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.

Development of a therapeutic vaccine targeting Merkel cell polyomavirus capsid protein VP1 against Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare but aggressive skin cancer with a high mortality rate, while Merkel cell polyomavirus (MCV) has been pointed as the causative agent of MCC. A better prognosis of MCC associated with a high level of antibodies against the capsid protein VP1 suggests that anti-VP1 immune response might be essential against MCC growth. In the current study, we developed a VP1-target vaccine formulated with CRA. Using a tumorigenic CMS5-VP1 tumor model, the vaccine-induced a potent antitumor efficacy in a dose-dependent manner was evidently demonstrated and mainly mediated by both VP1-specific CD4+ and CD8+ T-cell responses against the growth of CMS5-VP1 tumors in vaccinated BALB/c mice since the depletion of CD4+ and CD8+ T cells reverse the antitumor effects. Thus, immunotherapy with this vaccine represents a novel approach for the clinical treatment of aggressive MCV-related MCC in humans.

Molecular Characterization and Cross-Reactivity of Feline Calicivirus Circulating in Southwestern China

Feline calicivirus (FCV) is an important pathogen of cats that has two genogroups (GI and GII). To investigate the prevalence and molecular characteristics of FCVs in southwestern China, 162 nasal swab samples were collected from cats in animal shelters and pet hospitals. In total, 38 of the clinical samples (23.46%) were identified as FCV positive using nested RT-PCR. Phylogenetic analyses using 10 capsid protein VP1 sequences revealed that 8 GI and 2 GII strains formed two independent clusters. Additionally, three separated FCVs that were not clustered phylogenetically (two GI and one GII strains) were successfully isolated from clinical samples and their full-length genomes were obtained. Phylogenetic and recombinant analyses of a GI FCV revealed genomic breakpoints in ORF1 and ORF2 regions with evidence for recombinant events between GI sub-genogroups, which is reported in China for the first time. Furthermore, sera obtained from mice immunized independently with the three FCV isolates and a commercial vaccine were used to evaluate the cross-reactivity of neutralizing antibodies. The three separate FCVs were neutralized by each other at a 1:19 to 1:775 titer range, whereas the triple-inactivated vaccine was at a titer of 1:16, which suggested that different genogroup/sub-genogroup FCV strains exhibit significantly different titers of neutralizing antibodies, including the commercial FCV vaccine. Thus, our study revealed the genetic diversity and complex cross-reactivity levels of FCVs in southwestern China, which provides new insights for application in vaccination strategies.

Norovirus Structure and Classification

Norovirus are a major cause of acute gastroenteritis worldwide. Diarrheal disease is now the fourth common cause of mortality children under the age of 5 years but remain the 2nd most cause of morbidity. NoV are associated with 18% diarrheal diseases worldwide where rotavirus vaccinations has been successfully introduced. NoV has become major cause of gastroenteritis in children. NoV belong to family caliciviridae. They are non-enveloped, single stranded positive sense RNA Viruses. The genome consists of 3 Open reading frames, ORF-1 codes for non-structural protein, ORF-2 codes for major capsid protein VP1 and ORF-3 for minor capsid protein VP2. Based on sequence difference of the capsid gene (VP1), NoV have been classified in to seven genogroup GI-GVII with over 30 genotypes. Genogroups I, II, IV are associated with human infection. Despite this extensive diversity a single genotype GII.4 has been alone to be the more prevalent. Basic epidemiological disease burden data are generated from developing countries. NoV are considered fast evolving viruses and present an extensive diversity that is driven by acquisition of point mutations and recombinations. Immunity is strain or genotype specific with little or no protection conferred across genogroups. Majority of outbreaks and sporadic norovirus cases worldwide are associated with a single genotype, GII.4 which was responsible for 62% of reported NoV outbreaks in 5 continents from 2001 to 2007. GII.4 variants have been reported as major cause of global gastroenteritis pandemics starting in 1995 frequent emergence of novel GII.4 variants is known to be due to rapid evolution and antigenic variation in response to herd immunity. Novel GII.4 variants appear almost every 2 years. Recent GII.4 variant reported include Lordsdale 1996, Farmington Hills 2002, Hunter 2004, Yerseke 2006a, Den Haag 2006b, Apeldoon 2007, New Orleans 2009,most recently Sydney 2012. Detailed molecular epidemiologic investigation of NoV is associated for understanding the genetic diversity of NoV strain and emergence of novel NoV variants. However, reports have revealed that not all individuals develop symptoms and a significant proportion remains asymptomatic after NoV infections.

SNAP47 Interacts with ATG14 to Promote VP1 Conjugation and CVB3 Propagation

Coxsackievirus B3 (CVB3), an enterovirus (EV) in the family of Picornaviridae, is a global human pathogen for which effective antiviral treatments and vaccines are lacking. Previous research demonstrated that EV-D68 downregulated the membrane fusion protein SNAP47 (synaptosome associated protein 47) and SNAP47 promoted EV-D68 replication via regulating autophagy. In the current study, we investigated the interplay between CVB3 and cellular SNAP47 using HEK293T/HeLa cell models. We showed that, upon CVB3 infection, protein levels of SNAP47 decreased independent of the activity of virus-encoded proteinase 3C. We further demonstrated that the depletion of SNAP47 inhibited CVB3 infection, indicating a pro-viral function of SNAP47. Moreover, we found that SNAP47 co-localizes with the autophagy-related protein ATG14 on the cellular membrane fractions together with viral capsid protein VP1, and expression of SNAP47 or ATG14 enhanced VP1 conjugation. Finally, we revealed that disulfide interactions had an important role in strengthening VP1 conjugation. Collectively, our study elucidated a mechanism by which SNAP47 and ATG14 promoted CVB3 propagation through facilitating viral capsid assembly.

Molecular Epidemiology of Human Sapovirus Among Children with Acute Gastroenteritis in Western Canada

Objectives: Sapovirus is increasingly recognized as an important cause of acute gastroenteritis (AGE) worldwide, however studies of prevalence, genetic diversity and strain-specific clinical implications have been scarce. Methods: To fill this knowledge gap, we used reverse transcription real-time PCR and sequencing of the partial major capsid protein VP1 gene to analyze stool specimens and rectal swabs obtained from 3347 children with AGE and 1355 asymptomatic controls (all <18 years old) collected between December 2014 and August 2018 in Alberta, Canada. Results: Sapovirus was identified in 9.5% (317/3347) of the children with AGE and 2.9% of controls. GI.1 (36%) was the predominant genotype identified, followed by GI.2 (18%), GII.5 (8%) and GII.3 (6%). Rare genotypes GII.1, GII.2, GV.1, GII.4, GIV.1, GI.3 and GI.7 were also seen. Sapovirus was detected year-round, peaking during the winter months of November to January. The exception was the 2016-2017 season when GI.2 overtook GI.1 as the predominant strain with a high detection rate persisting into April. We did not observe significant difference in the severity of gastroenteritis by genogroup or genotype. Repeated infection by sapovirus of different genogroups occurred in three controls who developed AGE later. Conclusions: Our data suggests that sapovirus is a common cause of AGE in children with high genetic diversity.

Tradeoffs for a viral mutant with enhanced replication speed

RNA viruses exist as genetically heterogeneous populations due to high mutation rates, and many of these mutations reduce fitness and/or replication speed. However, it is unknown whether mutations can increase replication speed of a virus already well adapted to replication in cultured cells. By sequentially passaging coxsackievirus B3 in cultured cells and collecting the very earliest progeny, we selected for increased replication speed. We found that a single mutation in a viral capsid protein, VP1-F106L, was sufficient for the fast-replication phenotype. Characterization of this mutant revealed quicker genome release during entry compared to wild-type virus, highlighting a previously unappreciated infection barrier. However, this mutation also reduced capsid stability in vitro and reduced replication and pathogenesis in mice. These results reveal a tradeoff between overall replication speed and fitness. Importantly, this approach—selecting for the earliest viral progeny—could be applied to a variety of viral systems and has the potential to reveal unanticipated inefficiencies in viral replication cycles.

Protein Secondary Structure Affects Glycan Clustering in Native Mass Spectrometry

Infection by the human noroviruses (hNoV), for the vast majority of strains, requires attachment of the viral capsid to histo blood group antigens (HBGAs). The HBGA-binding pocket is formed by dimers of the protruding domain (P dimers) of the capsid protein VP1. Several studies have focused on HBGA binding to P dimers, reporting binding affinities and stoichiometries. However, nuclear magnetic resonance spectroscopy (NMR) and native mass spectrometry (MS) analyses yielded incongruent dissociation constants (KD) for the binding of HBGAs to P dimers and, in some cases, disagreed on whether glycans bind at all. We hypothesized that glycan clustering during electrospray ionization in native MS critically depends on the physicochemical properties of the protein studied. It follows that the choice of a reference protein is crucial. We analysed carbohydrate clustering using various P dimers and eight non-glycan binding proteins serving as possible references. Data from native and ion mobility MS indicate that the mass fraction of β-sheets has a strong influence on the degree of glycan clustering. Therefore, the determination of specific glycan binding affinities from native MS must be interpreted cautiously.