Host Receptor Recognition Research Articles

Molecular mechanism and structure-guided humanization of a broadly neutralizing antibody against SFTSV.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel tick-borne bunyavirus that causes severe fever with thrombocytopenia syndrome (SFTS), with a high mortality rate of up to 30%. The envelope glycoproteins of SFTSV, glycoprotein N (Gn) and glycoprotein C (Gc), facilitate the recognition of host receptors and the process of membrane fusion, allowing the virus to enter host cells. We previously reported a monoclonal antibody, mAb 40C10, capable of neutralizing different genotypes of SFTSV and SFTSV-related viruses. However, the specific neutralization mechanism is poorly understood. In this study, we elucidated the high-resolution structure of the SFTSV Gn head domain in complex with mAb 40C10, confirming that the binding epitope in the domain I region of SFTSV Gn, and it represented that a novel binding epitope of SFTSV Gn was identified. Through in-depth structural and sequence analyses, we found that the binding sites of mAb 40C10 are relatively conserved among different genotypes of SFTSV and SFTSV-related Heartland virus and Guertu virus, elucidating the molecular mechanism underlying the broad-spectrum neutralizing activity of mAb 40C10. Furthermore, we humanized of mAb 40C10, which is originally of murine origin, to reduce its immunogenicity. The resulting nine humanized antibodies maintained potent affinity and neutralizing activity. One of the humanized antibodies exhibited neutralizing activity at picomolar IC50 values and demonstrated effective therapeutic and protective effects in a mouse infection model. These findings provide a novel target for the future development of SFTSV vaccines or drugs and establish a foundation for the research and development of antibody therapeutics for clinical applications.

The long and sinuous road to phage-based therapy of Clostridioides difficile infections.

With the antibiotic crisis and the rise in antimicrobial resistance worldwide, new therapeutic alternatives are urgently needed. Phage therapy represents one of the most promising alternatives but for some pathogens, such as Clostridioides difficile, important challenges are being faced. The perspective of phage therapy to treat C. difficile infections is complicated by the fact that no strictly lytic phages have been identified so far, and current temperate phages generally have a narrow host range. C. difficile also harbors multiple antiphage mechanisms, and the bacterial genome is often a host of one or multiple prophages that can interfere with lytic phage infection. Nevertheless, due to recent advances in phage host receptor recognition and improvements in genetic tools to manipulate phage genomes, it is now conceivable to genetically engineer C. difficile phages to make them suitable for phage therapy. Other phage-based alternatives such as phage endolysins and phage tail-like bacteriocins (avidocins) are also being investigated but these approaches also have their own limitations and challenges. Last but not least, C. difficile produces spores that are resistant to phage attacks and all current antibiotics, and this complicates therapeutic interventions. This mini-review gives a brief historical overview of phage work that has been carried out in C. difficile, presents recent advances in the field, and addresses the most important challenges that are being faced, with potential solutions.

Omicron Spike Protein Has a Positive Electrostatic Surface That Promotes ACE2 Recognition and Antibody Escape

High transmissibility is a hallmark of the Omicron variant of SARS-CoV-2. Understanding the molecular determinants of Omicron’s transmissibility will impact development of intervention strategies. Here we map the electrostatic potential surface of the Spike protein to show that major SARS-CoV-2 variants have accumulated positive charges in solvent-exposed regions of the Spike protein, especially its ACE2-binding interface. Significantly, the Omicron Spike-ACE2 complex has complementary electrostatic surfaces. In contrast, interfaces between Omicron and neutralizing antibodies tend to have similar positively charged surfaces. Structural modeling demonstrates that the electrostatic property of Omicron’s Spike receptor binding domain (S RBD) plays a role in enhancing ACE2 recognition and destabilizing Spike-antibody complexes. Specifically, the Omicron S RBD has favorable electrostatic interaction energy with ACE2 that is 3-5 times greater than the Delta variant over a range of 20 Å, implying efficient recognition of host receptors. Computed binding affinities of six representative S RBD-antibody complexes show that Omicron can escape most antibodies targeting the ACE2-binding region of S RBD. Interestingly, a straightforward assessment of the electrostatic surfaces of 18 neutralizing antibodies correctly predicted the Omicron escape status of 80% of cases. Collectively, our structural analysis implies that Omicron S RBD interaction interfaces have been optimized to simultaneously promote access to human ACE2 receptors and evade antibodies. These findings suggest that electrostatic interactions are a major contributing factor for increased Omicron transmissibility relative to other variants.

HiSpike Method for High-Throughput Cost Effective Sequencing of the SARS-CoV-2 Spike Gene.

The changing nature of the SARS-CoV-2 pandemic poses unprecedented challenges to the world's health systems. Emerging spike gene variants jeopardize global efforts to produce immunity and reduce morbidity and mortality. These challenges require effective real-time genomic surveillance solutions that the medical community can quickly adopt. The SARS-CoV-2 spike protein mediates host receptor recognition and entry into the cell and is susceptible to generation of variants with increased transmissibility and pathogenicity. The spike protein is the primary target of neutralizing antibodies in COVID-19 patients and the most common antigen for induction of effective vaccine immunity. Tight monitoring of spike protein gene variants is key to mitigating COVID-19 spread and generation of vaccine escape mutants. Currently, SARS-CoV-2 sequencing methods are labor intensive and expensive. When sequence demands are high sequencing resources are quickly exhausted. Consequently, most SARS-CoV-2 strains are sequenced in only a few developed countries and rarely in developing regions. This poses the risk that undetected, dangerous variants will emerge. In this work, we present HiSpike, a method for high-throughput cost effective targeted next generation sequencing of the spike gene. This simple three-step method can be completed in < 30 h, can sequence 10-fold more samples compared to conventional methods and at a fraction of their cost. HiSpike has been validated in Israel, and has identified multiple spike variants from real-time field samples including Alpha, Beta, Delta and the emerging Omicron variants. HiSpike provides affordable sequencing options to help laboratories conserve resources for widespread high-throughput, near real-time monitoring of spike gene variants.

SARS-CoV-2 spike engagement of ACE2 primes S2′ site cleavage and fusion initiation

Significance The SARS-CoV-2 spike protein is responsible for host receptor recognition, membrane fusion, and viral infection. Understanding the cellular and inhibiting the molecular mechanisms of spike-driven viral entry is a research priority in curbing the ongoing pandemic and preventing future coronavirus outbreaks. Here, we highlight that the generation of SARS-CoV-2 S2′ fragments, a proteolytic event occurring within the S2 subunit, is a molecular switch coupled to membrane fusion. Downstream of host receptor recognition, spike-driven syncytia formation requires the presence of an S2′ cleavage site at arginine 815 but not 685. Hence, the proteolytic processing of spike at the S2′ site upon its engagement of host ACE2 may serve as a potential antiviral target against the current SARS-CoV-2 and related coronavirus strains.

Real-time SARS-CoV-2 diagnostic and variants tracking over multiple candidates using nanopore DNA sequencing

Since December 2019, a novel coronavirus responsible for a severe acute respiratory syndrome (SARS-CoV-2) is accountable for a major pandemic situation. The emergence of the B.1.1.7 strain, as a highly transmissible variant has accelerated the world-wide interest in tracking SARS-CoV-2 variants’ occurrence. Similarly, other extremely infectious variants, were described and further others are expected to be discovered due to the long period of time on which the pandemic situation is lasting. All described SARS-CoV-2 variants present several mutations within the gene encoding the Spike protein, involved in host receptor recognition and entry into the cell. Hence, instead of sequencing the whole viral genome for variants’ tracking, herein we propose to focus on the SPIKE region to increase the number of candidate samples to screen at once; an essential aspect to accelerate diagnostics, but also variants’ emergence/progression surveillance. This proof of concept study accomplishes both at once, population-scale diagnostics and variants' tracking. This strategy relies on (1) the use of the portable MinION DNA sequencer; (2) a DNA barcoding and a SPIKE gene-centered variant’s tracking, increasing the number of candidates per assay; and (3) a real-time diagnostics and variant’s tracking monitoring thanks to our software RETIVAD. This strategy represents an optimal solution for addressing the current needs on SARS-CoV-2 progression surveillance, notably due to its affordable implementation, allowing its implantation even in remote places over the world.

Molecular basis for higher affinity of SARS‐CoV‐2 spike RBD for human ACE2 receptor

Severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) has caused substantially more infections, deaths, and economic disruptions than the 2002‐2003 SARS‐CoV. The key to understanding SARS‐CoV‐2's higher infectivity lies partly in its host receptor recognition mechanism. Experiments show that the human angiotensin converting enzyme 2 (ACE2) protein, which serves as the primary receptor for both CoVs, binds to the receptor binding domain (RBD) of CoV‐2's spike protein stronger than SARS‐CoV's spike RBD. The molecular basis for this difference in binding affinity, however, remains unexplained from X‐ray structures. To go beyond insights gained from X‐ray structures and investigate the role of thermal fluctuations in structure, we employ all‐atom molecular dynamics simulations. Microseconds‐long simulations reveal that while CoV and CoV‐2 spike‐ACE2 interfaces have similar conformational binding modes, CoV‐2 spike interacts with ACE2 via a larger combinatorics of polar contacts, and on average, makes 45% more polar contacts. Correlation analysis and thermodynamic calculations indicate that these differences in the density and dynamics of polar contacts arise from differences in spatial arrangements of interfacial residues, and dynamical coupling between interfacial and non‐interfacial residues. These results recommend that ongoing efforts to design spike‐ACE2 peptide blockers will benefit from incorporating dynamical information as well as allosteric coupling effects.

Targeting SARS-CoV-2 RBD Interface: a Supervised Computational Data-Driven Approach to Identify Potential Modulators.

Coronavirus disease 2019 (COVID‐19) has spread out as a pandemic threat affecting over 2 million people. The infectious process initiates via binding of SARS‐CoV‐2 Spike (S) glycoprotein to host angiotensin‐converting enzyme 2 (ACE2). The interaction is mediated by the receptor‐binding domain (RBD) of S glycoprotein, promoting host receptor recognition and binding to ACE2 peptidase domain (PD), thus representing a promising target for therapeutic intervention. Herein, we present a computational study aimed at identifying small molecules potentially able to target RBD. Although targeting PPI remains a challenge in drug discovery, our investigation highlights that interaction between SARS‐CoV‐2 RBD and ACE2 PD might be prone to small molecule modulation, due to the hydrophilic nature of the bi‐molecular recognition process and the presence of druggable hot spots. The fundamental objective is to identify, and provide to the international scientific community, hit molecules potentially suitable to enter the drug discovery process, preclinical validation and development.

Evolutionary relationships and sequence-structure determinants in human SARS coronavirus-2 spike proteins for host receptor recognition.

Coronavirus disease 2019 (COVID‐19) is a pandemic infectious disease caused by novel severe acute respiratory syndrome coronavirus‐2 (SARS CoV‐2). The SARS CoV‐2 is transmitted more rapidly and readily than SARS CoV. Both, SARS CoV and SARS CoV‐2 via their glycosylated spike proteins recognize the human angiotensin converting enzyme‐2 (ACE‐2) receptor. We generated multiple sequence alignments and phylogenetic trees for representative spike proteins of SARS CoV and SARS CoV‐2 from various host sources in order to analyze the specificity in SARS CoV‐2 spike proteins required for causing infection in humans. Our results show that among the genomes analyzed, two sequence regions in the N‐terminal domain “MESEFR” and “SYLTPG” are specific to human SARS CoV‐2. In the receptor‐binding domain, two sequence regions “VGGNY“ and ”EIYQAGSTPCNGV” and a disulfide bridge connecting 480C and 488C in the extended loop are structural determinants for the recognition of human ACE‐2 receptor. The complete genome analysis of representative SARS CoVs from bat, civet, human host sources, and human SARS CoV‐2 identified the bat genome (GenBank code: MN996532.1) as closest to the recent novel human SARS CoV‐2 genomes. The bat SARS CoV genomes (GenBank codes: MG772933 and MG772934) are evolutionary intermediates in the mutagenesis progression toward becoming human SARS CoV‐2.

Study of combining virtual screening and antiviral treatments of the Sars-CoV-2 (Covid-19)

The recent epidemic outbreak of a novel human coronavirus called SARS-CoV-2 and causing the respiratory tract disease COVID-19 has reached worldwide resonance and a global effort is being undertaken to characterize the molecular features and evolutionary origins of this virus. Therefore, rapid and accurate identification of pathogenic viruses plays a vital role in selecting appropriate treatments, saving people's lives and preventing epidemics. Additionally, general treatments, coronavirus-specific treatments, and antiviral treatments useful in fighting COVID-19 are addressed. This review sets out to shed light on the SARS-CoV-2 and host receptor recognition, a crucial factor for successful virus infection and taking immune-informatics approaches to identify B- and T-cell epitopes for surface glycoprotein of SARS-CoV-2. A variety of improved or new approaches also have been developed. It is anticipated that this will assist researchers and clinicians in developing better techniques for timely and effective detection of coronavirus infection. Moreover, the genomic sequence of the virus responsible for COVID-19, as well as the experimentally determined three-dimensional structure of the Main protease (Mpro) is available. The reported structure of the target Mpro was described in this review to identify potential drugs for COVID-19 using virtual high throughput screening.

Design of a peptide-based subunit vaccine against novel coronavirus SARS-CoV-2

Coronavirus disease 2019 (COVID-19) is an emerging infectious disease that was first reported in Wuhan, China, and has subsequently spread worldwide. In the absence of any antiviral or immunomodulatory therapies, the disease is spreading at an alarming rate. A possibility of a resurgence of COVID-19 in places where lockdowns have already worked is also developing. Thus, for controlling COVID-19, vaccines may be a better option than drugs. An mRNA-based anti-COVID-19 candidate vaccine has entered a phase 1 clinical trial. However, its efficacy and potency have to be evaluated and validated. Since vaccines have high failure rates, as an alternative, we are presenting a new, designed multi-peptide subunit-based epitope vaccine against COVID-19. The recombinant vaccine construct comprises an adjuvant, cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes joined by linkers. The computational data suggest that the vaccine is non-toxic, non-allergenic, thermostable, with the capability to elicit a humoral and cell-mediated immune response. The stabilization of the vaccine construct is validated with molecular dynamics simulation studies. This unique vaccine is made up of 33 highly antigenic epitopes from three proteins that have a prominent role in host-receptor recognition, viral entry, and pathogenicity. We advocate this vaccine must be synthesized and tested urgently as a public health priority.

Master Regulator Analysis of the SARS-CoV-2/Human Interactome.

The recent epidemic outbreak of a novel human coronavirus called SARS-CoV-2 causing the respiratory tract disease COVID-19 has reached worldwide resonance and a global effort is being undertaken to characterize the molecular features and evolutionary origins of this virus. In this paper, we set out to shed light on the SARS-CoV-2/host receptor recognition, a crucial factor for successful virus infection. Based on the current knowledge of the interactome between SARS-CoV-2 and host cell proteins, we performed Master Regulator Analysis to detect which parts of the human interactome are most affected by the infection. We detected, amongst others, affected apoptotic and mitochondrial mechanisms, and a downregulation of the ACE2 protein receptor, notions that can be used to develop specific therapies against this new virus.

Pichia pastoris-Expressed Bivalent Virus-Like Particulate Vaccine Induces Domain III-Focused Bivalent Neutralizing Antibodies without Antibody-Dependent Enhancement in Vivo.

Dengue, a significant public health problem in several countries around the world, is caused by four different serotypes of mosquito-borne dengue viruses (DENV-1, -2, -3, and -4). Antibodies to any one DENV serotype which can protect against homotypic re-infection, do not offer heterotypic cross-protection. In fact, cross-reactive antibodies may augment heterotypic DENV infection through antibody-dependent enhancement (ADE). A recently launched live attenuated vaccine (LAV) for dengue, which consists of a mixture of four chimeric yellow-fever/dengue vaccine viruses, may be linked to the induction of disease-enhancing antibodies. This is likely related to viral interference among the replicating viral strains, resulting in an unbalanced immune response, as well as to the fact that the LAV encodes prM, a DENV protein documented to elicit ADE-mediating antibodies. This makes it imperative to explore the feasibility of alternate ADE risk-free vaccine candidates. Our quest for a non-replicating vaccine centered on the DENV envelope (E) protein which mediates virus entry into the host cell and serves as an important target of the immune response. Serotype-specific neutralizing epitopes and the host receptor recognition function map to E domain III (EDIII). Recently, we found that Pichia pastoris-expressed DENV E protein, of all four serotypes, self-assembled into virus-like particles (VLPs) in the absence of prM. Significantly, these VLPs displayed EDIII and elicited EDIII-focused DENV-neutralizing antibodies in mice. We now report the creation and characterization of a novel non-replicating recombinant particulate vaccine candidate, produced by co-expressing the E proteins of DENV-1 and DENV-2 in P. pastoris. The two E proteins co-assembled into bivalent mosaic VLPs (mVLPs) designated as mE1E2bv VLPs. The mVLP, which preserved the serotype-specific antigenic integrity of its two component proteins, elicited predominantly EDIII-focused homotypic virus-neutralizing antibodies in BALB/c mice, demonstrating its efficacy. In an in vivo ADE model, mE1E2bv VLP-induced antibodies lacked discernible ADE potential, compared to the cross-reactive monoclonal antibody 4G2, as evidenced by significant reduction in the levels of IL-6 and TNF-α, suggesting inherent safety. The results obtained with these bivalent mVLPs suggest the feasibility of incorporating the E proteins of DENV-3 and DENV-4 to create a tetravalent mVLP vaccine.

Protein Toxins That Utilize Gangliosides as Host Receptors.

Subsets of protein toxins utilize gangliosides as host receptors. Gangliosides are preferred receptors due to their extracellular localization on the eukaryotic cell and due to their essential nature in host physiology. Glycosphingolipids, including gangliosides, are mediators of signal transduction within and between eukaryotic cells. Protein toxins possess AB structure-function organization, where the A domain encodes a catalytic function for the posttranslational modification of a host macromolecule, including proteins and nucleic acids, and a B domain, which encodes host receptor recognition, including proteins and glycosphingolipids, alone or in combination. Protein toxins use similar strategies to bind glycans by pockets and loops, generally employing hydrogen bonding and aromatic stacking to stabilize interactions with sugars. In some cases, glycan binding facilitates uptake, while in other cases, cross-linking or a second receptor is necessary to stimulate entry. The affinity that protein toxins have for host glycans is necessary for tissue targeting, but not always sufficient to cause disease. In addition to affinity for binding the glycan, the lipid moiety also plays an important role in productive uptake and tissue tropism. Upon endocytosis, the protein toxin must escape to another intracellular compartment or into cytosol to modify a host substrate, modulating host signaling, often resulting in cytotoxic or apoptotic events in the cell, and a unique morbidity for the organism. The study of protein toxins that utilize gangliosides as host receptors has illuminated numerous eukaryotic cellular processes, identified the basis for developing interventions to prevent disease through vaccines and control bacterial diseases through therapies. In addition, subsets of these protein toxins have been utilized as therapeutic agents to treat numerous human inflictions.

Novel Fri1-like Viruses Infecting Acinetobacter baumannii-vB_AbaP_AS11 and vB_AbaP_AS12-Characterization, Comparative Genomic Analysis, and Host-Recognition Strategy.

Acinetobacter baumannii is a gram-negative, non-fermenting aerobic bacterium which is often associated with hospital-acquired infections and known for its ability to develop resistance to antibiotics, form biofilms, and survive for long periods in hospital environments. In this study, we present two novel viruses, vB_AbaP_AS11 and vB_AbaP_AS12, specifically infecting and lysing distinct multidrug-resistant clinical A. baumannii strains with K19 and K27 capsular polysaccharide structures, respectively. Both phages demonstrate rapid adsorption, short latent periods, and high burst sizes in one-step growth experiments. The AS11 and AS12 linear double-stranded DNA genomes of 41,642 base pairs (bp) and 41,402 bp share 86% nucleotide sequence identity with the most variable regions falling in host receptor–recognition genes. These genes encode tail spikes possessing depolymerizing activities towards corresponding capsular polysaccharides which are the primary bacterial receptors. We described AS11 and AS12 genome organization and discuss the possible regulation of transcription. The overall genomic architecture and gene homology analyses showed that the phages are new representatives of the recently designated Fri1virus genus of the Autographivirinae subfamily within the Podoviridae family.

Recombinant Dengue Virus 4 Envelope Glycoprotein Virus-Like Particles Derived from Pichia pastoris are Capable of Eliciting Homotypic Domain III-Directed Neutralizing Antibodies.

Dengue is a viral pandemic caused by four dengue virus serotypes (DENV-1, 2, 3, and 4) transmitted by Aedes mosquitoes. Reportedly, there has been a 2-fold increase in dengue cases every decade. An efficacious tetravalent vaccine, which can provide long-term immunity against all four serotypes in all target populations, is still unavailable. Despite the progress being made in the live virus-based dengue vaccines, the World Health Organization strongly recommends the development of alternative approaches for safe, affordable, and efficacious dengue vaccine candidates. We have explored virus-like particles (VLPs)-based nonreplicating subunit vaccine approach and have developed recombinant envelope ectodomains of DENV-1, 2, and 3 expressed in Pichia pastoris These self-assembled into VLPs without pre-membrane (prM) protein, which limits the generation of enhancing antibodies, and elicited type-specific neutralizing antibodies against the respective serotype. Encouraged by these results, we have extended this work further by developing P. pastoris-expressed DENV-4 ectodomain (DENV-4 E) in this study, which was found to be glycosylated and assembled into spherical VLPs without prM, and displayed critical neutralizing epitopes on its surface. These VLPs were found to be immunogenic in mice and elicited DENV-4-specific neutralizing antibodies, which were predominantly directed against envelope domain III, implicated in host-receptor recognition and virus entry. These observations underscore the potential of VLP-based nonreplicative vaccine approach as a means to develop a safe, efficacious, and tetravalent dengue subunit vaccine. This work paves the way for the evaluation of a DENV E-based tetravalent dengue vaccine candidate, as an alternative to live virus-based dengue vaccines.

Mutagenesis of Paramyxovirus Hemagglutinin-Neuraminidase Membrane-Proximal Stalk Region Influences Stability, Receptor Binding, and Neuraminidase Activity.

Paramyxoviridae consist of a large family of enveloped, negative-sense, nonsegmented single-stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN [also called H or G depending on virus type and substrate]), which acts as a receptor binding protein, and a fusion (F) protein, which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN 4-helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross-linking as well as functional assays to investigate the role of the structurally unresolved membrane-proximal stalk region (MPSR) (residues 37 to 58) of HN in the context of headless and full-length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F triggering. Paramyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F), to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane-anchored flexible stalk of ∼80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane-proximal stalk region (MPSR) (HN, residues 37 to 56) of the paramyxovirus parainfluenza virus (PIV5), a region of the HN stalk that has not had its structure determined by X-ray crystallography. Our data suggest that the MPSR influences receptor binding and neuraminidase activity via an indirect mechanism. Moreover, the receptor binding head group stabilizes the 4HB stalk as part of the general mechanism to fine-tune F-activation.

Characterization of a Novel Conformational GII.4 Norovirus Epitope: Implications for Norovirus-Host Interactions.

Human noroviruses (NoVs) are the main etiological agents of acute gastroenteritis worldwide. While NoVs are highly diverse (more than 30 genotypes have been detected in humans), during the last 40 years most outbreaks and epidemics have been caused by GII.4 genotype strains, raising questions about their persistence in the population. Among other potential explanations, immune evasion is considered to be a main driver of their success. In order to study antibody recognition and evasion in detail, we analyzed a conformational epitope recognized by a monoclonal antibody (3C3G3) by phage display, site-directed mutagenesis, and surface plasmon resonance. Our results show that the predicted epitope is composed of 11 amino acids within the P domain: P245, E247, I389, Q390, R397, R435, G443, Y444, P445, N446, and D448. Only two of them, R397 and D448, differ from the homologous variant (GII.4 Den-Haag_2006b) and from a previous variant (GII.4 VA387_1996) that is not recognized by the antibody. A double mutant derived from the VA387_1996 variant containing both changes, Q396R and N447D, is recognized by the 3C3G3 monoclonal antibody, confirming the participation of the two sites in the epitope recognized by the antibody. Furthermore, a single change, Q396R, is able to modify the histo-blood group antigen (HBGA) recognition pattern. These results provide evidence that the epitope recognized by the 3C3G3 antibody is involved in the virus-host interactions, both at the immunological and at the receptor levels. Human noroviruses are the main cause of viral diarrhea worldwide in people of all ages. Noroviruses can infect individuals who had been previously exposed to the same or different norovirus genotypes. Norovirus genotype GII.4 has been reported to be most prevalent during the last 40 years. In the present study, we describe a novel viral epitope identified by a monoclonal antibody and located within the highly diverse P domain of the capsid protein. The evolution of this epitope along with sequential GII.4 variants has allowed noroviruses to evade previously elicited antibodies, thus explaining how the GII.4 genotype can persist over long periods, reinfecting the population. Our results also show that the epitope participates in the recognition of host receptors that have evolved over time, as well.

Parallel Evolution of Chemokine Binding by Structurally Related Herpesvirus Decoy Receptors

A wide variety of pathogens targets chemokine signaling networks in order to disrupt host immune surveillance and defense. Here, we report a structural and mutational analysis of rodent herpesvirus Peru encoded R17, a potent chemokine inhibitor that sequesters CC and C chemokines with high affinity. R17 consists of a pair of β-sandwich domains linked together by a bridging sheet, which form an acidic binding cleft for the chemokine CCL3 on the opposite face of a basic surface cluster that binds glycosaminoglycans. R17 promiscuously engages chemokines primarily through the same N-loop determinants used for host receptor recognition while residues located in the chemokine 40s loop drive kinetically stable complex formation. The core fold adopted by R17 is unexpectedly similar to that of the M3 chemokine decoy receptor encoded by MHV-68, although, strikingly, neither the location of ligand engagement nor the stoichiometry of binding is conserved, suggesting that their functions evolved independently.

Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm.

We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.