Biology subjects

Green, T. J.

Publications and source records attributed to Green, T. J..

18 recordsLinked to original sources

bioRxiv2026

Intranasal immunization with live-attenuated RSV-vectored SARS-CoV-2 vaccines elicits antigen-specific systemic and mucosal immunity and protects against viral challenge and natural infection

The emergence of new SARS-CoV-2 variants and breakthrough infections underscores the need for next-generation vaccines capable of protecting from natural infection and/or preventing virus transmission to others. Intranasal vaccination offers a promising approach by eliciting local immune responses in the nasal mucosa, the primary site of infection and reservoir for transmissible virus. We evaluated two live-attenuated, respiratory syncytial virus vectored vaccines in which the RSV F and G surface glycoproteins were replaced with a chimeric SARS-CoV-2 Spike protein from either the ancestral USA/WA-1/2020 strain (MV-014-212) or the Delta variant (MV-014-212-delta). A single intranasal dose of either vaccine elicited systemic and mucosal immunity in K18-hACE2 mice, including serum neutralizing antibodies, Spike-specific memory B cells and plasmablasts, and Spike-specific CD8 lung-resident memory T cells. Although MV-014-212-delta vaccination provided the best protection against Delta variant virus challenge, both vaccines decreased viral loads in nasal discharge, lung and brain, and reduced weight loss and mortality. In naturally acquired infection studies, vaccinated hamsters exposed to infected cagemates exhibited minimal weight loss, limited viral replication within the nasal mucosa, and attenuated lung pathology. Therefore, intranasal RSV-vectored vaccines can elicit broad protective respiratory immunity, suggesting that this platform could be leveraged for other respiratory pathogens.

immunology↗

bioRxiv2026

Roseobacter enrichment early in life facilitates future colonization of Roseobacter bacteria and improves long-term survival against Vibrio aestuarianus in the Pacific oyster

Exposure to beneficial bacteria during early immune development may promote long-term survival against pathogens, known as "microbial education." As marine diseases intensify with ocean warming, methods to improve marine organisms robustness against disease will be valuable for mitigation. Here, we experimentally tested whether exposure to high temperature seawater, seawater enrichment with Roseobacter bacteria, or a combination of both during the first 24 hours of life enhances long-term survival of the Pacific oyster (Crassostrea gigas) against the widespread marine pathogen, Vibrio aestuarianus subsp. francensis, at high temperatures (24 {degrees}C). Exposure to high temperatures early in life did not improve future survival during disease challenges and caused high larval mortality. Conversely, Roseobacter enrichment during high temperature exposure resulted in a "microbial rescue effect," improving larval survival. We found that Roseobacter enrichment during the first 24 hours of life improved future survival against V. aestuarianus at high temperatures by up to [~]28% at 13 days post-fertilization (dpf) and [~]30% at 90 dpf. Ultimately, the supplemented Roseobacter species did not remain associated with the host microbiome but instead was replaced by a high abundance of other Roseobacter bacteria at 90 dpf. These findings suggest that early Roseobacter enrichment facilitates future colonization by other Roseobacter species, which may in turn protect against pathogenic V. aestuarianus, supporting the concept of microbial education in marine invertebrates and its potential use to combat marine diseases. ImportanceThis research identifies key aspects of early developmental processes in a marine invertebrate, Crassostrea gigas, and explores how these processes can be utilized to alter host-microbe interactions later in life.

microbiology↗

bioRxiv2025

An amplicon panel for high-throughput and low-cost genotyping of Yesso scallop Mizuhopecten yessoensis

The Yesso scallop Mizuhopecten yessoensis was imported from Japan to western Canada in the late 1980s to establish an economically viable scallop aquaculture industry. Since this time, the industry in Canada has operated with existing genetic diversity within the broodstock, which is considerably limited relative to wild populations. The sector has not been able to realise its full potential in part due to idiopathic hatchery failures and farm stock collapses due to disease outbreaks associated with the intracellular bacterial pathogen Francisella halioticida. To support Yesso scallop production and breeding, here we generate a low-density, genotyping-by-sequencing amplicon panel using single nucleotide polymorphism (SNP) markers that are evenly spaced across the M. yessoensis genome and that show high heterozygosity in Canada and Japan. The panel can also exploit the high genetic polymorphism of the M. yessoensis genome, with de novo SNP calling identifying over 2,500 high quality SNPs within the 579 sequenced amplicons. We demonstrate the utility and versatility of this new genotyping tool for breeding applications including parentage assignment, low density family-based genome-wide association study, trait heritability evaluation to determine potential for genomic selection, and species differentiation (against the weathervane scallop Patinopecten caurinus). We did not find any genomic regions significantly associated with F. halioticida resistance but did identify potential for genomic selection. We could separate the two species based on genotypes, and we did not see evidence of a past M. yessoensis x P. caurinus hybridization event within the M. yessoensis breeding population at Vancouver Island University. This low-cost genotyping panel is expected to accelerate selective breeding improvements for M. yessoensis in Canada and elsewhere.

genomics↗

bioRxiv2025

Identification of a broad and potent V3 glycan site bNAb targeting anN332gp120 glycan-independent epitope

Broadly neutralizing antibodies (bNAbs) against HIV-1 can suppress viremia in vivo and inform vaccine development. Here, we characterized 007, a V3 glycan site bNAb exhibiting high levels of antiviral activity against multiclade pseudovirus panels1-3 (GeoMean IC50 = 0.012 {micro}g/mL, breadth = 69%, 217 virus strains) by targeting a N332gp120 glycan-independent V3 epitope, a site of Env vulnerability to which only weakly neutralizing antibodies had previously been identified. Functional analyses demonstrated distinct binding and neutralization profiles compared to classical V3 glycan site bNAbs. A 007 Fab-Env cryo-EM structure revealed contacts with the V3 324GD/NIR327 motif and interactions with N156gp120 and N301gp120 glycans. In contrast to classical V3 bNAbs, 007 binding to Env does not depend on the N332gp120 glycan, rendering it resistant to common escape mutations. Structures of 007 IgG-Env trimer complexes showed two Env trimers crosslinked by three bivalent IgGs, and bivalent 007 IgG was up to [~]300-fold more potent than monovalent 007 IgG heterodimer, suggesting a role for avidity in potent neutralization. Finally, in HIV-1ADA-infected humanized mice, 007 caused transient decline of viremia and overcame classical V3 escape mutations, highlighting 007s potential for HIV-1 prevention, therapy, functional cure, and vaccine design.

immunology↗

bioRxiv2025

Pedigree-based genome-wide imputation using a low-density amplicon panel for the highly polymorphic Pacific oyster Crassostrea (Magallana) gigas

High-density genomic data are instrumental for selective breeding, but the costs associated with these approaches can hinder progress, as is the case for most aquaculture species. A strategy to reduce genotyping costs is to genotype a few select individuals at high-density (e.g., parents, grandparents), and many others at low density (e.g., offspring), then impute genotypes. This has been demonstrated in silico for Pacific oyster Crassostrea (Magallana) gigas but was particularly challenging relative to other species and has never been empirically tested. Here, four families of Pacific oysters, bred via marker-assisted selection for variation at a locus for field survival in an ostreid herpesvirus 1 (OsHV-1)-positive estuary, were exposed to OsHV-1 then genotyped using a low-density amplicon panel (n = 240 individuals). Parents were genotyped with the amplicon panel and by whole-genome resequencing. Offspring genotypes were imputed, and accuracy was determined by comparing against held-out whole-genome data for offspring. Imputation resulted in reduced minor allele frequencies and enriched homozygosity relative to empirical data. An in silico three-generation analysis was used to investigate the effect of deepening the pedigree, resulting in superior concordance in genotypes (GC = 84.5%) and allelic dosage (r = 0.73) compared to two-generation imputation (GC = 75.3%; r = 0.63). Genome-wide associations to OsHV-1 survivorship with imputed data identified significantly associated regions on the expected chromosome 8, but not at the expected position based on previous work, pointing to a potentially more complex genetic architecture for the trait. Our results empirically demonstrate the utility of amplicon panel-based genome-wide imputation in shellfish, and thus enable low-cost selective breeding techniques.

genomics↗

bioRxiv2024

The presence of the Pacific Oyster OSHV-1 resistance marker on chromosome 8 does not impact susceptibility to infection by Vibrio aestuarianus

The Pacific oyster, Crassostrea (Magallana) gigas, is an important species in aquaculture globally, but its production is threatened by pathogens including ostreid herpesvirus 1 (OsHV-1) and Vibrio aestuarianus. A genetic marker associated with resistance to OsHV-1 infection has been identified on chromosome 8 (Chr8) of the Pacific oyster genome. Marker-assisted selective breeding has used the Chr8 marker to produce oyster families with increased survivorship and resistance to OsHV-1 infection. The potential effect of the Chr8 marker on susceptibility to other pathogens remains largely unknown, but was recently associated with increased resistance of spat to Vibrio coralliilyticus. Here we assess the effect of the presence and allelic dosage of the Chr8 marker on the susceptibility of selectively bred juvenile Pacific oysters to V. aestuarianus infection. Sixteen families were produced with various Chr8 marker genotypes, and a V. aestuarianus disease challenge was conducted. Challenged oysters were individually genotyped at the Chr8 marker, and Vibrio susceptibility was evaluated among genotypes within and between families. Prior to the challenge trial, the Chr8 marker did not occur at the expected Mendelian ratios in families with heterozygous parents; in multiple families the homozygous alternate genotype occurred at a lower frequency than was expected. Mortality rates in the Vibrio challenge differed between families, ranging from 47.9% to 85.4%, but no association was observed between the Chr8 marker and survival to V. aestuarianus exposure. Therefore, we observed no pleiotropic effect (positive or negative) of the Chr8 marker on survivorship to V. aestuarianus at the evaluated life stage. Reduced-representation sequencing was used to genotype the challenged oysters and a genome-wide association study for V. aestuarianus survivorship was performed, but no significant associations were found, suggesting polygenic architecture for the trait.

genomics↗

bioRxiv2024

Potent neutralization by a receptor binding domain monoclonal antibody with broad specificity for SARS-CoV-2 JN.1 and other variants

SARS-CoV-2 continues to be a public health burden, driven in-part by its continued antigenic diversification and resulting emergence of new variants. While increasing herd immunity, current vaccines, and therapeutics have improved outcomes for some; prophylactic and treatment interventions that are not compromised by viral evolution of the Spike protein are still needed. Using a rationally designed SARS-CoV-2 Receptor Binding Domain (RBD) - ACE2 fusion protein and differential selection process with native Omicron RBD protein, we developed a recombinant human monoclonal antibody (hmAb) from a convalescent individual following SARS-CoV-2 Omicron infection. The resulting hmAb, 1301B7 potently neutralized a wide range of SARS-CoV-2 variants including the original Wuhan and more recent Omicron JN.1 strain, as well as SARS-CoV. Structure determination of the SARS-CoV-2 EG5.1 Spike/1301B7 Fab complex by cryo-electron microscopy at 3.1[A] resolution demonstrates 1301B7 contacts the ACE2 binding site of RBD exclusively through its VH1-69 heavy chain, making contacts using CDRs1-3, as well as framework region 3 (FR3). Broad specificity is achieved through 1301B7 binding to many conserved residues of Omicron variants including Y501 and H505. Consistent with its extensive binding epitope, 1301B7 is able to potently diminish viral burden in the upper and lower respiratory tract and protect mice from challenge with Omicron XBB1.5 and Omicron JN.1 viruses. These results suggest 1301B7 has broad potential to prevent or treat clinical SARS-CoV-2 infections and to guide development of RBD-based universal SARS-CoV-2 prophylactic vaccines and therapeutic approaches.

immunology↗

bioRxiv2023

An amplicon panel for high-throughput and low-cost genotyping of Pacific oyster

Maintaining genetic diversity in cultured shellfish can be challenging due to high variance in individual reproductive success, founder effects, and rapid genetic drift, but is important to retain adaptive potential and avoid inbreeding depression. To support broodstock management and selective breeding in cultured Pacific oysters (Crassostrea (Magallana) gigas), we developed an amplicon panel targeting 592 genomic regions and SNP variants with an average of 50 amplicons per chromosome. Target SNPs were selected based on elevated observed heterozygosity or differentiation in Pacific oyster populations in British Columbia (BC), Canada. The use of the panel for parentage applications was evaluated by genotyping three generations of oysters from a breeding program in BC (n = 181) and a set of families that were selected for Ostreid herpesvirus-1 (OSHV-1) resistance from the Molluscan Broodstock Program, a Pacific oyster breeding program in Oregon, USA (n = 136). Population characterization was evaluated using collections of wild, naturalized, farmed, or hatchery oysters sampled throughout the Northern Hemisphere (n = 190). Technical replicate samples showed high genotype concordance (97.5%; n = 68 replicates). Initial parentage analysis found instances of suspected pedigree and sample handling errors, demonstrating the panels value for quality control in breeding programs. Suspected null alleles were identified in parentage datasets and were found to reduce assignment success. Null alleles were largely population dependent, suggesting population-specific variation impacts target amplification. By taking an iterative approach, null alleles were identified using existing data without the need for pedigree information, and once null alleles were removed, assignments increased to 93.0% and 86.0% of possible assignments in the two breeding program datasets. A pipeline for analyzing the amplicon sequence data from sequencer output, amplitools, is also provided.

genetics↗

bioRxiv2023

The Prokaryotic and Eukaryotic Microbiome of Pacific Oyster Spat is Shaped by Ocean Warming but not Acidification

Pacific oysters (Magallana gigas, also known as Crassostrea gigas), the most widely farmed oysters, are under threat from climate change and emerging pathogens. In part, their resilience may be affected by their microbiome, which, in turn, may be influenced by ocean warming and acidification. Consequently, for three weeks, we exposed early-development Pacific oyster spat to different temperatures (18 and 24 {degrees}C) and pCO2 levels (800, 1600 and 2800 {micro}atm) in a fully crossed design. Under all conditions, the microbiome developed over time, with potentially pathogenic ciliates (Uronema marinum) greatly reduced in all treatments, suggesting that the spats microbiome undergoes adaptive shifts as the oysters age. The microbiome composition also differed significantly with temperature, but not acidification, indicating that M. gigas spat microbiomes can be altered by ocean warming but resilient to ocean acidification in our experiments. These findings highlight the spat microbiomes flexibility to environmental changes as well as its "protective" capability against potentially pathogenic microbes.

microbiology↗

bioRxiv2023

Genomic diversity of wild and cultured Yesso scallop Mizuhopecten yessoensis from Japan and Canada

The Yesso scallop Mizuhopecten yessoensis is an important aquaculture species that was introduced to Western Canada from Japan to establish an economically viable scallop farming industry. This highly fecund species has been propagated in Canadian aquaculture hatcheries for the past 40 years, raising questions about genetic diversity and genetic differences among hatchery stocks. In this study, we compare cultured Canadian and wild Japanese populations of Yesso scallop using double-digest restriction site-associated DNA (ddRAD)-sequencing to genotype 21,048 variants in 71 wild-caught scallops from Japan, 65 scallops from the Vancouver Island University breeding population, and 37 scallops obtained from a commercial farm off Vancouver Island, British Columbia. The wild scallops are largely comprised of equally unrelated individuals, whereas cultured scallops are comprised of multiple families of related individuals. The polymorphism rate estimated in wild scallops was 1.7%, whereas in the cultured strains it ranged between 1.35% and 1.07%. Interestingly, heterozygosity rates were highest in the cultured populations, which is likely due to shellfish hatchery practices of crossing divergent strains to gain benefits of heterosis and to avoid inbreeding. Evidence of founder effects and drift were observed in the cultured strains, including high genetic differentiation between cultured populations and between cultured populations and the wild population. Cultured populations had effective population sizes ranging from 9-26 individuals whereas the wild population was estimated at 25-50K individuals. Further, a depletion of low frequency variants was observed in the cultured populations. These results indicate significant genetic diversity losses in cultured scallops in Canadian breeding programs. Article SummaryYesso scallop was introduced to breeding programs in Canada around 40 years ago and has become a valuable aquaculture species in the country with little information regarding its genetic diversity. This work genotypes over 20K genetic variants in wild Yesso scallops from Japan and compares to a major broodstock collection in British Columbia, Canada, as well as a commercial farm in the same region. Reduced polymorphism but elevated heterozygosity indicates value of using genetic information to guide breeding programs.

genomics↗

bioRxiv2023

Alloreactivity and autoreactivity converge to support B cell epitope targeting in transplant rejection

Donor-specific antibody (DSA) responses against human leukocyte antigen (HLA) proteins mismatched between kidney transplant donors and recipients cause allograft loss. The rules governing the immunogenicity of non-self donor HLA are poorly understood. Using single-cell, molecular, structural, and proteomic techniques, we profiled the HLA-specific B cell response in the kidney and blood of a transplant recipient with antibody-mediated rejection (AMR). We observed an immunodominant B cell antibody response focused on topographically exposed, solvent-accessible mismatched HLA residues along the peptide-binding groove - a subregion comprising only 20% of the HLA molecule. We further demonstrated that, even within a diverse cohort of transplant recipients, the B cell alloresponse consistently converges on this same immunodominant subregion on the crown of the HLA molecule. Based on these findings, we propose that B cell immunodominance in transplant rejection relies on antigenic topography, and we suggest that this link could be exploited for organ matching and therapeutics.

immunology↗

bioRxiv2023

A high-throughput multiplex array for antigen-specific serology with automated analysis.

High-throughput customizable CBA automated script-based analysisThe utility of high-throughput systems to evaluate antigen-specific antibody (Ab) has been highlighted by the SARS-CoV-2 pandemic. Pathogen-specific Ab levels are often used to assess protection following vaccination and, in the case of novel pathogens, an indication of prior exposure. Several platforms exist to visualize antigen-specific Ab, however most are not quantitative and are difficult to scale for population level studies. Additionally, the sensitivity across platforms differs making direct comparisons between studies difficult. Cytometric bead arrays (CBA) are an attractive platform for antigen-specific Ab measurements as they can be used to assess Ab reactivity against several antigens and of several isotypes to be performed simultaneously. Additionally, CBAs exhibit high sensitivity and can be designed to provide quantitative measurements. Using commercially available particles, a biotin-Streptavidin bead loading strategy, and the inclusion of indirect standards, we describe a flexible system that can be modified to include a variety of antigens. Here we describe two arrays, focused on antigens derived from either {beta}-coronaviruses or influenza virus. To support the high-throughput capacity of this system, we developed a suit of automated software tools, the CBA Toolbox, to process raw data into antigen-reactive IgM, IgA, and IgG concentrations. We describe quality control requirements, assay performance, and normalizations to accurately quantitate antigen-specific Ig.

immunology↗

bioRxiv2021

Water-soluble tocopherol derivatives inhibit SARS-CoV-2 RNA-dependent RNA polymerase

The recent emergence of a novel coronavirus, SARS-CoV-2, has led to the global pandemic of the severe disease COVID-19 in humans. While efforts to quickly identify effective antiviral therapies have focused largely on repurposing existing drugs1-4, the current standard of care, remdesivir, remains the only authorized antiviral intervention of COVID-19 and provides only modest clinical benefits5. Here we show that water-soluble derivatives of -tocopherol have potent antiviral activity and synergize with remdesivir as inhibitors of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Through an artificial-intelligence-driven in silico screen and in vitro viral inhibition assay, we identified D--tocopherol polyethylene glycol succinate (TPGS) as an effective antiviral against SARS-CoV-2 and {beta}-coronaviruses more broadly that also displays strong synergy with remdesivir. We subsequently determined that TPGS and other water-soluble derivatives of -tocopherol inhibit the transcriptional activity of purified SARS-CoV-2 RdRp and identified affinity binding sites for these compounds within a conserved, hydrophobic interface between SARS-CoV-2 nonstructural protein 7 and nonstructural protein 8 that is functionally implicated in the assembly of the SARS-CoV-2 RdRp6. In summary, we conclude that solubilizing modifications to -tocopherol allow it to interact with the SARS-CoV-2 RdRp, making it an effective antiviral molecule alone and even more so in combination with remdesivir. These findings are significant given that many tocopherol derivatives, including TPGS, are considered safe for humans, orally bioavailable, and dramatically enhance the activity of the only approved antiviral for SARS-CoV-2 infection7-9.

microbiology↗

bioRxiv2021

Natural isolate and recombinant SARS-CoV-2 rapidly evolve in vitro to higher infectivity through more efficient binding to heparan sulfate and reduced S1/S2 cleavage

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant, cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increase the positive charge of the surface of this domain, ii) insertions into NTD of heterologous peptides, containing positively charged amino acids, and iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide and makes viruses less capable of syncytia formation. These viral adaptations result in higher affinity of viral particles to heparin sepharose, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers and two orders of magnitude lower GE:PFU ratios. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA+ viruses, evolution to HS binding may result in virus attenuation in vivo. IMPORTANCEThe spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to ACE2 receptor and later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the N-terminal domain (NTD) and in P1 position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells and decrease GE:PFU ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high affinity interaction of the spikes with the ACE2 receptor.

microbiology↗

bioRxiv2021

NAP1L1 and NAP1L4 binding to Hypervariable Domain of Chikungunya Virus nsP3 Protein is bivalent and requires phosphorylation

Chikungunya virus (CHIKV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. Within the last two decades, CHIKV has expanded its presence to both hemispheres and is currently circulating in both Old and New Worlds. Despite the severity and persistence of the arthritis it causes in humans, no approved vaccines or therapeutic means have been developed for CHIKV infection. Replication of alphaviruses, including CHIKV, is determined not only by their nonstructural proteins, but also by a wide range of host factors, which are indispensable components of viral replication complexes (vRCs). Alphavirus nsP3s contain hypervariable domains (HVDs), which encode multiple motifs that drive recruitment of cell- and virus-specific host proteins into vRCs. Our previous data suggested that NAP1 family members are a group of host factors that may interact with CHIKV nsP3 HVD. In this study, we performed a detailed investigation of the NAP1 function in CHIKV replication in vertebrate cells. Our data demonstrate that i) the NAP1-HVD interactions have strong stimulatory effects on CHIKV replication; ii) both NAP1L1 and NAP1L4 interact with the CHIKV HVD; iii) NAP1 family members interact with two motifs, which are located upstream and downstream of the G3BP-binding motifs of CHIKV HVD; iv) NAP1 proteins interact only with a phosphorylated form of CHIKV HVD and HVD phosphorylation is mediated by CK2 kinase; v) NAP1 and other families of host factors redundantly promote CHIKV replication and their bindings have additive stimulatory effects on viral replication. IMPORTANCECellular proteins play critical roles in the assembly of alphavirus replication complexes (vRCs). Their recruitment is determined by the viral nonstructural protein 3 (nsP3). This protein contains a long, disordered hypervariable domain (HVD), which encodes virus-specific combinations of short linear motifs interacting with host factors during vRC assembly. Our study defined the binding mechanism of NAP1 family members to CHIKV HVD and demonstrated a stimulatory effect of this interaction on viral replication. We showed that interaction with NAP1L1 is mediated by two HVD motifs and requires phosphorylation of HVD by CK2 kinase. Based on the accumulated data, we present a map of the binding motifs of the critical host factors currently known to interact with CHIKV HVD. It can be used to manipulate cell specificity of viral replication and pathogenesis, and to develop a new generation of vaccine candidates.

microbiology↗

bioRxiv2020

Structure of nonstructural protein 1 from SARS-CoV-2.

The periodic emergence of novel coronaviruses (CoVs) represents an ongoing public health concern with significant health and financial burden worldwide. The most recent occurrence originated in the city of Wuhan, China where a novel coronavirus (SARS-CoV-2) emerged causing severe respiratory illness and pneumonia. The continual emergence of novel coronaviruses underscores the importance of developing effective vaccines as well as novel therapeutic options that target either viral functions or host factors recruited to support coronavirus replication. The CoV nonstructural protein 1 (nsp1) has been shown to promote cellular mRNA degradation, block host cell translation, and inhibit the innate immune response to virus infection. Interestingly, deletion of the nsp1-coding region in infectious clones prevented the virus from productively infecting cultured cells. Because of nsp1s importance in the CoV lifecycle, it has been highlighted as a viable target for both antiviral therapy and vaccine development. However, the fundamental molecular and structural mechanisms that underlie nsp1 function remain poorly understood, despite its critical role in the viral lifecycle. Here we report the high-resolution crystal structure of the amino, globular portion of SARS-CoV-2 nsp1 (residues 10 - 127) at 1.77[A] resolution. A comparison of our structure with the SARS-CoV-1 nsp1 structure reveals how mutations alter the conformation of flexible loops, inducing the formation of novel secondary structural elements and new surface features. Paired with the recently published structure of the carboxyl end of nsp1 (residues 148 - 180), our results provide the groundwork for future studies focusing on SARS-CoV-2 nsp1 structure and function during the viral lifecycle. IMPORTANCEThe Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent for the COVID-19 pandemic. One protein known to play a critical role in the coronavirus lifecycle is nonstructural protein1 (nsp1). As such, it has been highlighted in numerous studies as a target for both the development of antivirals and for the design of live-attenuated vaccines. Here we report the high-resolution crystal structure of nsp1 derived from SARS-CoV-2 at 1.77[A] resolution. This structure will facilitate future studies focusing on understanding the relationship between structure and function for nsp1. In turn, understanding these structure-function relationships will allow nsp1 to be fully exploited as a target for both antiviral development and vaccine design.

microbiology↗

bioRxiv2020

Structural characterization of HIV-1 matrix mutants implicated in envelope incorporation

During the late phase of HIV-1 infection, viral Gag polyproteins are targeted to the plasma membrane (PM) for assembly. Gag localization at the PM is a prerequisite for the incorporation of the envelope protein (Env) into budding particles. Gag assembly and Env incorporation are mediated by the N-terminal myristoylated matrix (MA) domain of Gag. Nonconservative mutations in the trimer interface of MA (A45E, T70R, and L75G) were found to impair Env incorporation and infectivity, leading to the hypothesis that MA trimerization is an obligatory step for Env incorporation. Conversely, Env incorporation can be rescued by a compensatory mutation in the MA trimer interface (Q63R). The impact of these MA mutations on the structure and trimerization properties of MA is not known. In this study, we employed NMR spectroscopy, x-ray crystallography, and sedimentation techniques to characterize the structure and trimerization properties of HIV-1 MA A45E, Q63R, T70R, and L75G mutant proteins. NMR data revealed that these point mutations did not alter the overall structure and folding of MA but caused minor structural perturbations in the trimer interface. Analytical ultracentrifugation data indicated that mutations had a minimal effect on the MA monomer-trimer equilibrium. The high-resolution x-ray structure of the unmyristoylated MA Q63R protein revealed hydrogen bonding between the side chains of Arg-63 and Ser-67 located in the center of the trimer interface, providing the first structural evidence for a stabilization of the trimer form. These findings advance our knowledge of the interplay of MA trimerization and Env incorporation into HIV-1 particles.

biochemistry↗

bioRxiv2019

Micronutrient intake and prevalence of micronutrient inadequacy among women (15-49 y) and children (6-59 mo) in South Kivu and Kongo Central, Democratic Republic of Congo (DRC)

Iron biofortified beans and carotenoid enriched cassava are proposed as a solution to combat iron and vitamin A deficiencies, respectively, in the Democratic Republic of Congo (DRC). To inform the need for biofortified foods we conducted a survey in 2014, in two provinces of the DRC, South Kivu and Kongo Central. Unexpectedly, women of reproductive age (WRA; 15-49 y) and their children (6-59 m) had a low prevalence of biochemical iron and vitamin A deficiency, based on ferritin and retinol binding protein, respectively. However, to better understand the lack of biochemical deficiency of these nutrients we examined the prevalence of inadequate intake for these and other select nutrients. Dietary intake was assessed using 24-hour recalls (with repeats) among 744 mother-child dyads. In WRA the prevalence of inadequate iron intakes was 32% (10% bioavailability). The prevalence of inadequate vitamin A intakes was low in South Kivu (18%) and negligible in Kongo Central (1%). The prevalence of inadequate iron intake was 74% in infants (6-11 m) and ~22% in the older children (12-59 months). There was a high prevalence of inadequate zinc intake in women and children (i.e. 83% WRA) consistent with our findings of a high prevalence of low serum zinc in the same sample. Dietary data here corroborate the low prevalence of biochemical vitamin A deficiency but not iron. However, any change to the supply of red palm oil would dramatically reduce population vitamin A intakes, thus a carotenoid enriched cassava program may be beneficial as a safety net measure. Additionally, iron biofortified beans may be warranted given the high rates of dietary inadequacy and uncertainty around the validity of iron biomarkers. Crops biofortified with zinc also appear warranted. We caution that our findings cannot be extrapolated to the entire Congo where diverse agro-ecological landscape exist or when political and environmental shocks occur which challenge food production.

physiology↗