Subnanometer structure of medusavirus capsid during maturation using cryo-electron microscopy.

Medusavirus is a giant virus classified into an independent family of Mamonoviridae. Amoebae infected with medusavirus release immature particles in addition to virions. These particles were suggested to exhibit the maturation process of this virus, but the structure of these capsids during maturation remains unknown. Here, we apply a block-based reconstruction method in cryo-electron microscopy (cryo-EM) single particle analysis to these viral capsids, extending the resolution to 7-10 Å. The maps reveal a novel network composed of minor capsid proteins (mCPs) supporting major capsid proteins (MCPs). A predicted molecular model of the MCP fitted into the cryo-EM maps clarified the boundaries between the MCP and the underlining mCPs, as well as between the MCP and the outer spikes, and identified molecular interactions between the MCP and these components. Several structural changes of the mCPs under the fivefold vertices of the immature particles were observed, depending on the presence or absence of the underlying internal membrane. In addition, the lower part of the penton proteins on the fivefold vertices was also missing in mature virions. These dynamic conformational changes of mCPs indicate an important function in the maturation process of medusavirus.IMPORTANCEThe structural changes of giant virus capsids during maturation have not thus far been well clarified. Medusavirus is a unique giant virus in which infected amoebae release immature particles in addition to mature virus particles. In this study, we used cryo-electron microscopy to investigate immature and mature medusavirus particles and elucidate the structural changes of the viral capsid during the maturation process. In DNA-empty particles, the conformation of the minor capsid proteins changed dynamically depending on the presence or absence of the underlying internal membranes. In DNA-full particles, the lower part of the penton proteins was lost. This is the first report of structural changes of the viral capsid during the maturation process of giant viruses.

Cellular homologs of the double jelly-roll major capsid proteins clarify the origins of an ancient virus kingdom.

Viruses are a distinct type of replicators that encode structural proteins encasing virus genomes in virions. For some of the widespread virus capsid proteins and other major components of virions, likely ancestors encoded by cellular life forms are identifiable. In particular, one of the most common capsid proteins, with the single jelly-roll (SJR) fold, appears to have evolved from a particular family of cellular carbohydrate-binding proteins. However, the double jelly-roll major capsid protein (DJR-MCP), the hallmark of the enormously diverse viruses of the kingdom Bamfordvirae within the realm Varidnaviria, which includes bacterial and archaeal icosahedral viruses as well as eukaryotic giant viruses, has been perceived as a virus innovation that evolved by duplication and fusion of the SJR capsid proteins. Here we employ protein structure comparison to show that the DJR fold is represented in several widespread families of cellular proteins, including several groups of carbohydrate-active enzymes. We show that DJR-MCPs share a common ancestry with a distinct family of bacterial DJR proteins (DUF2961) involved in carbohydrate metabolism. Based on this finding, we propose a scenario in which bamfordviruses evolved from nonviral replicators, in particular plasmids, by recruiting a host protein for capsid formation. This sequence of events appears to be the general route of virus origin. The results of this work indicate that virus kingdoms Bamfordvirae, with the DJR-MCPs, and Helvetiavirae that possess two SJR-MCPs, have distinct origins, suggesting a reappraisal of the realm Varidnaviria.

A Membrane-Associated Light-Harvesting Model is Enabled by Functionalized Assemblies of Gene-Doubled TMV Proteins.

Photosynthetic light harvesting requires efficient energy transfer within dynamic networks of light-harvesting complexes embedded within phospholipid membranes. Artificial light-harvesting models are valuable tools for understanding the structural features underpinning energy absorption and transfer within chromophore arrays. Here, a method for attaching a protein-based light-harvesting model to a planar, fluid supported lipid bilayer (SLB) is developed.  The protein model consists of the tobacco mosaic viral capsid proteins that are gene-doubled to create a tandem dimer (dTMV). Assemblies of dTMV break the facial symmetry of the double disk to allow for differentiation between the disk faces. A single reactive lysine residue is incorporated into the dTMV assemblies for the site-selective attachment of chromophores for light absorption. On the opposing dTMV face, a cysteine residue is incorporated for the bioconjugation of a peptide containing a polyhistidine tag for association with SLBs. The dual-modified dTMV complexes show significant association with SLBs and exhibit mobility on the bilayer. The techniques used herein offer a new method for protein-surface attachment and provide a platform for evaluating excited state energy transfer events in a dynamic, fully synthetic artificial light-harvesting system.

Development of a highly sensitive imaged cIEF immunoassay for studying AAV capsid protein charge heterogeneity.

Post-translational modifications (PTMs) of adeno-associated virus (AAV) capsid proteins tune and regulate the AAV infective life cycle, which can impact the safety and efficacy of AAV gene therapy products. Many of these PTMs induce changes in protein charge heterogeneity, including deamidation, oxidation, glycation, and glycosylation. To characterize the charge heterogeneity of a protein, imaged capillary isoelectric focusing (icIEF) has become the gold standard method. We have previously reported an icIEF method with native fluorescence detection for denatured AAV capsid protein charge heterogeneity analysis. Although well suited for final products, the method does not have sufficient sensitivity for upstream, low-concentration AAV samples, and lacks the specificity for capsid protein detection in complex samples like cell culture supernatants and cell lysates. In contrast, the combination of icIEF, protein capture, and immunodetection affords significantly higher sensitivity and specificity, addressing the challenges of the icIEF method. By leveraging different primary antibodies, the icIEF immunoassay provides additional selectivity and affords a detailed characterization of individual AAV capsid proteins. In this study, we describe an icIEF immunoassay method for AAV analysis that is 90 times more sensitive than native fluorescence icIEF. This icIEF immunoassay provides AAV stability monitoring, where changes in individual capsid protein charge heterogeneity can be observed in response to heat stress. When applied to different AAV serotypes, this method also provides serotype identity with reproducible quantification of VP protein peak areas and apparent isoelectric point (pI). Overall, the described icIEF immunoassay is a sensitive, reproducible, quantitative, specific, and selective tool that can be used across the AAV biomanufacturing process, especially in upstream process development where complex sample types are often encountered.

Direct deamidation analysis of intact adeno-associated virus serotype 9 capsid proteins using reversed-phase liquid chromatography.

Recombinant adeno-associated viral (AAV) vectors have taken center stage as gene delivery vehicles for gene therapy. Asparagine deamidation of AAV capsid proteins has been reported to reduce vector stability and potency of AAV gene therapy products. Deamidation of asparagine residue is a common post-translational modification of proteins that is detected and quantified by liquid chromatography-tandem mass spectrometry (LC-MS)-based peptide mapping. However, artificial deamidation can be spontaneously induced during sample preparation for peptide mapping prior to LC-MS analysis. We have developed an optimized sample preparation method to reduce and minimize deamidation artifacts induced during sample preparation for peptide mapping, which typically takes several hours to complete. To shorten turnaround time of deamidation results and to avoid artificial deamidation, we developed orthogonal RPLC-MS and RPLC-fluorescence detection methods for direct deamidation analysis at the intact AAV9 capsid protein level to routinely support downstream purification, formulation development, and stability testing. Similar trends of increasing deamidation of AAV9 capsid proteins in stability samples were observed at the intact protein level and peptide level, indicating that the developed direct deamidation analysis of intact AAV9 capsid proteins is comparable to the peptide mapping-based deamidation analysis and both methods are suitable for deamidation monitoring of AAV9 capsid proteins.

Viral Capsid and Polymerase in Reoviridae.

The members of the family Reoviridae (reoviruses) consist of 9-12 discrete double-stranded RNA (dsRNA) segments enclosed by single, double, or triple capsid layers. The outer capsid proteins of reoviruses exhibit the highest diversity in both sequence and structural organization. By contrast, the conserved RNA-dependent RNA polymerase (RdRp) structure in the conserved innermost shell in all reoviruses suggests that they share common transcriptional regulatory mechanisms. After reoviruses are delivered into the cytoplasm of a host cell, their inner capsid particles (ICPs) remain intact and serve as a stable nanoscale machine for RNA transcription and capping performed using enzymes in ICPs. Advances in cryo-electron microscopy have enabled the reconstruction at near-atomic resolution of not only the icosahedral capsid, including capping enzymes, but also the nonicosahedrally distributed complexes of RdRps within the capsid at different transcriptional stages. These near-atomic resolution structures allow us to visualize highly coordinated structural changes in the related enzymes, genomic RNA, and capsid protein during reovirus transcription. In addition, reoviruses encode their own enzymes for nascent RNA capping before RNA releasing from their ICPs.

Multiple origins of viral capsid proteins from cellular ancestors.

Viruses are the most abundant biological entities on earth and show remarkable diversity of genome sequences, replication and expression strategies, and virion structures. Evolutionary genomics of viruses revealed many unexpected connections but the general scenario(s) for the evolution of the virosphere remains a matter of intense debate among proponents of the cellular regression, escaped genes, and primordial virus world hypotheses. A comprehensive sequence and structure analysis of major virion proteins indicates that they evolved on about 20 independent occasions, and in some of these cases likely ancestors are identifiable among the proteins of cellular organisms. Virus genomes typically consist of distinct structural and replication modules that recombine frequently and can have different evolutionary trajectories. The present analysis suggests that, although the replication modules of at least some classes of viruses might descend from primordial selfish genetic elements, bona fide viruses evolved on multiple, independent occasions throughout the course of evolution by the recruitment of diverse host proteins that became major virion components.

Structural Homology Between Nucleoproteins of ssRNA Viruses.

Our understanding of the viral world changed just after the first structures of icosahedral viral particles were unveiled. The structural similarities between capsid proteins of distant viral groups were not anticipated, and the findings suggested the existence of common ancestors for viruses with different host range, genomic structure and multiplication strategies. This way, diverse viruses with icosahedral particles can now be grouped based on the structural homology between their capsid proteins. In the last years, the presence of conserved folds between viral proteins in non-icosahedral viruses has also emerged. Viral particles with radically different morphologies, ranging from naked and filamentous to enveloped and pleomorphic, have shown structural homology between the nucleoproteins that bind directly to their genomes. This chapter overviews recent findings regarding the similar structure found between nucleoproteins of eukaryotic ssRNA viruses. The structural homology includes the coat proteins from all known families of flexible filamentous plant viruses, a group with monopartite (+)ssRNA genomes. Their coat proteins share a core domain with nucleoproteins of previously unrelated families of enveloped viruses that have segmented (-)ssRNA genomes. This last group consists of mostly animals viruses, including influenza virus.

Papillomaviruses and Endocytic Trafficking.

Endocytic trafficking plays a major role in transport of incoming human papillomavirus (HPVs) from plasma membrane to the trans Golgi network (TGN) and ultimately into the nucleus. During this infectious entry, several cellular sorting factors are recruited by the viral capsid protein L2, which plays a critical role in ensuring successful transport of the L2/viral DNA complex to the nucleus. Later in the infection cycle, two viral oncoproteins, E5 and E6, have also been shown to modulate different aspects of endocytic transport pathways. In this review, we highlight how HPV makes use of and perturbs normal endocytic transport pathways, firstly to achieve infectious virus entry, secondly to produce productive infection and the completion of the viral life cycle and, finally, on rare occasions, to bring about the development of malignancy.

Easy-to-Attach/Detach Solubilizing-Tag-Aided Chemical Synthesis of an Aggregative Capsid Protein.

A solubilizing Trt-K10 tag was developed for the effective chemical preparation of peptides/proteins with low solubility. The Trt-K10 tag comprises a hydrophilic oligo-Lys sequence and a trityl anchor, and can be selectively introduced to a side chain thiol of Cys of deprotected peptides/proteins with a trityl alcohol-type introducing reagent Trt(OH)-K10 under acidic conditions. Significantly, the ligation product in the reaction mixture of a thiol-additive-free native chemical ligation can be modified directly in a one-pot manner to facilitate the isolation of the product by high-performance liquid chromatography. Finally, the Trt-K10 tag can be readily removed with a standard trifluoroacetic acid cocktail. Using this easy-to-attach/detach tag-aided method, a hepatitis B virus capsid protein that is usually difficult to handle was synthesized successfully.

Expression and characterization of the major capsid protein derived from a GII.6 norovirus strain isolated in China.

The objective of this study was to express and characterize the major capsid protein (VP1) of a GII.6 Norovirus (NoV)strain isolated in China. The newly identified GII.6 NoV strain was isolated from a five-year old boy presenting acute gastroenteritis. The genome of the GII.6 strain was 7550 nucleotides in length, excluding the poly-adenylation tail. Multiple sequence alignment and phylogenetic analysis based on deduced VP1 amino acid sequences from different genotypes indicated close relationship between GII.3 and GII.6 NoVs, as demonstrated by the presence of a short sequence insertion in the P2 domain and clustering in the same subgroup. Expression of GII.6 VP1 led to assembly of virus like particles (VLPs). In vitro VLP-salivary histo-blood group antigens (HBGAs) binding assay demonstrated wide-spectrum binding activities of assembled VLPs to blood type A, B, AB and O salivary HBGAs with highest binding capacity to type A salivary HBGAs and lowest to type AB and O salivary HBGAs. In vitro VLP-salivary HBGAs binding blockade assay indicated absence of cross-blocking effects for hyperimmune sera produced against different genotypes. In conclusion, our results suggest a rational VLPs-based multivalent NoV vaccine should contain capsid proteins of a GII.6 strain.

Construction and Cloning of Reporter-Tagged Replicon cDNA for an In Vitro Replication Study of Murine Norovirus-1 (MNV-1).

BACKGROUND: A norovirus maintains its viability, infectivity and virulence by its ability to replicate. However, the biological mechanisms of the process remain to be explored. In this work, the NanoLuc™ Luciferase gene was used to develop a reporter-tagged replicon system to study norovirus replication. METHODS: The NanoLuc™ Luciferase reporter protein was engineered to be expressed as a fusion protein for MNV-1 minor capsid protein, VP2. The foot-and-mouth disease virus 2A (FMDV2A) sequence was inserted between the 3'end of the reporter gene and the VP2 start sequence to allow co-translational 'cleavage' of fusion proteins during intracellular transcript expression. Amplification of the fusion gene was performed using a series of standard and overlapping polymerase chain reactions. The resulting amplicon was then cloned into three readily available backbones of MNV-1 cDNA clones. RESULTS: Restriction enzyme analysis indicated that the NanoLucTM Luciferase gene was successfully inserted into the parental MNV-1 cDNA clone. The insertion was further confirmed by using DNA sequencing. CONCLUSION: NanoLuc™ Luciferase-tagged MNV-1 cDNA clones were successfully engineered. Such clones can be exploited to develop robust experimental assays for in vitro assessments of viral RNA replication.

Structure based sequence analysis of viral and cellular protein assemblies.

It is well accepted that, in general, protein structural similarity is strongly related to the amino acid sequence identity. To analyze in great detail the correlation, distribution and variation levels of conserved residues in the protein structure, we analyzed all available high-resolution structural data of 5245 cellular complex-forming proteins and 293 spherical virus capsid proteins (VCPs). We categorized and compare them in terms of protein structural regions. In all cases, the buried core residues are the most conserved, followed by the residues at the protein-protein interfaces. The solvent-exposed surface shows greater sequence variations. Our results provide evidence that cellular monomers and VCPs could be two extremes in the quaternary structural space, with cellular dimers and oligomers in between. Moreover, based on statistical analysis, we detected a distinct group of icosahedral virus families whose capsid proteins seem to evolve much slower than the rest of the protein complexes analyzed in this work.

Assembly, Engineering and Applications of Virus-Based Protein Nanoparticles.

Viruses and their protein capsids can be regarded as biologically evolved nanomachines able to perform multiple, complex biological functions through coordinated mechano-chemical actions during the infectious cycle. The advent of nanoscience and nanotechnology has opened up, in the last 10 years or so, a vast number of novel possibilities to exploit engineered viral capsids as protein-based nanoparticles for multiple biomedical, biotechnological or nanotechnological applications. This chapter attempts to provide a broad, updated overview on the self-assembly and engineering of virus capsids, and on applications of virus-based nanoparticles. Different sections provide outlines on: (i) the structure, functions and properties of virus capsids; (ii) general approaches for obtaining assembled virus particles; (iii) basic principles and events related to virus capsid self-assembly; (iv) genetic and chemical strategies for engineering virus particles; (v) some applications of engineered virus particles being developed; and (vi) some examples on the engineering of virus particles to modify their physical properties, in order to improve their suitability for different uses.

Characterization of Adeno-Associated Virus Capsid Proteins by Microflow Liquid Chromatography Coupled with Mass Spectrometry.

Adeno-associated virus (AAV) has been widely used to treat various human diseases as an important delivery vector for gene therapy due to its low immunogenicity and safety. AAV capsids proteins are comprised of three capsid viral proteins (VP; VP1, VP2, VP3). The capsid proteins play a key role in viral vector infectivity and transduction efficiency. To ensure the safety and efficacy of AAV gene therapy products, the quality of AAV vector capsid proteins during development and production should be carefully monitored and controlled. Microflow liquid chromatography coupled with mass spectrometry provides superior sensitivity and fast analysis capability. It showed significant advantages in the analysis of low- concentration and large numbers of AAV samples. The intact mass of capsid protein can be accurately determined using high-resolution mass spectrometry (MS). And MS also provides highly confident confirmation of sequence coverage and post-translational modifications site identification and quantitation. In this study, we used microflow liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the characterization of AAV2 capsid protein. we obtained nearly 100% sequence coverage of low-concentration AAV2 capsid protein (8 × 1011 GC/mL). More than 30 post-translational modifications (PTMs) sites were identified, the PTMs types included deamidation, oxidation and acetylation. From this study, the proposed microflow LC-MS/MS method provides a sensitive and high throughput approach in the characterization of AAVs and other biological products with low abundance.

Characterizing Glycosylation of Adeno-Associated Virus Serotype 9 Capsid Proteins Generated from HEK293 Cells through Glycopeptide Mapping and Released Glycan Analysis.

Recombinant adeno-associated viral (AAV) vectors have emerged as prominent gene delivery vehicles for gene therapy. AAV capsid proteins determine tissue specificity and immunogenicity and play important roles in receptor binding, the escape of the virus from the endosome, and the transport of the viral DNA to the nuclei of target cells. Therefore, the comprehensive characterization of AAV capsid proteins is necessary for a better understanding of the vector assembly, stability, and transduction efficiency of AAV gene therapies. Glycosylation is one of the most common post-translational modifications (PTMs) and may affect the tissue tropism of AAV gene therapy. However, there are few studies on the characterization of the N- and O-glycosylation of AAV capsid proteins. In this study, we identified the N- and O-glycosylation sites and forms of AAV9 capsid proteins generated from HEK293 cells using liquid chromatography-tandem mass spectrometry (LC-MS)-based glycopeptide mapping and identified free N-glycans released from AAV9 capsid proteins by PNGase F using hydrophilic interaction (HILIC) LC-MS and HILIC LC-fluorescence detection (FLD) methods. This study demonstrates that AAV9 capsids are sprinkled with sugars, including N- and O-glycans, albeit at low levels. It may provide valuable information for a better understanding of AAV capsids in supporting AAV-based gene therapy development.

Phylogenomic analysis expands the known repertoire of single-stranded DNA viruses in benthic zones of the South Indian Ocean.

Single-stranded (ss) DNA viruses are ubiquitous and constitute some of the most diverse entities on Earth. Most studies have focused on ssDNA viruses from terrestrial environments resulting in a significant deficit in benthic ecosystems including aphotic zones of the South Indian Ocean (SIO). Here, we assess the diversity and phylogeny of ssDNA in deep waters of the SIO using a combination of established viral taxonomy tools and a Hidden Markov Model based approach. Replication initiator protein-associated (Rep) phylogenetic reconstruction and sequence similarity networks were used to show that the SIO hosts divergent and as yet unknown circular Rep-encoding ssDNA viruses. Several sequences appear to represent entirely novel families, expanding the repertoire of known ssDNA viruses. Results suggest that a small proportion of these viruses may be circular genetic elements, which may strongly influence the diversity of both eukaryotes and prokaryotes in the SIO. Taken together, our data show that the SIO harbours a diverse assortment of previously unknown ssDNA viruses. Due to their potential to infect a variety of hosts, these viruses may be crucial for marine nutrient recycling through their influence of the biological carbon pump.

NRP1 is a receptor for mammalian orthoreovirus engaged by distinct capsid subunits.

Mammalian orthoreovirus (reovirus) is a nonenveloped virus that establishes primary infection in the intestine and disseminates to sites of secondary infection, including the CNS. Reovirus entry involves multiple engagement factors, but how the virus disseminates systemically and targets neurons remains unclear. In this study, we identified murine neuropilin 1 (mNRP1) as a receptor for reovirus. mNRP1 binds reovirus with nanomolar affinity using a unique mechanism of virus-receptor interaction, which is coordinated by multiple interactions between distinct reovirus capsid subunits and multiple NRP1 extracellular domains. By exchanging essential capsid protein-encoding gene segments, we determined that the multivalent interaction is mediated by outer-capsid protein σ3 and capsid turret protein λ2. Using capsid mutants incapable of binding NRP1, we found that NRP1 contributes to reovirus dissemination and neurovirulence in mice. Collectively, our results demonstrate that NRP1 is an entry receptor for reovirus and uncover mechanisms by which NRPs promote viral entry and pathogenesis.

Expanding the Plant Virome: Umbra-Like Viruses Use Host Proteins for Movement.

Before the very recent discovery of umbra-like viruses (ULVs), the signature defining feature of all plant RNA viruses was the encoding of specialized RNA-binding movement proteins (MPs) for transiting their RNA genomes through gated plasmodesmata to establish systemic infections. The vast majority of ULVs share umbravirus-like RNA-dependent RNA polymerases and 3'-terminal structures, but they differ by not encoding cell-to-cell and long-distance MPs and by not relying on a helper virus for trans-encapsidation and plant-to-plant transmission. The recent finding that two groups of ULVs do not necessarily encode MPs is expanding our understanding of the minimum requirements for modern plant RNA viruses. ULV CY1 from citrus uses host protein PHLOEM PROTEIN 2 (PP2) for systemic movement, and related ULVs encode a capsid protein, thereby providing an explanation for the lack of helper viruses present in many ULV-infected plants. ULVs thus resemble the first viruses that infected plants, which were likely deposited from feeding organisms and would have similarly required the use of host proteins such as PP2 to exit initially infected cells.

Cell membrane-bound toll-like receptor-1/2/4/6 monomers and -2 heterodimer inhibit enterovirus 71 replication by activating the antiviral innate response.

Host immune activation is critical for enterovirus 71 (EV71) clearance and immunopathogenesis. However, the mechanism of innate immune activation, especially of cell membrane-bound toll-like receptors (TLRs), against EV71 remains unknown. We previously demonstrated that TLR2 and its heterodimer inhibit EV71 replication. In this study, we systematically investigated the effects of TLR1/2/4/6 monomers and TLR2 heterodimer (TLR2/TLR1, TLR2/TLR6, and TLR2/TLR4) on EV71 replication and innate immune activation. We found that the overexpression of human- or mouse-derived TLR1/2/4/6 monomers and TLR2 heterodimer significantly inhibited EV71 replication and induced the production of interleukin (IL)-8 via activation of the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase (MAPK) pathways. Furthermore,human-mouse chimeric TLR2 heterodimer inhibited EV71 replication and activated innate immunity. Dominant-negative TIR-less (DN)-TLR1/2/4/6 did not exert any inhibitory effects, whereas DN-TLR2 heterodimer inhibited EV71 replication. Prokaryotic expression of purified recombinant EV71 capsid proteins (VP1, VP2, VP3, and VP4) or overexpression of EV71 capsid proteins induced the production of IL-6 and IL-8 via activation of the PI3K/AKT and MAPK pathways. Notably, two types of EV71 capsid proteins served as pathogen-associated molecular patterns for TLR monomers (TLR2 and TLR4) and TLR2 heterodimer (TLR2/TLR1, TLR2/TLR6, and TLR2/TLR4) and activated innate immunity. Collectively, our results revealed that membrane TLRs inhibited EV71 replication via activation of the antiviral innate response, providing insights into the EV71 innate immune activation mechanism.