closely related to a parent model peptide, HA2-Del, which we previously reported adopts a parallel trimer; HA2-Del-L2seM differs by only hydrophobic leucine to selenomethione mutations and thus this subtle difference is sufficient to switch both relative α-helical topology and number of α-helices participating in the coiled-coil. Comparison of the X-ray structures of HA2-Del-L2seM (reported here) with the HA2-Del parent (reported previously) reveals novel interactions involving the selenomethionine residues that promote antiparallel coiled-coil configuration and preclude parallel trimer formation. These novel atomic insights are instructive for understanding subtle features that can affect coiled-coil topology and provide additional information for design of antiparallel coiled-coils.