N-terminal Receptor-binding Domain Research Articles

Antigenic Characterization of Circulating and Emerging SARS-CoV-2 Variants in the U.S. throughout the Delta to Omicron Waves.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into numerous lineages with unique spike mutations and caused multiple epidemics domestically and globally. Although COVID-19 vaccines are available, new variants with the capacity for immune evasion continue to emerge. To understand and characterize the evolution of circulating SARS-CoV-2 variants in the U.S., the Centers for Disease Control and Prevention (CDC) initiated the National SARS-CoV-2 Strain Surveillance (NS3) program and has received thousands of SARS-CoV-2 clinical specimens from across the nation as part of a genotype to phenotype characterization process. Focus reduction neutralization with various antisera was used to antigenically characterize 143 SARS-CoV-2 Delta, Mu and Omicron subvariants from selected clinical specimens received between May 2021 and February 2023, representing a total of 59 unique spike protein sequences. BA.4/5 subvariants BU.1, BQ.1.1, CR.1.1, CQ.2 and BA.4/5 + D420N + K444T; BA.2.75 subvariants BM.4.1.1, BA.2.75.2, CV.1; and recombinant Omicron variants XBF, XBB.1, XBB.1.5 showed the greatest escape from neutralizing antibodies when analyzed against post third-dose original monovalent vaccinee sera. Post fourth-dose bivalent vaccinee sera provided better protection against those subvariants, but substantial reductions in neutralization titers were still observed, especially among BA.4/5 subvariants with both an N-terminal domain (NTD) deletion and receptor binding domain (RBD) substitutions K444M + N460K and recombinant Omicron variants. This analysis demonstrated a framework for long-term systematic genotype to antigenic characterization of circulating and emerging SARS-CoV-2 variants in the U.S., which is critical to assessing their potential impact on the effectiveness of current vaccines and antigen recommendations for future updates.

Evidence of antigenic drift in the fusion machinery core of SARS-CoV-2 spike

Antigenic drift of SARS-CoV-2 is typically defined by mutations in the N-terminal domain and receptor binding domain of spike protein. In contrast, whether antigenic drift occurs in the S2 domain remains largely elusive. Here, we perform a deep mutational scanning experiment to identify S2 mutations that affect binding of SARS-CoV-2 spike to three S2 apex public antibodies. Our results indicate that spatially diverse mutations, including D950N and Q954H, which are observed in Delta and Omicron variants, respectively, weaken the binding of spike to these antibodies. Although S2 apex antibodies are known to be nonneutralizing, we show that they confer protection in vivo through Fc-mediated effector functions. Overall, this study indicates that the S2 domain of SARS-CoV-2 spike can undergo antigenic drift, which represents a potential challenge for the development of more universal coronavirus vaccines.

Mutations in S2 subunit of SARS-CoV-2 Omicron spike strongly influence its conformation, fusogenicity, and neutralization sensitivity.

The Omicron subvariants have substantially evaded host-neutralizing antibodies and adopted an endosomal route of entry. The virus has acquired several mutations in the receptor binding domain and N-terminal domain of S1 subunit, but remarkably, also incorporated mutations in S2 which are fixed in Omicron sub-lineage. Here, we found that the mutations in the S2 subunit affect the structural and biological properties such as neutralization escape, entry route, fusogenicity, and protease requirement. In vivo, these mutations may have significant roles in tropism and replication. A detailed understanding of the effects of S2 mutations on Spike function, immune evasion, and viral entry would inform the vaccine design, as well as therapeutic interventions aiming to block the essential proteases for virus entry. Thus, our study has identified the crucial role of S2 mutations in stabilizing the Omicron spike and modulating neutralization resistance to antibodies targeting the S1 subunit.

Molecular evolution and targeted recombination of SARS-CoV-2 in South Korea

SARS-CoV-2 variants have continuously emerged globally, including in South Korea. To characterize the molecular evolution of SARS-CoV-2 in South Korea, we performed phylogenetic and genomic recombination analyses using more than 12,000 complete genome sequences collected until October 2022. The variants in South Korea originated from globally identified variants of concern and harbored genetic clade-common and clade-specific amino acid mutations mainly around the N-terminal domain (NTD) or receptor binding domain (RBD) in the spike protein. Several point mutation residues in key antigenic sites were under positive selection persistently with changing genetic clades of SARS-CoV-2. Furthermore, we detected 17 potential genomic recombinants and 76.4% (13/17) retained the mosaic NTD or RBD genome. Our results suggest that point mutations and genomic recombination in the spike contributed to the molecular evolution of SARS-CoV-2 in South Korea, which will form an integral part of global prevention and control measures against SARS-CoV-2.

Stabilized SARS-CoV-2 spike S2 immunogens designed using insights from enhanced sampling molecular dynamics simulations.

The SARS-CoV-2 spike protein is an important molecular machine that facilitates membrane fusion to host cells and is the major antigenic component of vaccines. Notably, full-length prefusion-stabilized spike proteins produce a strong neutralizing antibody response against S1, which contains the N-terminal domain (NTD) and receptor binding domain (RBD). Unfortunately, emerging variants of concern contain numerous mutations, insertions, and deletions within S1 that impact the binding of neutralizing antibodies which may diminish the efficacy of current vaccines. In contrast, S2 is the most conserved region of the spike and is capable of eliciting antibodies that bind broadly to diverse β-coronavirus spikes. Although S2-based immunogens may have potential benefits, a challenge to its usage as an alternative vaccine strategy is the general instability of S2 in the absence of S1. Using enhanced sampling molecular dynamics simulations, we observed that the S2 head adopts an open conformation where individual protomers separate from one another at the apex. Extensive contact map analysis reveals that the formation and breakage of contacts between three helices mediates these opening motions. Through introducing mutations in these helices, we designed a stabilized S2 construct that maintains a closed conformation and has enhanced thermal stability and protein expression. These studies represent the first simulations characterizing S2 conformational dynamics and demonstrate the potential of using enhanced sampling molecular dynamics simulations in the design of stabilized SARS-CoV-2 S2 immunogens.

Effect on the conformations of the spike protein of SARS-CoV-2 due to mutation.

The spike protein of SARS-CoV-2 mediates receptor binding and cell entry and is the key immunogenic target for virus neutralization and the present attention of many vaccine layouts. It exhibits significant conformational flexibility. We study the structural fluctuations of spike protein among the most common mutations that appeared in the variant of concerns (VOC). We report the thermodynamics of conformational changes in mutant spike protein with respect to the wild-type from the distributions of the dihedral angles obtained from the equilibrium configurations generated via all-atom molecular dynamics simulations. We find that the mutation causes the increase in distance between the N-terminal domain and receptor binding domain, leading to an obtuse angle cosine θ distribution in the trimeric structure in spike protein. Thus, an increase in open state is conferred to the more infectious variants of SARS-CoV-2. The thermodynamically destabilized and disordered residues of receptor binding motif among the mutant variants of spike protein are proposed to serve as better binding sites for the host factor. We identify a short stretch of region connecting the N-terminal domain and receptor binding domain forming a linker loop where many residues undergo stabilization in the open state compared to the closed one.

A DNA vaccine candidate delivered by an electroacupuncture machine provides protective immunity against SARS-CoV-2 infection

A major challenge in the use of DNA vaccines is efficient DNA delivery in vivo. Establishing a safe and efficient electric transfer method is the key to developing rapid DNA vaccines against emerging infectious diseases. To overcome the complexity of designing new electric transfer machines for DNA delivery, a clinically approved electric transfer machine could be considered as an alternative. Here, we report an electroacupuncture machine-based method for DNA vaccine delivery after intramuscular injection of the COVID-19 DNA vaccine. The S gene of SARS-CoV-2 in the pVAX1 plasmid (pSARS2-S) was used as an antigen in this study. We optimized the clinically used electroacupuncture machine settings for efficient induction of the neutralizing antibody titer after intramuscular injection of pSARS2-S in mice. We found that pSARS2-S immunization at 40 Vpp for 3–5 s could induce high neutralizing antibody titers and Th1-biased immune responses. IFN-γ/TNF-α-secreting CD4+ and CD8+ T cells were also observed in the DNA vaccination group but not in the recombinant protein vaccination group. T-cell epitope mapping shows that the major reactive epitopes were located in the N-terminal domain (a.a. 261–285) and receptor-binding domain (a.a. 352–363). Importantly, pSARS2-S immunization in hamsters could induce protective immunity against SARS-CoV-2 challenge in vivo. In the preclinical toxicology study, blood biochemistry, hematology, and DNA persistence analysis reveal that the DNA delivery method is safe. Furthermore, the raised antisera could also cross-neutralize different variants of concern. These findings suggest that DNA vaccination using an electroacupuncture machine is feasible for use in humans in the future.

Conserved Pattern and Potential Role of Recurrent Deletions in SARS-CoV-2 Evolution.

ABSTRACTSARS-CoV-2 continues adapting to human hosts during the current worldwide pandemic since 2019. This virus evolves through multiple means, such as single nucleotide mutations and structural variations, which has brought great difficulty to disease prevention and control of COVID-19. Structural variation, including multiple nucleotide changes like insertions and deletions, has a greater impact relative to single nucleotide mutation on both genome structures and protein functions. In this study, we found that deletion occurred frequently in not only SARS-CoV-2 but also in other SARS-related coronaviruses. These deletions showed obvious location bias and formed 45 recurrent deletion regions in the viral genome. Some of these deletions showed proliferation advantages, including four high-frequency deletions (nsp6 Δ106-109, S Δ69-70, S Δ144, and Δ28271) that were detected in around 50% of SARS-CoV-2 genomes and other 19 median-frequency deletions. In addition, the association between deletions and the WHO reported variants of concern (VOC) and variants of interest (VOI) of SARS-CoV-2 indicated that these variants had a unique combination of deletion patterns. In the spike (S) protein, the deletions in SARS-CoV-2 were mainly in the N-terminal domain. Some deletions, such as S Δ144/145 and S Δ243-244, have been confirmed to block the binding sites of neutralizing antibodies. Overall, this study revealed a conservative regional pattern and the potential effect of some deletions in SARS-CoV-2 over the whole genome, providing important evidence for potential epidemic control and vaccine development.IMPORTANCE Mutations in SARS-CoV-2 were studied extensively, while only the structure variations on the spike protein were discussed well in previous studies. To study the role of structural variations in virus evolution, we described the distribution of structure variations on the whole genome. Conserved patterns were found of deletions among SARS-CoV-2, SARS-CoV-2-like, and SARS-CoV-like viruses. There were 45 recurrent deletion regions (RDRs) in SARS-CoV-2 generated through the integration of deleted positions. In these regions, four high-frequency deletions parallelly appeared in multiple strains. Furthermore, in the spike protein, the deletions in SARS-CoV-2 were mainly in the N-terminal domain, blocking the binding sites of some neutralizing antibodies, while the structural variations in SARS-related coronavirus were mainly in the N-terminal domain and receptor binding domain. The receptor binding domain is highly related to hosting recognition. The deletions in the receptor binding domain may play a role in host adaption.

Dynamic Asymmetry Exposes 2019-nCoV Prefusion Spike.

The novel coronavirus (2019-nCoV) spike protein is a smart molecular machine that instigates the entry of coronavirus to the host cell causing the COVID-19 pandemic. In this study, a symmetry-information-loaded structure-based Hamiltonian is developed using recent Cryo-EM structural data to explore the complete conformational energy landscape of the full-length prefusion spike protein. The study finds the 2019-nCoV prefusion spike to adopt a unique strategy by undertaking a dynamic conformational asymmetry that results in two prevalent asymmetric structures of spike where one or two spike heads rotate up to provide better exposure to the host-cell receptor. A few unique interchain interactions are identified at the interface of closely associated N-terminal domain (NTD) and receptor binding domain (RBD) playing a crucial role in the thermodynamic stabilization of the up conformation of the RBD in the case of the 2019-nCoV spike. The interaction-level information decoded in this study may provide deep insight into developing effective therapeutic targets.

Role of the GTNGTKR motif in the N-terminal receptor-binding domain of the SARS-CoV-2 spike protein

The 2019 novel coronavirus disease (COVID-19) that emerged in China has been declared as public health emergency of international concern by the World Health Organization and the causative pathogen was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this report, we analyzed the structural characteristics of the N-terminal domain of the S1 subunit (S1-NTD) of the SARS-CoV-2 spike protein in comparison to the SARS-CoV in particular, and to other viruses presenting similar characteristic in general. Given the severity and the wide and rapid spread of the SARS-CoV-2 infection, it is very likely that the virus recognizes other receptors/co-receptors besides the ACE2. The NTD of the SARS-CoV-2 contains a receptor-binding motif different from that of SARS-CoV, with some insertions that could confer to the new coronavirus new receptor binding abilities. In particular, motifs similar to the insertion 72GTNGTKR78 have been found in structural proteins of other viruses; and these motifs were located in putative regions involved in recognizing protein and sugar receptors, suggesting therefore that similar binding abilities could be displayed by the SARS-CoV-2 S1-NTD. Moreover, concerning the origin of these NTD insertions, our findings point towards an evolutionary acquisition rather than the hypothesis of an engineered virus.

COVID-19 and comorbidities: A role for dipeptidyl peptidase 4 (DPP4) in disease severity?

The coronavirus disease 2019 (COVID-19) pandemic is caused by a novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), similar to SARS-CoV and Middle East respiratory syndrome (MERS-CoV), which cause acute respiratory distress syndrome and case fatalities. COVID-19 disease severity is worse in older obese patients with comorbidities such as diabetes, hypertension, cardiovascular disease, and chronic lung disease. Cell binding and entry of betacoronaviruses is via their surface spike glycoprotein; SARS-CoV binds to the metalloprotease angiotensin-converting enzyme 2 (ACE2), MERS-CoV utilizes dipeptidyl peptidase 4 (DPP4), and recent modeling of the structure of SARS-CoV-2 spike glycoprotein predicts that it can interact with human DPP4 in addition to ACE2. DPP4 is a ubiquitous membrane-bound aminopeptidase that circulates in plasma; it is multifunctional with roles in nutrition, metabolism, and immune and endocrine systems. DPP4 activity differentially regulates glucose homeostasis and inflammation via its enzymatic activity and nonenzymatic immunomodulatory effects. The importance of DPP4 for the medical community has been highlighted by the approval of DPP4 inhibitors, or gliptins, for the treatment of type 2 diabetes mellitus. This review discusses the dysregulation of DPP4 in COVID-19 comorbid conditions; DPP4 activity is higher in older individuals and increased plasma DPP4 is a predictor of the onset of metabolic syndrome. DPP4 upregulation may be a determinant of COVID-19 disease severity, which creates interest regarding the use of gliptins in management of COVID-19. Also, knowledge of the chemistry and biology of DPP4 could be utilized to develop novel therapies to block viral entry of some betacoronaviruses, potentially including SARS-CoV-2.

Structural characterization of the HCoV-229E fusion core

HCoV-229E spike (S) protein mediates virion attachment to cells and subsequent fusion of the viral and cellular membranes. This protein is composed of an N-terminal receptor-binding domain (S1) and a C-terminal trans-membrane fusion domain (S2). S2 contains a highly conserved heptad repeat 1 and 2 (HR1 and HR2). In this study, the HRs sequences were designed and connected with a flexible linker. The recombinant fusion core protein was crystallized and its structure was solved at a resolution of 2.45 Å. Then we characterized the binding of HR1s and HR2s via both sequence alignment and structural analysis. The overall structures, especially the residues in some positions of HR2 are highly conserved. Fourteen hydrophobic and three polar residues from each HR1 peptide are packed in layers at the coiled-coil interface. These core amino acids can be grouped into seven heptad repeats. Analysis of hydrophobic and hydrophilic interactions between HR2 helix and HR1 helices, shows that the HR1 and HR2 polypeptides are highly complementary in both shape and chemical properties. Furthermore, the available knowledge concerning HCoV-229E fusion core may make it possible to design small molecule or polypeptide drugs targeting membrane fusion, a crucial step of HCoV-229E infection.

The VP1u Receptor Restricts Parvovirus B19 Uptake to Permissive Erythroid Cells.

Parvovirus B19 (B19V) is a small non-enveloped virus and known as the causative agent for the mild childhood disease erythema infectiosum. B19V has an extraordinary narrow tissue tropism, showing only productive infection in erythroid precursor cells in the bone marrow. We recently found that the viral protein 1 unique region (VP1u) contains an N-terminal receptor-binding domain (RBD), which mediates the uptake of the virus into cells of the erythroid lineage. To further investigate the role of the RBD in connection with a B19V-unrelated capsid, we chemically coupled the VP1u of B19V to the bacteriophage MS2 capsid and tested the internalization capacity of the bioconjugate on permissive cells. In comparison, we studied the cellular uptake and infection of B19V along the erythroid differentiation. The results showed that the MS2-VP1u bioconjugate mimicked the specific internalization of the native B19V into erythroid precursor cells, which further coincides with the restricted infection profile. The successful mimicry of B19V uptake demonstrates that the RBD in the VP1u is sufficient for the endocytosis of the viral capsid. Furthermore, the recombinant VP1u competed with B19V uptake into permissive cells, thus excluding a significant alternative uptake mechanism by other receptors. Strikingly, the VP1u receptor appeared to be expressed only on erythropoietin-dependent erythroid differentiation stages that also provide the necessary intracellular factors for a productive infection. Taken together, these findings suggest that the VP1u binds to a yet-unknown erythroid-specific cellular receptor and thus restricts the virus entry to permissive cells.

Cognitive Deficits and Disruption of Neurogenesis in a Mouse Model of Apolipoprotein E4 Domain Interaction

Apolipoprotein E4 (apoE4) allele is the major genetic risk factor for sporadic Alzheimer disease (AD) due to the higher prevalence and earlier onset of AD in apoE4 carriers. Accumulating data suggest that the interaction between the N- and the C-terminal domains in the protein may be the main pathologic feature of apoE4. To test this hypothesis, we used Arg-61 mice, a model of apoE4 domain interaction, by introducing the domain interaction feature of human apoE4 into native mouse apoE. We carried out hippocampus-dependent learning and memory tests and related cellular and molecular assays on 12- and 3-month-old Arg-61 and age-matched background C57BL/6J mice. Learning and memory task performance were impaired in Arg-61 mice at both old and young ages compared with C57BL/6J mice. Surprisingly, young Arg-61 mice had more mitotic doublecortin-positive cells in the subgranular zone; mRNA levels of brain-derived neurotrophic factor (BDNF) and TrkB were also higher in 3-month-old Arg-61 hippocampus compared with C57BL/6J mice. These early-age neurotrophic and neurogenic (proliferative) effects in the Arg-61 mouse may be an inadequate compensatory but eventually detrimental attempt by the system to "repair" itself. This is supported by the higher cleaved caspase-3 levels in the young animals that not only persisted, but increased in old age, and the lower levels of doublecortin at old age in the hippocampus of Arg-61 mice. These results are consistent with human apoE4-dependent cognitive and neuro-pathologic changes, supporting the principal role of domain interaction in the pathologic effect of apoE4. Domain interaction is, therefore, a viable therapeutic/prophylactic target for cognitive impairment and AD in apoE4 subjects.

The soluble pyocins S2 and S4 from Pseudomonas aeruginosa bind to the same FpvAI receptor

Soluble (S-type) pyocins are Pseudomonas aeruginosa bacteriocins that kill nonimmune P. aeruginosa cells by gaining entry via a specific receptor, which, in the case of pyocin S2, is the siderophore pyoverdine receptor FpvAI, and in the case of pyocin S3, FpvAII. The nucleic acid sequence at the positions 4327697–4327359 of P. aeruginosa PAO1 genome was not annotated, but it was predicted to encode the immunity gene of the flanking pyocin S4 gene (PA3866) based on our analysis of the genome sequence. Using RT-PCR, the expression of the immunity gene was detected, confirming the existence of an immunity gene overlapping the S4 pyocin gene. The PA3866 coding for pyocin S4 and the downstream gene coding for the immunity protein were cloned and expressed in Escherichia coli and the His-tagged S4 pyocin was obtained in pure form. Forty-three P. aeruginosa strains were typed via PCR to identify their ferripyoverdine receptor gene (fpvAI–III) and were tested for their sensitivity to pyocin S4. All S4-sensitive strains had the type I ferripyoverdine receptor fpvA gene. Some S4-resistant type I fpvA-positive strains were detected, but all of them had the S4 immunity gene, and, following the deletion of the immunity gene, became S4-sensitive. The fpvAI receptor gene was deleted in a S4-sensitive strain, and, as expected, the mutant became resistant to S4. The N-terminal receptor binding domain (RBD) of pyocin S2, which also uses the FpvAI receptor to enter the cell, was cloned in the pET-15b vector, and expressed in E. coli. When the purified RBD was mixed with pyocin S4 at different ratios, an inhibition of killing was observed, indicating that S2 RBD competes with the pyocin S4 for the binding to the FpvAI receptor. The S2 RBD was also shown to enhance the expression of the pvdA pyoverdine gene, suggesting that it, like pyoverdine, works via the known siderophore-mediated signalization pathway.

Antibodies to the Envelope Glycoprotein of Human T Cell Leukemia Virus Type 1 Robustly Activate Cell-Mediated Cytotoxic Responses and Directly Neutralize Viral Infectivity at Multiple Steps of the Entry Process

Infection of human cells by human T cell leukemia virus type 1 (HTLV-1) is mediated by the viral envelope glycoproteins. The gp46 surface glycoprotein binds to cell surface receptors, including heparan sulfate proteoglycans, neuropilin 1, and glucose transporter 1, allowing the transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. The envelope glycoproteins are recognized by neutralizing Abs and CTL following a protective immune response, and therefore, represent attractive components for a HTLV-1 vaccine. To begin to explore the immunological properties of potential envelope-based subunit vaccine candidates, we have used a soluble recombinant surface glycoprotein (gp46, SU) fused to the Fc region of human IgG (sRgp46-Fc) as an immunogen to vaccinate mice. The recombinant SU protein is highly immunogenic and induces high titer Ab responses, facilitating selection of hybridomas that secrete mAbs targeting SU. Many of these mAbs recognize envelope displayed on the surface of HTLV-1-infected cells and virions and several of the mAbs robustly antagonize envelope-mediated membrane fusion and neutralize pseudovirus infectivity. The most potently neutralizing mAbs recognize the N-terminal receptor-binding domain of SU, though there is considerable variation in neutralizing proficiency of the receptor-binding domain-targeted mAbs. By contrast, Abs targeting the C-terminal domain of SU tend to lack robust neutralizing activity. Importantly, we find that both neutralizing and poorly neutralizing Abs strongly stimulate neutrophil-mediated cytotoxic responses to HTLV-1-infected cells. Our data demonstrate that recombinant forms of SU possess immunological features that are of significant utility to subunit vaccine design.

Robust neutralising activity and activation of neutrophil cytotoxic responses mediated by antibodies targeting the HTLV-1 envelope glycoprotein

Infection of human cells by Human T-cell leukaemia virus (HTLV-1) is mediated by the viral envelope glycoproteins. The gp46 surface glycoprotein binds to a cell surface receptors, including heparan sulphate proteoglycans, neuropilin-1, and Glut-1, allowing the transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. The envelope glycoproteins are recognised by neutralising antibodies and cytotoxic T lymphocytes following a protective immune response, and therefore represent attractive components for an HTLV-1 vaccine. Here we begin to explore the immunological properties of recombinant SU as a potential subunit vaccine candidate. We have used a soluble recombinant gp46 fused to the Fc-region of human IgG (sRgp46-Fc) as an immunogen to vaccinate mice and generate monoclonal antibodies (mAbs) targeting SU. The recombinant SU protein is highly immunogenic, inducing high titre antibody responses to the immunogen and facilitating selection of hybridomas that secrete anti-SU mAbs. Many of these mAbs recognise envelope expressed by HTLV-1 infected cells and these antibodies strongly stimulate neutrophil-mediated cytotoxic responses. Moreover, several of the mAbs directly and robustly antagonise envelope-mediated fusion and neutralize pseudovirus infectivity. While antibodies targeting either the receptor-binding domain or C-terminal domain of SU exhibit anti-viral activity, the most potently neutralizing mAbs tend to recognise epitopes that cluster in the N-terminal receptor-binding domain of SU. Thus, our data demonstrate that recombinant forms of SU possess immunological features that are of direct utility to subunit vaccine design.

The C-Terminal α-Helix Domain of Apolipoprotein E Is Required for Interaction with Nonstructural Protein 5A and Assembly of Hepatitis C Virus

We have recently demonstrated that human apolipoprotein E (apoE) is required for the infectivity and assembly of hepatitis C virus (HCV) (K. S. Chang, J. Jiang, Z. Cai, and G. Luo, J. Virol. 81:13783-13793, 2007; J. Jiang and G. Luo, J. Virol. 83:12680-12691, 2009). In the present study, we have determined the molecular basis underlying the importance of apoE in HCV assembly. Results derived from mammalian two-hybrid studies demonstrate a specific interaction between apoE and HCV nonstructural protein 5A (NS5A). The C-terminal third of apoE per se is sufficient for interaction with NS5A. Progressive deletion mutagenesis analysis identified that the C-terminal α-helix domain of apoE is important for NS5A binding. The N-terminal receptor-binding domain and the C-terminal 20 amino acids of apoE are dispensable for the apoE-NS5A interaction. The NS5A-binding domain of apoE was mapped to the middle of the C-terminal α-helix domain between amino acids 205 and 280. Likewise, deletion mutations disrupting the apoE-NS5A interaction resulted in blockade of HCV production. These findings demonstrate that the specific apoE-NS5A interaction is required for assembly of infectious HCV. Additionally, we have determined that using different major isoforms of apoE (E2, E3, and E4) made no significant difference in the apoE-NS5A interaction. Likewise, these three major isoforms of apoE are equally compatible with infectivity and assembly of infectious HCV, suggesting that apoE isoforms do not differentially modulate the infectivity and/or assembly of HCV in cell culture.

Core Structure of S2 from the Human Coronavirus NL63 Spike Glycoprotein,

Human coronavirus NL63 (HCoV-NL63) has recently been identified as a causative agent of acute respiratory tract illnesses in infants and young children. The HCoV-NL63 spike (S) protein mediates virion attachment to cells and subsequent fusion of the viral and cellular membranes. This viral entry process is a primary target for vaccine and drug development. HCoV-NL63 S is expressed as a single-chain glycoprotein and consists of an N-terminal receptor-binding domain (S1) and a C-terminal transmembrane fusion domain (S2). The latter contains two highly conserved heptad-repeat (HR) sequences that are each extended by 14 amino acids relative to those of the SARS coronavirus or the prototypic murine coronavirus, mouse hepatitis virus. Limited proteolysis studies of the HCoV-NL63 S2 fusion core identify an alpha-helical domain composed of a trimer of the HR segments N57 and C42. The crystal structure of this complex reveals three C42 helices entwined in an oblique and antiparallel manner around a central triple-stranded coiled coil formed by three N57 helices. The overall geometry comprises distinctive high-affinity conformations of interacting cross-sectional layers of the six helices. As a result, this structure is unusually stable, with an apparent melting temperature of 78 degrees C in the presence of the denaturant guanidine hydrochloride at 5 M concentration. The extended HR regions may therefore be required to prime the group 1 S glycoproteins for their fusion-activating conformational changes during viral entry. Our results provide an initial basis for understanding an intriguing interplay between the presence or absence of proteolytic maturation among the coronavirus groups and the membrane fusion activity of their S glycoproteins. This study also suggests a potential strategy for the development of improved HCoV-NL63 fusion inhibitors.

N-terminal truncation enables crystallization of the receptor-binding domain of the FedF bacterial adhesin.

FedF is the two-domain tip adhesin of F18 fimbriae from enterotoxigenic Escherichia coli. Bacterial adherence, mediated by the N-terminal receptor-binding domain of FedF to carbohydrate receptors on intestinal microvilli, causes diarrhoea and oedema disease in newly weaned piglets and induces the secretion of Shiga toxins. A truncate containing only the receptor-binding domain of FedF was found to be further cleaved at its N-terminus. Reconstruction of this N-terminal truncate rendered FedF amenable to crystallization, resulting in crystals with space group P2(1)2(1)2(1) and unit-cell parameters a = 36.20, b = 74.64, c = 99.03 A that diffracted to beyond 2 A resolution. The binding specificity of FedF was screened for on a glycan array, exposing 264 glycoconjugates, to identify specific receptors for cocrystallization with FedF.