The SARS-CoV-2 spike (S) protein is the target of vaccine design efforts to end the COVID-19 pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S protein appeared early during the pandemic, and are now the dominant form worldwide. Here, we explore spike conformational changes and the effects of the D614G mutation on a soluble S ectodomain construct. Cryo-EM structures reveal altered RBD disposition; antigenicity and proteolysis experiments reveal structural changes and enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage alters the up/down ratio of the Receptor Binding Domains (RBD) in the G614 S ectodomain, demonstrating an allosteric effect on RBD positioning triggered by changes in the SD2 region, that harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 spike conformational landscape and allostery, and have implications for vaccine design.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with multiple spike mutations enable increased transmission and antibody resistance. We combined cryo–electron microscopy (cryo-EM), binding, and computational analyses to study variant spikes, including one that was involved in transmission between minks and humans, and others that originated and spread in human populations. All variants showed increased angiotensin-converting enzyme 2 (ACE2) receptor binding and increased propensity for receptor binding domain (RBD)–up states. While adaptation to mink resulted in spike destabilization, the B.1.1.7 (UK) spike balanced stabilizing and destabilizing mutations. A local destabilizing effect of the RBD E484K mutation was implicated in resistance of the B.1.1.28/P.1 (Brazil) and B.1.351 (South Africa) variants to neutralizing antibodies. Our studies revealed allosteric effects of mutations and mechanistic differences that drive either interspecies transmission or escape from antibody neutralization.

The SARS-CoV-2 spike (S) protein is the target of vaccine design efforts to end the COVID-19 pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S protein appeared early during the pandemic, and are now the dominant form worldwide. Here, we analyze the D614G mutation in the context of a soluble S ectodomain construct. Cryo-EM structures, antigenicity and proteolysis experiments suggest altered conformational dynamics resulting in enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage alters the conformational dynamics of the Receptor Binding Domains (RBD) in the G614 S ectodomain, demonstrating an allosteric effect on the RBD dynamics triggered by changes in the SD2 region, that harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 spike conformational dynamics and allostery, and have implications for vaccine design.

New SARS-CoV-2 variants that have accumulated multiple mutations in the spike (S) glycoprotein enable increased transmission and resistance to neutralizing antibodies. Here, we study the antigenic and structural impacts of the S protein mutations from four variants, one that was involved in transmission between minks and humans, and three that rapidly spread in human populations and originated in the United Kingdom, Brazil or South Africa. All variants either retained or improved binding to the ACE2 receptor. The B.1.1.7 (UK) and B.1.1.28 (Brazil) spike variants showed reduced binding to neutralizing NTD and RBD antibodies, respectively, while the B.1.351 (SA) variant showed reduced binding to both NTD- and RBD-directed antibodies. Cryo-EM structural analyses revealed allosteric effects of the mutations on spike conformations and revealed mechanistic differences that either drive inter-species transmission or promotes viral escape from dominant neutralizing epitopes.

A detailed study of the pathogenesis of herpetic eye disease in the guinea pig was undertaken to further develop this animal model. Several well-known HSV-1 strains were tested for their ability to produce disease and cause acute and latent infections of the trigeminal ganglion: McKrae, KOS, McIntyre, RE, and Shealey. Two HSV-2 strains failed to cause eye infections. The Shealey strain [HSV-1 (Sh)] produced the most severe eye infections, characterized by epithelial and stromal disease, corneal vascularization and ulcerative blepharitis. Consequently, HSV-1 (Sh) was selected as the prototype strain for this study. The frequency and severity of HSV-1 (Sh) eye disease patterns was determined by a semi-quantitative rating scale, which permitted accurate monitoring of the temporal development of the disease patterns cited above. Virus shedding from infected eyes was also quantified. All of the HSV-1 strains tested established trigeminal ganglionic latency with varying frequency, although HSV-1 (Sh) latency approached 100 percent. The kinetics of acute ganglionic infection by HSV-1 (Sh) was determined, and peak virus titers occurred on the third day after corneal inoculation. This study emphasizes the usefulness of the Guinea pig model for investigations on the pathogenesis, prevention and treatment of herpetic eye infections.

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Background The DHVI Division of Structural Biology seeks to use atomic level structural information for design of an effective HIV-1 vaccine. Through visualization of the HIV-1 envelope (Env) and its interactions with the human immune system, we obtain structural information that we translate into the rational development vaccine immunogens Methods We use negative stain electron microscopy (NSEM), cryo-electron microscopy (cryo-EM), and x-ray crystallography as the major structural techniques for visualization of HIV-1 Env, and combine these with biochemical and biophysical studies, as well as computational methods to obtain a basic understanding of the functions and interactions of the HIV-1 Env. Results: The DHVI NSEM pipeline runs on a daily basis to quality control vaccine immunogens for animal studies and other applications. Offering rapid sample turnover and economical operations, the NSEM pipeline is the most widely utilized resource of the DHVI Division of Structural Biology. Over the last year, the NSEM team has focused efforts on improving operational speed and data processing allowing high-quality visualization of a large variety of samples including HIV-1 Env immunogens, antibodies, nanoparticles, and VLPs. In the last year we have also expanded our NSEM studies to the analyses of serum samples and mucosal fluids. To understand the mechanism of HIV-1 entry we have determined structures of HIV-1 entry intermediates. We have determined a 3.8 Å resolution structure of a single CD4 bound to a closed HIV-1 Env trimer revealing new contacts of CD4 with Env. We have also structurally characterized an Env designed to prevent CD4-induced rearrangements by targeted disruption of an allosteric network modulating Env conformational changes. We have structurally characterized the HIV-1 glycan-V3 targeting DH270 Broadly Neutralizing Antibody Lineage. The structures revealed movements in the V1 loop and interactive glycans, shifts in antibody orientations, antibody VH-VL orientations, and antibody elbow angles, as the lineage progressed to maturation. We have solved a structure in complex with the HIV-1 Env immunogen Man5-enriched CH505.N279K.G458Y.SOSIP.664 of the unmutated common ancestor (UCA) of the HIV-1 CD4-binding site targeting CH235 Broadly Neutralizing Antibody Lineage. The structure revealed interactions of the N279K and G458Y mutations with the CDR L3 loop of CH235 UCA thus providing a structural understanding of the role of these mutations in facilitating binding to the CH235 UCA. (see also Henderson et al abstract) Using NSEM and cryo-EM we have characterized the structural properties of a novel class of 2G12-mimetic, yet non domain-swapped Fab dimer glycan-reactive (FDG) antibodies. These studies showed that the Fab-dimerized 2G12-like motif is more common than previously thought, and that creation of a Fab-dimerized paratope for an HIV-1 neutralizing antibody does not require VH domain-swapping. Finally, the structural team is an integral part of the CHAVD Kalma Immunogen Design Team, wherein ...