Audray K. Harris

Influenza virus pleiomorphy characterized by cryoelectron tomography.

Influenza virus remains a global health threat, with millions of infections annually and the impending threat that a strain of avian influenza may develop into a human pandemic. Despite its importance as a pathogen, little is known about the virus structure, in part because of its intrinsic structural variability (pleiomorphy): the primary distinction is between spherical and elongated particles, but both vary in size. Pleiomorphy has thwarted structural analysis by image reconstruction of electron micrographs based on averaging many identical particles. In this study, we used cryoelectron tomography to visualize the 3D structures of 110 individual virions of the X-31 (H3N2) strain of influenza A. The tomograms distinguish two kinds of glycoprotein spikes [hemagglutinin (HA) and neuraminidase (NA)] in the viral envelope, resolve the matrix protein layer lining the envelope, and depict internal configurations of ribonucleoprotein (RNP) complexes. They also reveal the stems that link the glycoprotein ectodomains to the membrane and interactions among the glycoproteins, the matrix, and the RNPs that presumably control the budding of nascent virions from host cells. Five classes of virions, four spherical and one elongated, are distinguished by features of their matrix layer and RNP organization. Some virions have substantial gaps in their matrix layer (“molecular fontanels”), and others appear to lack a matrix layer entirely, suggesting the existence of an alternative budding pathway in which matrix protein is minimally involved.

Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection

The antibody response to influenza is primarily focused on the head region of the hemagglutinin (HA) glycoprotein, which in turn undergoes antigenic drift, thus necessitating annual updates of influenza vaccines. In contrast, the immunogenically subdominant stem region of HA is highly conserved and recognized by antibodies capable of binding multiple HA subtypes. Here we report the structure-based development of an H1 HA stem–only immunogen that confers heterosubtypic protection in mice and ferrets. Six iterative cycles of structure-based design (Gen1–Gen6) yielded successive H1 HA stabilized-stem (HA–SS) immunogens that lack the immunodominant head domain. Antigenic characterization, determination of two HA–SS crystal structures in complex with stem-specific monoclonal antibodies and cryo-electron microscopy analysis of HA–SS on ferritin nanoparticles (H1–SS–np) confirmed the preservation of key structural elements. Vaccination of mice and ferrets with H1–SS–np elicited broadly cross-reactive antibodies that completely protected mice and partially protected ferrets against lethal heterosubtypic H5N1 influenza virus challenge despite the absence of detectable H5N1 neutralizing activity in vitro. Passive transfer of immunoglobulin from H1–SS–np–immunized mice to naive mice conferred protection against H5N1 challenge, indicating that vaccine-elicited HA stem–specific antibodies can protect against diverse group 1 influenza strains.

Rapid development of a DNA vaccine for Zika virus

A DNA vaccine candidate for Zika The ongoing Zika epidemic in the Americas and the Caribbean urgently needs a protective vaccine. Two DNA vaccines composed of the genes that encode the structural premembrane and envelope proteins of Zika virus have been tested in monkeys. Dowd et al. show that two doses of vaccine given intramuscularly completely protected 17 of 18 animals against Zika virus challenge. A single low dose of vaccine was not protective but did reduce viral loads. Protection correlated with serum antibody neutralizing activity. Phase I clinical trials testing these vaccines are already ongoing. Science, this issue p. 237 DNA-vaccine–induced neutralizing antibodies largely protect monkeys after experimental challenge by virus infection. Zika virus (ZIKV) was identified as a cause of congenital disease during the explosive outbreak in the Americas and Caribbean that began in 2015. Because of the ongoing fetal risk from endemic disease and travel-related exposures, a vaccine to prevent viremia in women of childbearing age and their partners is imperative. We found that vaccination with DNA expressing the premembrane and envelope proteins of ZIKV was immunogenic in mice and nonhuman primates, and protection against viremia after ZIKV challenge correlated with serum neutralizing activity. These data not only indicate that DNA vaccination could be a successful approach to protect against ZIKV infection, but also suggest a protective threshold of vaccine-induced neutralizing activity that prevents viremia after acute infection.

Trimeric HIV-1 glycoprotein gp140 immunogens and native HIV-1 envelope glycoproteins display the same closed and open quaternary molecular architectures

The initial step in HIV-1 infection occurs with the binding of cell surface CD4 to trimeric HIV-1 envelope glycoproteins (Env), a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120). The design of soluble versions of trimeric Env that display structural and functional properties similar to those observed on intact viruses is highly desirable from the viewpoint of designing immunogens that could be effective as vaccines against HIV/AIDS. Using cryoelectron tomography combined with subvolume averaging, we have analyzed the structure of SOSIP gp140 trimers, which are cleaved, solubilized versions of the ectodomain of trimeric HIV-1 Env. We show that unliganded gp140 trimers adopt a quaternary arrangement similar to that displayed by native unliganded trimers on the surface of intact HIV-1 virions. When complexed with soluble CD4, Fab 17b, which binds to gp120 at its chemokine coreceptor binding site, or both soluble CD4 and 17b Fab, gp140 trimers display an open conformation in which there is an outward rotation and displacement of each gp120 protomer. We demonstrate that the molecular arrangements of gp120 trimers in the closed and open conformations of the soluble trimer are the same as those observed for the closed and open states, respectively, of trimeric gp120 on intact HIV-1 BaL virions, establishing that soluble gp140 trimers can be designed to mimic the quaternary structural transitions displayed by native trimeric Env.

Structure, Assembly, and Antigenicity of Hepatitis B Virus Capsid Proteins

Publisher Summary This chapter reviews current information pertaining to the structure and assembly properties of hepatitis B virus (HBV) capsid protein, as well as the insights into its antigenicity and other interactions. HBV has a small (3.2 kb) DNA genome, although this modest genetic endowment is amplified by a variety of strategies, including alternative expression products of the same gene. In the replication cycle of HBV, the genome is initially incorporated into the assembling virus particle as a single-stranded RNA molecule—the pregenome—that is subsequently retrotranscribed in situ by the viral reverse transcriptase (RT). The DNA-containing nucleocapsid subsequently becomes enveloped by a membrane containing the viral glycoprotein—surface antigen (sAg), of which there are three size variants called S, M, and L, respectively—to yield the completely assembled and infectious virion. The capsid protein of HBV has several unexpected properties. It was found to have a novel fold, rich in a helix, and quite distinct from the eight-stranded b barrel that was common to the first dozen or so capsid proteins to be solved (from plant, animal, and bacterial viruses) and the other capsid protein folds that have been determined more recently.

Structure and accessibility of HA trimers on intact 2009 H1N1 pandemic influenza virus to stem region-specific neutralizing antibodies

Rapid antigenic variation of HA, the major virion surface protein of influenza A virus, remains the principal challenge to the development of broader and more effective vaccines. Some regions of HA, such as the stem region proximal to the viral membrane, are nevertheless highly conserved across strains and among most subtypes. A fundamental question in vaccine design is the extent to which HA stem regions on the surface of the virus are accessible to broadly neutralizing antibodies. Here we report 3D structures derived from cryoelectron tomography of HA on intact 2009 H1N1 pandemic virions in the presence and absence of the antibody C179, which neutralizes viruses expressing a broad range of HA subtypes, including H1, H2, H5, H6, and H9. By fitting previously derived crystallographic structures of trimeric HA into the density maps, we deduced the locations of the molecular surfaces of HA involved in interaction with C179. Using computational methods to distinguish individual unliganded HA trimers from those that have bound C179 antibody, we demonstrate that ∼75% of HA trimers on the surface of the virus have C179 bound to the stem domain. Thus, despite their close packing on the viral membrane, the majority of HA trimers on intact virions are available to bind anti-stem antibodies that target conserved HA epitopes, establishing the feasibility of universal influenza vaccines that elicit such antibodies.

HIV-1 Envelope Glycoprotein Trimers Display Open Quaternary Conformation When Bound to the gp41 Membrane-Proximal External-Region-Directed Broadly Neutralizing Antibody Z13e1

ABSTRACT We describe cryo-electron microscopic studies of the interaction between the ectodomain of the trimeric HIV-1 envelope glycoprotein (Env) and Z13e1, a broadly neutralizing antibody that targets the membrane-proximal external region (MPER) of the gp41 subunit. We show that Z13e1-bound Env displays an open quaternary conformation similar to the CD4-bound conformation. Our results support the idea that MPER-directed antibodies, such as Z13e1, block viral entry by interacting with Env at a step after CD4 activation.

Molecular Basis for the High Degree of Antigenic Cross-Reactivity between Hepatitis B Virus Capsids (HBcAg) and Dimeric Capsid-Related Protein (HBeAg): Insights into the Enigmatic Nature of the e-Antigen

The hepatitis B virus core gene codes for two closely related antigens: a 21-kDa protein that forms dimers that assemble as multimegadalton capsids, and a 17-kDa protein that also forms dimers but that do not assemble. The proteins, respectively referred to as core antigen (HBcAg) and e-antigen (HBeAg), share a sequence of 149 residues but have different amino- and carboxy-termini. Their structural and serological relationship has long been unclear. With insights gained from recent structural studies on immune complexes of the capsids, the relationship was reassessed using recombinant forms of the antigens and a panel of monoclonal antibodies (mAbs) commonly believed to discriminate between core and e-antigen. Surface plasmon resonance (SPR) was used to measure the affinities, in contrast to previous studies that used more error-prone and less sensitive plate-type assays. Four of the six mAbs did not discriminate between core and e-antigen, nor did they discriminate between e-antigen and dimers of dissociated core antigen capsids. One mAb (3120) was specific for assembled capsids and one (e6) was specific for unassembled dimers. Epitope valency of the e-antigen was also studied, using a sandwich SPR assay where e-antigen was captured with one mAb and probed with a second. The e-antigen is often considered to be a monomeric protein on the basis of monovalent reactivity with antibody pairs specific for either an alpha or beta epitope (in a prior nomenclature for e-antigen specificity). This model, however, is incorrect, because recombinant e-antigen is a stable dimer and its apparent monovalency is due to steric blockage. This was proven by the formation of a 2:1 Fab e6-e-antigen complex. These results suggest new approaches for the isolation of the authentic e-antigen, its biological assay, and its stabilization as an immune complex for structural studies.

Structural studies of influenza virus RNPs by electron microscopy indicate molecular contortions within NP supra-structures

Ribonucleoprotein (RNP) complexes of influenza viruses are composed of multiple copies of the viral nucleoprotein (NP) that can form filamentous supra-structures. RNPs package distinct viral genomic RNA segments of different lengths into pleomorphic influenza virions. RNPs also function in viral RNA transcription and replication. Different RNP segments have varying lengths, but all must be incorporated into virions during assembly and then released during viral entry for productive infection cycles. RNP structures serve varied functions in the viral replication cycle, therefore understanding their molecular organization and flexibility is essential to understanding these functions. Here, we show using electron tomography and image analyses that isolated RNP filaments are not rigid helical structures, but instead display variations in lengths, curvatures, and even tolerated kinks and local unwinding. Additionally, we observed NP rings within RNP preparations, which were commonly composed of 5, 6, or 7 NP molecules and were of similar widths to filaments, suggesting plasticity in NP-NP interactions mediate RNP structural polymorphism. To demonstrate that NP alone could generate rings of variable oligomeric state, we performed 2D single particle image analysis on recombinant NP and found that rings of 4 and 5 protomers dominated, but rings of all compositions up to 7 were directly observed with variable frequency. This structural flexibility may be needed as RNPs carry out the interactions and conformational changes required for RNP assembly and genome packaging as well as virus uncoating.

Characterization of Influenza Vaccine Hemagglutinin Complexes by Cryo-Electron Microscopy and Image Analyses Reveals Structural Polymorphisms

ABSTRACT Influenza virus afflicts millions of people worldwide on an annual basis. There is an ever-present risk that animal viruses will cross the species barrier to cause epidemics and pandemics resulting in great morbidity and mortality. Zoonosis outbreaks, such as the H7N9 outbreak, underscore the need to better understand the molecular organization of viral immunogens, such as recombinant influenza virus hemagglutinin (HA) proteins, used in influenza virus subunit vaccines in order to optimize vaccine efficacy. Here, using cryo-electron microscopy and image analysis, we show that recombinant H7 HA in vaccines formed macromolecular complexes consisting of variable numbers of HA subunits (range, 6 to 8). In addition, HA complexes were distributed across at least four distinct structural classes (polymorphisms). Three-dimensional (3D) reconstruction and molecular modeling indicated that HA was in the prefusion state and suggested that the oligomerization and the structural polymorphisms observed were due to hydrophobic interactions involving the transmembrane regions. These experiments suggest that characterization of the molecular structures of influenza virus HA complexes used in subunit vaccines will lead to better understanding of the differences in vaccine efficacy and to the optimization of subunit vaccines to prevent influenza virus infection.