A human monoclonal antibody binds within the poliovirus receptor-binding site to neutralize all three serotypes.

Global eradication of poliovirus remains elusive, and it is critical to develop next generation vaccines and antivirals. In support of this goal, we map the epitope of human monoclonal antibody 9H2 which is able to neutralize the three serotypes of poliovirus. Using cryo-EM we solve the near-atomic structures of 9H2 fragments (Fab) bound to capsids of poliovirus serotypes 1, 2, and 3. The Fab-virus complexes show that Fab interacts with the same binding mode for each serotype and at the same angle of interaction relative to the capsid surface. For each of the Fab-virus complexes, we find that the binding site overlaps with the poliovirus receptor (PVR) binding site and maps across and into a depression in the capsid called the canyon. No conformational changes to the capsid are induced by Fab binding for any complex. Competition binding experiments between 9H2 and PVR reveal that 9H2 impedes receptor binding. Thus, 9H2 outcompetes the receptor to neutralize poliovirus. The ability to neutralize all three serotypes, coupled with the critical importance of the conserved receptor binding site make 9H2 an attractive antiviral candidate for future development.

Viral Capsid, Antibody, and Receptor Interactions: Experimental Analysis of the Antibody Escape Evolution of Canine Parvovirus.

Canine parvovirus (CPV) is a small nonenveloped single-stranded DNA virus that causes serious diseases in dogs worldwide. The original strain of the virus (CPV-2) emerged in dogs during the late 1970s due to a host range switch of a virus similar to the feline panleukopenia virus that infected another host. The virus that emerged in dogs had altered capsid receptor and antibody binding sites, with some changes affecting both functions. Further receptor and antibody binding changes arose when the virus became better adapted to dogs or to other hosts. Here, we used in vitro selection and deep sequencing to reveal how two antibodies with known interactions select for escape mutations in CPV. The antibodies bound two distinct epitopes, and one largely overlapped the host receptor binding site. We also generated mutated antibody variants with altered binding structures. Viruses were passaged with wild-type (WT) or mutated antibodies, and their genomes were deep sequenced during the selective process. A small number of mutations were detected only within the capsid protein gene during the first few passages of selection, and most sites remained polymorphic or were slow to go to fixation. Mutations arose both within and outside the antibody binding footprints on the capsids, and all avoided the transferrin receptor type 1 binding footprint. Many selected mutations matched those that have arisen in the natural evolution of the virus. The patterns observed reveal the mechanisms by which these variants have been selected in nature and provide a better understanding of the interactions between antibody and receptor selections. IMPORTANCE Antibodies protect animals against infection by many different viruses and other pathogens, and we are gaining new information about the epitopes that induce antibody responses against viruses and the structures of the bound antibodies. However, less is known about the processes of antibody selection and antigenic escape and the constraints that apply in this system. Here, we used an in vitro model system and deep genome sequencing to reveal the mutations that arose in the virus genome during selection by each of two monoclonal antibodies or their mutated variants. High-resolution structures of each of the Fab:capsid complexes revealed their binding interactions. The wild-type antibodies or their mutated variants allowed us to examine how changes in antibody structure influence the mutational selection patterns seen in the virus. The results shed light on the processes of antibody binding, neutralization escape, and receptor binding, and they likely have parallels for many other viruses.

Viral capsid, antibody, and receptor interactions: experimental analysis of the antibody escape evolution of canine parvovirus.

Canine parvovirus (CPV) is a small non-enveloped single-stranded DNA virus that causes serious diseases in dogs worldwide. The original strain of the virus (CPV-2) emerged in dogs during the late-1970s due to a host range switch of a virus similar to the feline panleukopenia virus (FPV) that infected another host. The virus that emerged in dogs had altered capsid receptor- and antibody-binding sites, with some changes affecting both functions. Further receptor and antibody binding changes arose when the virus became better adapted to dogs or to other hosts. Here, we use in vitro selection and deep sequencing to reveal how two antibodies with known interactions select for escape mutations in CPV. The antibodies bind two distinct epitopes, and one largely overlaps the host receptor binding site. We also engineered antibody variants with altered binding structures. Viruses were passaged with the wild type or mutated antibodies, and their genomes deep sequenced during the selective process. A small number of mutations were detected only within the capsid protein gene during the first few passages of selection, and most sites remained polymorphic or were slow to go to fixation. Mutations arose both within and outside the antibody binding footprints on the capsids, and all avoided the TfR-binding footprint. Many selected mutations matched those that have arisen in the natural evolution of the virus. The patterns observed reveal the mechanisms by which these variants have been selected in nature and provide a better understanding of the interactions between antibody and receptor selections. IMPORTANCE: Antibodies protect animals against infection by many different viruses and other pathogens, and we are gaining new information about the epitopes that induce antibody responses against viruses and the structures of the bound antibodies. However, less is known about the processes of antibody selection and antigenic escape and the constraints that apply in this system. Here, we use an in vitro model system and deep genome sequencing to reveal the mutations that arise in the virus genome during selection by each of two monoclonal antibodies or their engineered variants. High-resolution structures of each of the Fab: capsid complexes revealed their binding interactions. The engineered forms of the wild-type antibodies or mutant forms allowed us to examine how changes in antibody structure influence the mutational selection patterns seen in the virus. The results shed light on the processes of antibody binding, neutralization escape, and receptor binding, and likely have parallels for many other viruses.

A Comprehensive Review of Neuromuscular Manifestations of COVID-19 and Management of Pre-Existing Neuromuscular Disorders in Children.

Since the emergence of SARS-CoV-2, several studies have been published describing neuromuscular manifestations of the disease, as well as management of pre-existing pediatric neuromuscular disorders during the COVID-19 pandemic. These disorders include muscular dystrophies, myasthenic syndromes, peripheral nerve disorders, and spinal muscular atrophy. Such patients are a vulnerable population due to frequent complications such as scoliosis, cardiomyopathy, and restrictive lung disease that put them at risk of severe complications of COVID-19. In this review, neuromuscular manifestations of COVID-19 in children and the management of pre-existing pediatric neuromuscular disorders during the COVID-19 pandemic are discussed. We also review strategies to alleviate pandemic-associated disruptions in clinical care and research, including the emerging role of telemedicine and telerehabilitation to address the continued special needs of these patients.

Cryo EM Analysis Reveals Inherent Flexibility of Authentic Murine Papillomavirus Capsids.

Human papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. However, studies have been hampered due to restricted tropism that makes production and purification of high titer virus problematic. This issue has been overcome by developing alternative HPV production methods such as virus-like particles (VLPs), which are devoid of a native viral genome. Structural studies have been limited in resolution due to the heterogeneity, fragility, and stability of the VLP capsids. The mouse papillomavirus (MmuPV1) presented here has provided the opportunity to study a native papillomavirus in the context of a common laboratory animal. Using cryo EM to solve the structure of MmuPV1, we achieved 3.3 Å resolution with a local symmetry refinement method that defined smaller, symmetry related subparticles. The resulting high-resolution structure allowed us to build the MmuPV1 asymmetric unit for the first time and identify putative L2 density. We also used our program ISECC to quantify capsid flexibility, which revealed that capsomers move as rigid bodies connected by flexible linkers. The MmuPV1 flexibility was comparable to that of a HPV VLP previously characterized. The resulting MmuPV1 structure is a promising step forward in the study of papillomavirus and will provide a framework for continuing biochemical, genetic, and biophysical research for papillomaviruses.

High-resolution asymmetric structure of a Fab-virus complex reveals overlap with the receptor binding site.

Canine parvovirus is an important pathogen causing severe diseases in dogs, including acute hemorrhagic enteritis, myocarditis, and cerebellar disease. Overlap on the surface of parvovirus capsids between the antigenic epitope and the receptor binding site has contributed to cross-species transmission, giving rise to closely related variants. It has been shown that Mab 14 strongly binds and neutralizes canine but not feline parvovirus, suggesting this antigenic site also controls species-specific receptor binding. To visualize the conformational epitope at high resolution, we solved the cryogenic electron microscopy (cryo-EM) structure of the Fab-virus complex. We also created custom software, Icosahedral Subparticle Extraction and Correlated Classification, to solve a Fab-virus complex with only a few Fab bound per capsid and visualize local structures of the Fab-bound and -unbound antigenic sites extracted from the same complex map. Our results identified the antigenic epitope that had significant overlap with the receptor binding site, and the structures revealed that binding of Fab induced conformational changes to the virus. We were also able to assign the order and position of attached Fabs to allow assessment of complementarity between the Fabs bound to different positions. This approach therefore provides a method for using cryo-EM to investigate complementarity of antibody binding.

High resolution cryo EM analysis of HPV16 identifies minor structural protein L2 and describes capsid flexibility.

Human papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. HPV is epitheliotropic and its replication is tightly associated with terminal keratinocyte differentiation making production and purification of high titer virus preparations for research problematic, therefore alternative HPV production methods have been developed for virological and structural studies. In this study we use HPV16 quasivirus, composed of HPV16 L1/L2 capsid proteins with a packaged cottontail rabbit papillomavirus genome. We have achieved the first high resolution, 3.1 Å, structure of HPV16 by using a local subvolume refinement approach. The high resolution enabled us to build L1 unambiguously and identify L2 protein strands. The L2 density is incorporated adjacent to conserved L1 residues on the interior of the capsid. Further interpretation with our own software for Icosahedral Subvolume Extraction and Correlated Classification revealed flexibility, on the whole-particle level through diameter analysis and local movement with inter-capsomer analysis. Inter-capsomer expansion or contraction, governed by the connecting arms, showed no bias in the magnitude or direction of capsomer movement. We propose that papillomavirus capsids are dynamic and capsomers move as rigid bodies connected by flexible linkers. The resulting virus structure will provide a framework for continuing biochemical, genetic and biophysical research for papillomaviruses. Furthermore, our approach has allowed insight into the resolution barrier that has previously been a limitation in papillomavirus structural studies.

Identification of a pocket factor that is critical to Zika virus assembly.

Zika virus (ZIKV) is an emerging mosquito borne flavivirus and a major public health concern causing severe disease. Due to the presence of a lipid membrane and structural heterogeneity, attaining an atomic resolution structure is challenging, but important to understand virus assembly and life cycle mechanisms that offer distinct targets for therapeutic intervention. We here use subvolume refinement to achieve a 3.4 Å resolution structure and identify two distinct lipid moieties. The first arises from the inner leaflet and is coordinated by hydrophobic residues of the M and E transmembrane helices that form a binding pocket not previously characterized. The second lipid arises from the outer leaflet coordinate between two E protein helices. Structure-based mutagenesis identifies critical hydrophobic interactions and their effect on the virus life cycle. Results show that lipids play an essential role in the ZIKV assembly pathway revealing a potential target of lipid based antiviral drug development.

Antibody escape by polyomavirus capsid mutation facilitates neurovirulence.

JCPyV polyomavirus, a member of the human virome, causes progressive multifocal leukoencephalopathy (PML), an oft-fatal demyelinating brain disease in individuals receiving immunomodulatory therapies. Mutations in the major viral capsid protein, VP1, are common in JCPyV from PML patients (JCPyV-PML) but whether they confer neurovirulence or escape from virus-neutralizing antibody (nAb) in vivo is unknown. A mouse polyomavirus (MuPyV) with a sequence-equivalent JCPyV-PML VP1 mutation replicated poorly in the kidney, a major reservoir for JCPyV persistence, but retained the CNS infectivity, cell tropism, and neuropathology of the parental virus. This mutation rendered MuPyV resistant to a monoclonal Ab (mAb), whose specificity overlapped the endogenous anti-VP1 response. Using cryo-EM and a custom sub-particle refinement approach, we resolved an MuPyV:Fab complex map to 3.2 Å resolution. The structure revealed the mechanism of mAb evasion. Our findings demonstrate convergence between nAb evasion and CNS neurovirulence in vivo by a frequent JCPyV-PML VP1 mutation.

CryoEM reconstruction approaches to resolve asymmetric features.

Although icosahedral viruses have highly symmetrical capsid features, asymmetric structural elements are also present since the genome and minor structural proteins are usually incorporated without adhering to icosahedral symmetry. Besides this inherent asymmetry, interactions with the host during the virus life cycle are also asymmetric. However, until recently it was impossible to resolve high resolution asymmetric features during single-particle cryoEM image processing. This review summarizes the current approaches that can be used to visualize asymmetric structural features. We have included examples of advanced structural strategies developed to reveal unique features and asymmetry in icosahedral viruses.

Asymmetry in icosahedral viruses.

Although icosahedral viruses have obvious and highly symmetrical features, asymmetric structural elements are also present. Asymmetric features may be inherent since the genome and location of minor capsid proteins are typically incorporated without adhering to icosahedral symmetry. Asymmetry also develops during the virus life cycle in order to accomplish key functions such as genome packaging, release, and organization. However, resolving asymmetric features complicates image processing during single-particle cryoEM analysis. This review summarizes the current state of knowledge regarding asymmetric structural features with specific examples drawn from members of picornaviridae, parvoviradae, microviradae, and leviviridae.

Patronin-mediated minus end growth is required for dendritic microtubule polarity.

Microtubule minus ends are thought to be stable in cells. Surprisingly, in Drosophila and zebrafish neurons, we observed persistent minus end growth, with runs lasting over 10 min. In Drosophila, extended minus end growth depended on Patronin, and Patronin reduction disrupted dendritic minus-end-out polarity. In fly dendrites, microtubule nucleation sites localize at dendrite branch points. Therefore, we hypothesized minus end growth might be particularly important beyond branch points. Distal dendrites have mixed polarity, and reduction of Patronin lowered the number of minus-end-out microtubules. More strikingly, extra Patronin made terminal dendrites almost completely minus-end-out, indicating low Patronin normally limits minus-end-out microtubules. To determine whether minus end growth populated new dendrites with microtubules, we analyzed dendrite development and regeneration. Minus ends extended into growing dendrites in the presence of Patronin. In sum, our data suggest that Patronin facilitates sustained microtubule minus end growth, which is critical for populating dendrites with minus-end-out microtubules.

Bilaterian Giant Ankyrins Have a Common Evolutionary Origin and Play a Conserved Role in Patterning the Axon Initial Segment.

In vertebrate neurons, the axon initial segment (AIS) is specialized for action potential initiation. It is organized by a giant 480 Kd variant of ankyrin G (AnkG) that serves as an anchor for ion channels and is required for a plasma membrane diffusion barrier that excludes somatodendritic proteins from the axon. An unusually long exon required to encode this 480Kd variant is thought to have been inserted only recently during vertebrate evolution, so the giant ankyrin-based AIS scaffold has been viewed as a vertebrate adaptation for fast, precise signaling. We re-examined AIS evolution through phylogenomic analysis of ankyrins and by testing the role of ankyrins in proximal axon organization in a model multipolar Drosophila neuron (ddaE). We find giant isoforms of ankyrin in all major bilaterian phyla, and present evidence in favor of a single common origin for giant ankyrins and the corresponding long exon in a bilaterian ancestor. This finding raises the question of whether giant ankyrin isoforms play a conserved role in AIS organization throughout the Bilateria. We examined this possibility by looking for conserved ankyrin-dependent AIS features in Drosophila ddaE neurons via live imaging. We found that ddaE neurons have an axonal diffusion barrier proximal to the cell body that requires a giant isoform of the neuronal ankyrin Ank2. Furthermore, the potassium channel shal concentrates in the proximal axon in an Ank2-dependent manner. Our results indicate that the giant ankyrin-based cytoskeleton of the AIS may have evolved prior to the radiation of extant bilaterian lineages, much earlier than previously thought.

Kinesin-2 and Apc function at dendrite branch points to resolve microtubule collisions.

In Drosophila neurons, kinesin-2, EB1 and Apc are required to maintain minus-end-out dendrite microtubule polarity, and we previously proposed they steer microtubules at branch points. Motor-mediated steering of microtubule plus ends could be accomplished in two ways: 1) by linking a growing microtubule tip to the side of an adjacent microtubule as it navigates the branch point (bundling), or 2) by directing a growing microtubule after a collision with a stable microtubule (collision resolution). Using live imaging to distinguish between these two mechanisms, we found that reduction of kinesin-2 did not alter the number of microtubules that grew along the edge of the branch points where stable microtubules are found. However, reduction of kinesin-2 or Apc did affect the number of microtubules that slowed down or depolymerized as they encountered the side of the branch opposite to the entry point. These results are consistent with kinesin-2 functioning with Apc to resolve collisions. However, they do not pinpoint stable microtubules as the collision partner as stable microtubules are typically very close to the membrane. To determine whether growing microtubules were steered along stable ones after a collision, we analyzed the behavior of growing microtubules at dendrite crossroads where stable microtubules run through the middle of the branch point. In control neurons, microtubules turned in the middle of the crossroads. However, when kinesin-2 was reduced some microtubules grew straight through the branch point and failed to turn. We propose that kinesin-2 functions to steer growing microtubules along stable ones following collisions.

Γ-tubulin controls neuronal microtubule polarity independently of Golgi outposts.

Neurons have highly polarized arrangements of microtubules, but it is incompletely understood how microtubule polarity is controlled in either axons or dendrites. To explore whether microtubule nucleation by γ-tubulin might contribute to polarity, we analyzed neuronal microtubules in Drosophila containing gain- or loss-of-function alleles of γ-tubulin. Both increased and decreased activity of γ-tubulin, the core microtubule nucleation protein, altered microtubule polarity in axons and dendrites, suggesting a close link between regulation of nucleation and polarity. To test whether nucleation might locally regulate polarity in axons and dendrites, we examined the distribution of γ-tubulin. Consistent with local nucleation, tagged and endogenous γ-tubulins were found in specific positions in dendrites and axons. Because the Golgi complex can house nucleation sites, we explored whether microtubule nucleation might occur at dendritic Golgi outposts. However, distinct Golgi outposts were not present in all dendrites that required regulated nucleation for polarity. Moreover, when we dragged the Golgi out of dendrites with an activated kinesin, γ-tubulin remained in dendrites. We conclude that regulated microtubule nucleation controls neuronal microtubule polarity but that the Golgi complex is not directly involved in housing nucleation sites.