Intranasal SARS-CoV-2 RBD decorated nanoparticle vaccine enhances viral clearance in the Syrian hamster model.

Multiple vaccines have been developed and licensed for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). While these vaccines reduce disease severity, they do not prevent infection. To prevent infection and limit transmission, vaccines must be developed that induce immunity in the respiratory tract. Therefore, we performed proof-of-principle studies with an intranasal nanoparticle vaccine against SARS-CoV-2. The vaccine candidate consisted of the self-assembling 60-subunit I3-01 protein scaffold covalently decorated with the SARS-CoV-2 receptor-binding domain (RBD) using the SpyCatcher-SpyTag system. We verified the intended antigen display features by reconstructing the I3-01 scaffold to 3.4 A using cryogenicelectron microscopy. Using this RBD-grafted SpyCage scaffold (RBD + SpyCage), we performed two intranasal vaccination studies in the "gold-standard" pre-clinical Syrian hamster model. The initial study focused on assessing the immunogenicity of RBD + SpyCage combined with the LTA1 intranasal adjuvant. These studies showed RBD + SpyCage vaccination induced an antibody response that promoted viral clearance but did not prevent infection. Inclusion of the LTA1 adjuvant enhanced the magnitude of the antibody response but did not enhance protection. Thus, in an expanded study, in the absence of an intranasal adjuvant, we evaluated if covalent bonding of RBD to the scaffold was required to induce an antibody response. Covalent grafting of RBD was required for the vaccine to be immunogenic, and animals vaccinated with RBD + SpyCage more rapidly cleared SARS-CoV-2 from both the upper and lower respiratory tract. These findings demonstrate the intranasal SpyCage vaccine platform can induce protection against SARS-CoV-2 and, with additional modifications to improve immunogenicity, is a versatile platform for the development of intranasal vaccines targeting respiratory pathogens.IMPORTANCEDespite the availability of efficacious COVID vaccines that reduce disease severity, SARS-CoV-2 continues to spread. To limit SARS-CoV-2 transmission, the next generation of vaccines must induce immunity in the mucosa of the upper respiratory tract. Therefore, we performed proof-of-principle, intranasal vaccination studies with a recombinant protein nanoparticle scaffold, SpyCage, decorated with the RBD of the S protein (SpyCage + RBD). We show that SpyCage + RBD was immunogenic and enhanced SARS-CoV-2 clearance from the nose and lungs of Syrian hamsters. Moreover, covalent grafting of the RBD to the scaffold was required to induce an immune response when given via the intranasal route. These proof-of-concept findings indicate that with further enhancements to immunogenicity (e.g., adjuvant incorporation and antigen optimization), the SpyCage scaffold has potential as a versatile, intranasal vaccine platform for respiratory pathogens.

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.

Cryo EM structures map a post vaccination polyclonal antibody response to canine parvovirus.

Canine parvovirus (CPV) is an important pathogen that emerged by cross-species transmission to cause severe disease in dogs. To understand the host immune response to vaccination, sera from dogs immunized with parvovirus are obtained, the polyclonal antibodies are purified and used to solve the high resolution cryo EM structures of the polyclonal Fab-virus complexes. We use a custom software, Icosahedral Subparticle Extraction and Correlated Classification (ISECC) to perform subparticle analysis and reconstruct polyclonal Fab-virus complexes from two different dogs eight and twelve weeks post vaccination. In the resulting polyclonal Fab-virus complexes there are a total of five distinct Fabs identified. In both cases, any of the five antibodies identified would interfere with receptor binding. This polyclonal mapping approach identifies a specific, limited immune response to the live vaccine virus and allows us to investigate the binding of multiple different antibodies or ligands to virus capsids.

The Structures and Functions of Parvovirus Capsids and Missing Pieces: the Viral DNA and Its Packaging, Asymmetrical Features, Nonprotein Components, and Receptor or Antibody Binding and Interactions.

Parvoviruses are among the smallest and superficially simplest animal viruses, infecting a broad range of hosts, including humans, and causing some deadly infections. In 1990, the first atomic structure of the canine parvovirus (CPV) capsid revealed a 26-nm-diameter T=1 particle made up of two or three versions of a single protein, and packaging about 5,100 nucleotides of single-stranded DNA. Our structural and functional understanding of parvovirus capsids and their ligands has increased as imaging and molecular techniques have advanced, and capsid structures for most groups within the Parvoviridae family have now been determined. Despite those advances, significant questions remain unanswered about the functioning of those viral capsids and their roles in release, transmission, or cellular infection. In addition, the interactions of capsids with host receptors, antibodies, or other biological components are also still incompletely understood. The parvovirus capsid's apparent simplicity likely conceals important functions carried out by small, transient, or asymmetric structures. Here, we highlight some remaining open questions that may need to be answered to provide a more thorough understanding of how these viruses carry out their various functions. The many different members of the family Parvoviridae share a capsid architecture, and while many functions are likely similar, others may differ in detail. Many of those parvoviruses have not been experimentally examined in detail (or at all in some cases), so we, therefore, focus this minireview on the widely studied protoparvoviruses, as well as the most thoroughly investigated examples of adeno-associated viruses.

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.

Cryo-EM Structure of Gokushovirus ΦEC6098 Reveals a Novel Capsid Architecture for a Single-Scaffolding Protein, Microvirus Assembly System.

Ubiquitous and abundant in ecosystems and microbiomes, gokushoviruses constitute a Microviridae subfamily, distantly related to bacteriophages ΦX174, α3, and G4. A high-resolution cryo-EM structure of gokushovirus ΦEC6098 was determined, and the atomic model was built de novo. Although gokushoviruses lack external scaffolding and spike proteins, which extensively interact with the ΦX174 capsid protein, the core of the ΦEC6098 coat protein (VP1) displayed a similar structure. There are, however, key differences. At each ΦEC6098 icosahedral 3-fold axis, a long insertion loop formed mushroom-like protrusions, which have been noted in lower-resolution gokushovirus structures. Hydrophobic interfaces at the bottom of these protrusions may confer stability to the capsid shell. In ΦX174, the N-terminus of the capsid protein resides directly atop the 3-fold axes of symmetry; however, the ΦEC6098 N-terminus stretched across the inner surface of the capsid shell, reaching nearly to the 5-fold axis of the neighboring pentamer. Thus, this extended N-terminus interconnected pentamers on the inside of the capsid shell, presumably promoting capsid assembly, a function performed by the ΦX174 external scaffolding protein. There were also key differences between the ΦX174-like DNA-binding J proteins and its ΦEC6098 homologue VP8. As seen with the J proteins, C-terminal VP8 residues were bound into a pocket within the major capsid protein; however, its N-terminal residues were disordered, likely due to flexibility. We show that the combined location and interaction of VP8's C-terminus and a portion of VP1's N-terminus are reminiscent of those seen with the ΦX174 and α3 J proteins. IMPORTANCE There is a dramatic structural and morphogenetic divide within the Microviridae. The well-studied ΦX174-like viruses have prominent spikes at their icosahedral vertices, which are absent in gokushoviruses. Instead, gokushovirus major coat proteins form extensive mushroom-like protrusions at the 3-fold axes of symmetry. In addition, gokushoviruses lack an external scaffolding protein, the more critical of the two ΦX174 assembly proteins, but retain an internal scaffolding protein. The ΦEC6098 virion suggests that key external scaffolding functions are likely performed by coat protein domains unique to gokushoviruses. Thus, within one family, different assembly paths have been taken, demonstrating how a two-scaffolding protein system can evolve into a one-scaffolding protein system, or vice versa.

Asymmetry in icosahedral viruses.

Asymmetric structural elements are typically not readily visualized in icosahedral viruses that have other obvious symmetrical features and most asymmetry has gone unresolved for decades. Asymmetric features may be incorporated during assembly or maturation or develop during key steps in the infectious cycle of the virus. However, resolving asymmetric features requires abandoning capsid-wide symmetry averaging and relying on special applications during single-particle cryogenic electron microscopy (cryo-EM) analysis. Thanks to the advances in the cryo-EM field, we are learning more about asymmetry of viruses. Here we summarize some of what is currently known about asymmetric structural features using as examples members of the Togaviridae, Flaviviridae, Herpesviridae, Parvoviridae, and Papillomaviridae.

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.

Activating Sphingosine-1-phospahte signaling in endothelial cells increases myosin light chain phosphorylation to decrease endothelial permeability thereby inhibiting cancer metastasis.

Targeting the metastatic process to prevent disease dissemination in cancer remains challenging. One step in the metastatic cascade involves cancer cells transiting through the vascular endothelium after inflammation has increased the permeability of this cellular layer. Reducing inflammation-mediated gaps in the vascular endothelium could potentially be used to retard metastasis. This study describes the development of a novel ASR396-containing nanoparticle designed to activate the Sphingosine-1-Phosphate Receptor 1 (S1PR1) in order to tighten the junctions between the endothelial cells lining the vascular endothelium thereby inhibiting metastasis. ASR396 was derived from the S1PR1 agonist SEW2871 through chemical modification enabling the new compound to be loaded into a nanoliposome. ASR396 retained S1PR1 binding activity and the nanoliposomal formulation (nanoASR396) made it systemically bioavailable upon intravenous injection. Studies conducted in microvessels demonstrated that nanoASR396 significantly attenuated inflammatory mediator-induced permeability increase through the S1PR1 activation. Similarly, nanoASR396 inhibited gap formation mediated by inflammatory agents on an endothelial cell monolayer by decreasing levels of phosphorylated myosin light chain protein thereby inhibiting cellular contractility. In animal models, nanoASR396 inhibited lung metastasis by up to 80%, indicating its potential for retarding melanoma metastasis. Thus, a novel bioavailable nanoparticle-based S1PR1 agonist has been developed to negate the effects of inflammatory mediators on the vascular endothelium in order to reduce the metastatic dissemination of cancer cells.

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.

Cryo-EM structure of catalytic ribonucleoprotein complex RNase MRP.

RNase MRP is an essential eukaryotic ribonucleoprotein complex involved in the maturation of rRNA and the regulation of the cell cycle. RNase MRP is related to the ribozyme-based RNase P, but it has evolved to have distinct cellular roles. We report a cryo-EM structure of the S. cerevisiae RNase MRP holoenzyme solved to 3.0 Å. We describe the structure of this 450 kDa complex, interactions between its components, and the organization of its catalytic RNA. We show that some of the RNase MRP proteins shared with RNase P undergo an unexpected RNA-driven remodeling that allows them to bind to divergent RNAs. Further, we reveal how this RNA-driven protein remodeling, acting together with the introduction of new auxiliary elements, results in the functional diversification of RNase MRP and its progenitor, RNase P, and demonstrate structural underpinnings of the acquisition of new functions by catalytic RNPs.

Limited Intrahost Diversity and Background Evolution Accompany 40 Years of Canine Parvovirus Host Adaptation and Spread.

Canine parvovirus (CPV) is a highly successful pathogen that has sustained pandemic circulation in dogs for more than 40 years. Here, integrating full-genome and deep-sequencing analyses, structural information, and in vitro experimentation, we describe the macro- and microscale features that accompany CPV's evolutionary success. Despite 40 years of viral evolution, all CPV variants are more than ∼99% identical in nucleotide sequence, with only a limited number (<40) of substitutions becoming fixed or widespread during this time. Notably, most substitutions in the major capsid protein (VP2) gene are nonsynonymous, altering amino acid residues that fall within, or adjacent to, the overlapping receptor footprint or antigenic regions, suggesting that natural selection has channeled much of CPV evolution. Among the limited number of variable sites, CPV genomes exhibit complex patterns of variation that include parallel evolution, reversion, and recombination, compromising phylogenetic inference. At the intrahost level, deep sequencing of viral DNA in original clinical samples from dogs and other host species sampled between 1978 and 2018 revealed few subconsensus single nucleotide variants (SNVs) above ∼0.5%, and experimental passages demonstrate that substantial preexisting genetic variation is not necessarily required for rapid host receptor-driven adaptation. Together, these findings suggest that although CPV is capable of rapid host adaptation, a relatively low mutation rate, pleiotropy, and/or a lack of selective challenges since its initial emergence have inhibited the long-term accumulation of genetic diversity. Hence, continuously high levels of inter- and intrahost diversity are not necessarily required for virus host adaptation.IMPORTANCE Rapid mutation rates and correspondingly high levels of intra- and interhost diversity are often cited as key features of viruses with the capacity for emergence and sustained transmission in a new host species. However, most of this information comes from studies of RNA viruses, with relatively little known about evolutionary processes in viruses with single-stranded DNA (ssDNA) genomes. Here, we provide a unique model of virus evolution, integrating both long-term global-scale and short-term intrahost evolutionary processes of an ssDNA virus that emerged to cause a pandemic in a new host animal. Our analysis reveals that successful host jumping and sustained transmission does not necessarily depend on a high level of intrahost diversity nor result in the continued accumulation of high levels of long-term evolution change. These findings indicate that all aspects of the biology and ecology of a virus are relevant when considering their adaptability.

Transferrin receptor binds virus capsid with dynamic motion.

Canine parvovirus (CPV) is an important pathogen causing severe diseases in dogs, including acute hemorrhagic enteritis, myocarditis, and cerebellar disease. Cross-species transmission of CPV occurs as a result of mutations on the viral capsid surface that alter the species-specific binding to the host receptor, transferrin receptor type-1 (TfR). The interaction between CPV and TfR has been extensively studied, and previous analyses have suggested that the CPV-TfR complex is asymmetric. To enhance the understanding of the underlying molecular mechanisms, we determined the CPV-TfR interaction using cryo-electron microscopy to solve the icosahedral (3.0-Å resolution) and asymmetric (5.0-Å resolution) complex structures. Structural analyses revealed conformational variations of the TfR molecules relative to the binding site, which translated into dynamic molecular interactions between CPV and TfR. The precise footprint of the receptor on the virus capsid was identified, along with the identity of the amino acid residues in the virus-receptor interface. Our "rock-and-roll" model provides an explanation for previous findings and gives insights into species jumping and the variation in host ranges associated with new pandemics in dogs.

Filling Adeno-Associated Virus Capsids: Estimating Success by Cryo-Electron Microscopy.

Adeno-associated viruses (AAVs) have been employed successfully as gene therapy vectors in treating various genetic diseases for almost two decades. However, transgene packaging is usually imperfect, and developing a rapid and accurate method for measuring the proportion of DNA encapsidation is an important step for improving the downstream process of large scale vector production. In this study, we used two-dimensional class averages and three-dimensional classes, intermediate outputs in the single particle cryo-electron microscopy (cryo-EM) image reconstruction pipeline, to determine the proportion of DNA-packaged and empty capsid populations. Two different preparations of AAV3 were analyzed to estimate the minimum number of particles required to be sampled by cryo-EM in order for robust calculation of the proportion of the full versus empty capsids in any given sample. Cost analysis applied to the minimum amount of data required for a valid ratio suggests that cryo-EM is an effective approach to analyze vector preparations.

Examination and Reconstruction of Three Ancient Endogenous Parvovirus Capsid Protein Gene Remnants Found in Rodent Genomes.

Parvovirus-derived endogenous viral elements (EVEs) have been found in the genomes of many different animal species, resulting from integration events that may have occurred from more than 50 million years ago to much more recently. Here, we further investigate the properties of autonomous parvovirus EVEs and describe their relationships to contemporary viruses. While we did not find any intact capsid protein open reading frames in the integrated viral sequences, we examined three EVEs that were repaired to form full-length sequences with relatively few changes. These sequences were found in the genomes of Rattus norvegicus (brown rat), Mus spretus (Algerian mouse), and Apodemus sylvaticus (wood mouse). The R. norvegicus sequence was not present in the genomes of the closely related species R. rattus, R. tanezumi, R. exulans, and R. everetti, indicating that it was less than 2 million years old, and the M. spretus and A. sylvaticus sequences were not found in the published genomes of other mouse species, also indicating relatively recent insertions. The M. spretus VP2 sequence assembled into capsids, which had high thermal stability, bound the sialic acid N-acetylneuraminic acid, and entered murine L cells. The 3.89-Å structure of the M. spretus virus-like particles (VLPs), determined using cryo-electron microscopy, showed similarities to rodent and porcine parvovirus capsids. The repaired VP2 sequences from R. norvegicus and A. sylvaticus did not assemble as first prepared, but chimeras combining capsid surface loops from R. norvegicus with canine parvovirus assembled, allowing some of that capsid's structures and functions to be examined.IMPORTANCE Parvovirus endogenous viral elements (EVEs) that have been incorporated into the genomes of different animals represent remnants of the DNA sequences of ancient viruses that infected the ancestors of those animals millions of years ago, but we know little about their properties or how they differ from currently circulating parvoviruses. By expressing the capsid proteins of different parvovirus EVEs that were found integrated into the genomes of three different rodents, we can examine their structures and functions. A VP2 (major capsid protein) EVE sequence from a mouse genome assembled into capsids that had a similar structure and biophysical properties to extant parvoviruses and also bound sialic acids and entered rodent cells. Chimeras formed from combinations of canine parvovirus and portions of the parvovirus sequences from the brown rat genome allowed us to examine the structures and functions of the surface loops of that EVE capsid.

Complex and Dynamic Interactions between Parvovirus Capsids, Transferrin Receptors, and Antibodies Control Cell Infection and Host Range.

Antibody and receptor binding are key virus-host interactions that control host range and determine the success of infection. Canine and feline parvovirus capsids bind the transferrin receptor type 1 (TfR) to enter host cells, and specific structural interactions appear necessary to prepare the stable capsids for infection. Here, we define the details of binding, competition, and occupancy of wild-type and mutant parvovirus capsids with purified receptors and antibodies. TfR-capsid binding interactions depended on the TfR species and varied widely, with no direct relationship between binding affinity and infection. Capsids bound feline, raccoon, and black-backed jackal TfRs at high affinity but barely bound canine TfRs, which mediated infection efficiently. TfRs from different species also occupied capsids to different levels, with an estimated 1 to 2 feline TfRs but 12 black-backed jackal TfRs binding each capsid. Multiple alanine substitutions within loop 1 on the capsid surface reduced TfR binding but substitutions within loop 3 did not, suggesting that loop 1 directly engaged the TfR and loop 3 sterically affected that interaction. Binding and competition between different TfRs and/or antibodies showed complex relationships. Both antibodies 14 and E competed capsids off TfRs, but antibody E could also compete capsids off itself and antibody 14, likely by inducing capsid structural changes. In some cases, the initial TfR or antibody binding event affected subsequent TfR binding, suggesting that capsid structure changes occur after TfR or antibody binding and may impact infection. This shows that precise, host-specific TfR-capsid interactions, beyond simple attachment, are important for successful infection.IMPORTANCE Host receptor binding is a key step during viral infection and may control both infection and host range. In addition to binding, some viruses require specific interactions with host receptors in order to infect, and anti-capsid antibodies can potentially disrupt these interactions, leading to neutralization. Here, we examine the interactions between parvovirus capsids, the receptors from different hosts, and anti-capsid antibodies. We show that interactions between parvovirus capsids and host-specific TfRs vary in both affinity and in the numbers of receptors bound, with complex effects on infection. In addition, antibodies binding to two sites on the capsids had different effects on TfR-capsid binding. These experiments confirm that receptor and antibody binding to parvovirus capsids are complex processes, and the infection outcome is not determined simply by the affinity of attachment.