Norovirus–glycan interactions — how strong are they really?

Infection with human noroviruses requires attachment to histo blood group antigens (HBGAs) via the major capsid protein VP1 as a primary step. Several crystal structures of VP1 protruding domain dimers, so called P-dimers, complexed with different HBGAs have been solved to atomic resolution. Corresponding binding affinities have been determined for HBGAs and other glycans exploiting different biophysical techniques, with mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy being most widely used. However, reported binding affinities are inconsistent. At the extreme, for the same system MS detects binding whereas NMR spectroscopy does not, suggesting a fundamental source of error. In this short essay, we will explain the reason for the observed differences and compile reliable and reproducible binding affinities. We will then highlight how a combination of MS techniques and NMR experiments affords unique insights into the process of HBGA binding by norovirus capsid proteins.

Mass Spectrometry-Based System for Identifying and Typing Norovirus Major Capsid Protein VP1

Norovirus-associated diseases are the most common foodborne illnesses worldwide. Polymerase chain reaction-based methods are the primary diagnostics for clinical samples; however, the high mutation rate of norovirus makes viral amplification and genotyping challenging. Technological advances in mass spectrometry (MS) make it a promising tool for identifying disease markers. Besides, the superior sensitivity of MS and proteomic approaches may enable the detection of all variants. Thus, this study aimed to establish an MS-based system for identifying and typing norovirus. We constructed three plasmids containing the major capsid protein VP1 of the norovirus GII.4 2006b, 2006a, and 2009a strains to produce virus-like particles for use as standards. Digested peptide signals were collected using a nano-flow ultra-performance liquid chromatography mass spectrometry (nano-UPLC/MSE) system, and analyzed by ProteinLynx Global SERVER and TREE-PUZZLE software. Results revealed that the LC/MSE system had an excellent coverage rate: the system detected more than 94% of amino acids of 3.61 femtomole norovirus VP1 structural protein. In the likelihood-mapping analysis, the proportions of unresolved quartets were 2.9% and 4.9% in the VP1 and S domains, respectively, which is superior to the 15.1% unresolved quartets in current PCR-based methodology. In summary, the use of LC/MSE may efficiently monitor genotypes, and sensitively detect structural and functional mutations of noroviruses.

Bcl-xL Reduces Chinese Giant Salamander Iridovirus-Induced Mitochondrial Apoptosis by Interacting with Bak and Inhibiting the p53 Pathway

Chinese giant salamander iridovirus (GSIV) infection could lead to mitochondrial apoptosis in this animal, a process that involves B-cell lymphoma-2 (BCL-2) superfamily molecules. The mRNA expression level of Bcl-xL, a crucial antiapoptotic molecule in the BCL-2 family, was reduced in early infection and increased in late infection. However, the molecular mechanism remains unknown. In this study, the function and regulatory mechanisms of Chinese giant salamander (Andrias davidianus) Bcl-xL (AdBcl-xL) during GSIV infection were investigated. Western blotting assays revealed that the level of Bcl-xL protein was downregulated markedly as the infection progressed. Plasmids expressing AdBcl-xL or AdBcl-xL short interfering RNAs were separately constructed and transfected into Chinese giant salamander muscle cells. Confocal microscopy showed that overexpressed AdBcl-xL was translocated to the mitochondria after infection with GSIV. Additionally, flow cytometry analysis demonstrated that apoptotic progress was reduced in both AdBcl-xL-overexpressing cells compared with those in the control, while apoptotic progress was enhanced in cells silenced for AdBcl-xL. A lower number of copies of virus major capsid protein genes and a reduced protein synthesis were confirmed in AdBcl-xL-overexpressing cells. Moreover, AdBcl-xL could bind directly to the proapoptotic molecule AdBak with or without GSIV infection. In addition, the p53 level was inhibited and the mRNA expression levels of crucial regulatory molecules in the p53 pathway were regulated in AdBcl-xL-overexpressing cells during GSIV infection. These results suggest that AdBcl-xL plays negative roles in GSIV-induced mitochondrial apoptosis and virus replication by binding to AdBak and inhibiting p53 activation.

Structural basis for genome packaging, retention, and ejection in human cytomegalovirus

How the human cytomegalovirus (HCMV) genome—the largest among human herpesviruses—is packaged, retained, and ejected remains unclear. We present the in situ structures of the symmetry-mismatched portal and the capsid vertex-specific components (CVSCs) of HCMV. The 5-fold symmetric 10-helix anchor—uncommon among known portals—contacts the portal-encircling DNA, which is presumed to squeeze the portal as the genome packaging proceeds. We surmise that the 10-helix anchor dampens this action to delay the portal reaching a “head-full” packaging state, thus facilitating the large genome to be packaged. The 6-fold symmetric turret, latched via a coiled coil to a helix from a major capsid protein, supports the portal to retain the packaged genome. CVSCs at the penton vertices—presumed to increase inner capsid pressure—display a low stoichiometry, which would aid genome retention. We also demonstrate that the portal and capsid undergo conformational changes to facilitate genome ejection after viral cell entry.

The 4.4 Å structure of the giant Melbournevirus virion belonging to the Marseilleviridae family

Members of Marseilleviridae, one family of icosahedral giant viruses classified in 2012 have been identified worldwide in all types of environments. The virion shows a characteristic internal membrane extrusion at the five-fold vertices of the capsid, but its structural details need to be elucidated. We now report the 4.4 Å cryo-electron microscopy structure of the Melbournevirus capsid. An atomic model of the major capsid protein (MCP) shows a unique cup structure on the trimer that accommodates additional proteins. A polyalanine model of the penton base protein shows internally extended N- and C-terminals, which indirectly connect to the internal membrane extrusion. The Marseilleviruses share the same orientational organisation of the MCPs as PBCV-1 and CroV, but do not appear to possess a protein akin to the ″tape measure″ of these viruses. Minor capsid proteins named PC-β, zipper, and scaffold are proposed to control the dimensions of the capsid during assembly.

Identification of hidden N4-like viruses and their interactions with hosts in global metagenomes

Background: N4-like viruses, with specific genomic features and propagation signatures, comprise a unique viral clade within the Podoviridae family. N4-like viruses are commonly characterized by the N4-like major capsid protein (MCP) and a giant virion-encapsulated RNA polymerase (N4-like RNAP) with a size of approximately 3,500-aa, which is the largest viral protein so far described. To date, our understanding of N4-like viruses is largely derived from 80 viral isolates that infect bacteria. Thus, it is necessary to expand the diversity of N4-like viruses in culturing-independent methods.Methods: A Hidden-Markov-Module based method was designed based on two characterized N4-specific marker genes, major capsid protein and N4-like virion-encapsulated RNA polymerase. Viral sub-clades were classified based on the monophyly presented in phylogenic tree and the results of pangenome analysis. Further analysis assessed different distribution patterns, genomic properties, hosts’ metabolism reprogramming potentialities, significance of viral tRNA and horizontal gene transfer landscape.Results: We identified 1,000 N4-like virus sequences from genomes and metagenomes representing diverse habitats from around the world. N4-like viruses have been classified into 27 sub-clades and detected in almost all habitats from pole to pole, including some novel habitats, such as oral mucosa and Antarctica. Virulent factors might be crucial for some human-associated N4-like viruses to reprogram the metabolism of host cells and mediate their pathogenic ability through horizontal gene transfer. From the pangenome analysis, the protein diversity was expended over 7-fold and 17 conserved house-keeping genes were identified. Transcriptional compensation of tRNA indicates that producing progeny virion might be the main significance of viral tRNAs. From the horizontal gene transfer network, some N4-like viral sub-clades were observed that potentially infect some important human pathogens, such as Campylobacteria and Veillonella , which have not been considered as potential hosts of N4-like virus or even any virus.Conclusion: This study expands the knowledge of N4-like viruses via global metagenomic datasets, reveals the novel ecological and genomic signatures of these viruses and will provide the backbone for further N4-like virus studies.

Head-to-Head Comparison of Modular Vaccines Developed Using Different Capsid Virus-Like Particle Backbones and Antigen Conjugation Systems

Capsid virus-like particles (cVLPs) are used as molecular scaffolds to increase the immunogenicity of displayed antigens. Modular platforms have been developed whereby antigens are attached to the surface of pre-assembled cVLPs. However, it remains unknown to what extent the employed cVLP backbone and conjugation system may influence the immune response elicited against the displayed antigen. Here, we performed a head-to-head comparison of antigen-specific IgG responses elicited by modular cVLP-vaccines differing by their employed cVLP backbone or conjugation system, respectively. Covalent antigen conjugation (i.e., employing the SpyTag/SpyCatcher system) resulted in significantly higher antigen-specific IgG titers compared to when using affinity-based conjugation (i.e., using biotin/streptavidin). The cVLP backbone also influenced the antigen-specific IgG response. Specifically, vaccines based on the bacteriophage AP205 cVLP elicited significantly higher antigen-specific IgG compared to corresponding vaccines using the human papillomavirus major capsid protein (HPV L1) cVLP. In addition, the AP205 cVLP platform mediated induction of antigen-specific IgG with a different subclass profile (i.e., higher IgG2a and IgG2b) compared to HPV L1 cVLP. These results demonstrate that the cVLP backbone and conjugation system can individually affect the IgG response elicited against a displayed antigen. These data will aid the understanding and process of tailoring modular cVLP vaccines to achieve improved immune responses.

Free Chlorine and Peroxynitrite Alter the Capsid Structure of Human Norovirus GII.4 and Its Capacity to Bind Histo-Blood Group Antigens

Human noroviruses (HuNoVs) are one of the leading causes of acute gastroenteritis worldwide. HuNoVs are frequently detected in water and foodstuffs. Free chlorine and peroxynitrite (ONOO−) are two oxidants commonly encountered by HuNoVs in humans or in the environment during their natural life cycle. In this study, we defined the effects of these two oxidants on GII.4 HuNoVs and GII.4 virus-like particles (VLPs). The impact on the capsid structure, the major capsid protein VP1 and the ability of the viral capsid to bind to histo-blood group antigens (HBGAs) following oxidative treatments were analyzed. HBGAs are attachment factors that promote HuNoV infection in human hosts. Overall, our results indicate that free chlorine acts on regions involved in the stabilization of VP1 dimers in VLPs and affects their ability to bind to HBGAs. These effects were confirmed in purified HuNoVs. Some VP1 cross-links also take place after free chlorine treatment, albeit to a lesser extent. Not only ONOO− mainly produced VP1 cross-links but can also dissociate VLPs depending on the concentration applied. Nevertheless, ONOO− has less effect on HuNoV particles.