Biology subjects

Khayat, R.

Publications and source records attributed to Khayat, R..

5 recordsLinked to original sources

bioRxiv2025

Understanding the infection mechanism of {Phi}8 using infection method in Pseudomonas syringae pv. Phaseolicola strain: In-situ CryoET analysis

The Cystoviridae family strain {Phi}8 is an ideal model for studying the virus-host interactions and viral assembly mechanisms. In this study, we explored {Phi}8 infection kinetics in P. syringae pv. phaseolicola host cells using a combination of host lysis assays, transmission electron microscopy (TEM), cryo-focused ion beam (Cryo-FIB) milling, and in-situ cryo-electron tomography (Cryo-ET). Purified {Phi}8 was infected with host cells and observed at different time points. The host-pathogen infection was detected using TEM studies. The infected sample underwent Cryo-FIB milling. Our findings indicated that the infected host cell membrane changes its shape during infection. We were able to visualize the various stages of {Phi}8 assembly using subtomogram averaging. These stages included the unpackaged procapsid (PC) having a size of [~]41 nm and the double-stranded RNA (dsRNA)-packaged procapsid having a size of [~]54 nm. The packaged PC represented the most prevalent intermediate stages during viral assembly. Notably, the majority of complete virions isolated from the host cell were found outside the host cell, revealing the extracellular predominance of fully constructed particles. These findings emphasize the importance of stable intermediate stages in viral assembly and maturation of {Phi}8. This work advances our understanding of viral assembly and complex host-cell interactions. HighlightsO_LISuccessfully isolated and infected Pseudomonas syringae pv. phaseolicola (Pph) host cells with {Phi}8. C_LIO_LILysis assay performed to quantify host lysis to evaluate viral infection efficiency. C_LIO_LICryo-focused ion beam milling enabled precise preparation of cellular cross-sections for structural analysis. This allows identification of key stages of the {Phi}8 life cycle during the infection cycle. C_LIO_LIThe majority of complete virions were localized extracellularly, emphasizing the role of extracellular stages in the {Phi}8 lifecycle. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/678632v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@6ba760org.highwire.dtl.DTLVardef@1b99d2dorg.highwire.dtl.DTLVardef@fdfe72org.highwire.dtl.DTLVardef@14c077e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

The structure of the Human Adenovirus 7 virus-like particles reveals that pentons and core-genome promote hexon-pIIIa interactions during capsid maturation

Members of the Adenoviridae family routinely infect humans, exhibit significant genetic diversity, and are associated with a variety of illnesses. Types 4 and 7 frequently circulate in the United States and are major causes of respiratory disease. Infections can result in hospitalization and, in severe cases, death. Although a live wild-type-virus vaccine targeting these two types exists, its use is restricted to military personnel due to concerns about viral-shedding and the potential for genetic recombination. To overcome these limitations, we recently developed a virus-like particle (VLP) platform as an alternative vaccination strategy. These VLPs are stable, lack genomic material, and elicit a potent humoral immune response in mice, effectively neutralizing adenoviral infection. Here, we describe the cryo-EM structure of these VLPs at near atomic resolution. Structural insights are essential to ensure that the neutralizing antigens displayed on the VLPs accurately mimic those of the infectious virion, guide the design of particles with improved stability and efficacy, and enable engineering of VLPs with antigenic properties targeting multiple adenovirus types. The structure confirms that the key epitopes capable of eliciting neutralizing antibodies are appropriately displayed for antibody recognition. It also reveals previously unobserved interactions critical for capsid maturation. The penton base adopts multiple conformations when interacting with hexons, and its proper positioning is necessary to facilitate hexon-pIIIa interactions that stabilize the capsid. A comparison of the VLP with immature and mature adenovirus structures shows that core-genome packaging strengthens interaction between cementing proteins pIIIa and pVIII and the hexon shell. Significance StatementAdenoviruses are large and morphologically complex family of viruses that undergo a sophisticated process of assembly and maturation. These viruses are responsible for ocular, respiratory, and enteric infections, posing a particular risk to children and the immunocompromised. Despite their clinical significance, there are no adenovirus vaccines available to the public. Here we report the cryo-electron microscopy structure of human adenovirus 7 virus-like particles, which have shown strong potential as a vaccine candidate. Our analysis provides critical insights for the design of particles as vaccines or carriers of genetic therapeutics. The structure reveals how interaction between hexon and penton promote cement protein pIIIa for increased capsid interaction, and how core-genome packaging contributes to capsid maturation and enhanced capsid stability.

biophysics↗

bioRxiv2024

Bacteriophages Phi 8 and Phi 12 host infection are inhibited by OMVs and LPS purified from P. pseudoalcaligenes strain: East River Isolate A

Cystoviridae is a family of double stranded RNA (dsRNA) phage that infects various strains of Pseudomonas syringae, a Gram-negative soil bacteria known to infect various crops. Surrounding the icosahedral capsids of these phages is a bacterial derived phospholipid membrane. Embedded within this membrane is a multi-component protein complex, referred to as the spike complex. The spike complex is responsible for host recognition and membrane fusion. We studied the ability of two members of the Cystivirdae family to infect cells in the presence of purified outer membrane vesicles (OMVs) and lipopolysaccharide (LPS) derived from distinct sources. In this study we determined that OMVs from the host Pseudomonas pseudoalcaligenes strain: East River isolate A (ERA) inhibit Phi 8 and Phi 12 host infection. These OMVs range in size from 30 to 60 nm and bind to Phi 8 and Phi 12. However, OMV purified from P. syringae pv. phaseolicola LM2691 and E. coli {Delta}yciB {Delta}dcrB did not inhibit Phi 8 or Phi 12 host infection. However, LPS derived from ERA and LM2691 inhibited Phi 8 and Phi 12 infection, demonstrating that LPS is the receptor for these two viruses, and that OMV biogenesis is selective of LPS. LPS derived from other non-Cystoviridae Gram-negative bacteria, did not inhibit infection. We confirmed that host proteins are not required for Phi 8 or Phi 12 host interaction. Our results also suggest that differences in lipid A and the core polysaccharide in LPS may influence Phi 8 and Phi 12 host binding. IMPORTANCEMost phage families studied to date use a tailed appendage, composed of a multitude of proteins, for cellular recognition, membrane penetration, and genome injection. This contrasts with members of the Cystoviridae family which possess a phospholipid membrane bilayer with embedded proteins responsible for cellular recognition and membrane fusion. Thus, the Cystoviridae are akin to enveloped viruses which also use protein complexes embedded into their membrane for cellular recognition and membrane fusion. Examples of such viruses include the Retroviridae, Coronoviridae, Herpesviridae, and Orthomyxoviridae families. The binding specifics of Cystoviridae to the host outer membrane are unknown. Using Cystoviridae-OMV interaction we began to uncover the host requirements for binding Cystoviridae. The results presented determine that only lipid A and the core polysaccharide of LPS are required for Cystoviridae outer membrane binding.

microbiology↗

bioRxiv2019

Structural characterization of the PCV2d genotype at 3.3 A resolution reveals differences to PCV2a and PCV2b genotypes, a tetranucleotide, and an N-terminus near the icosahedral 3-fold axes

Porcine circovirus 2 (PCV2) is a T=1 non-enveloped icosahedral virus that has a major impact on the swine industry as an agent of porcine circovirus associate disease. PCV2 capsid protein sequences have been employed by others to provide a temporal description of the emerging genotypes. PCV2a is believed to be the earliest genotype and responsible for giving rise to PCV2b, which gives rise to PCV2d. The underlying mechanism responsible for the emerging genotypes is not understood. To determine if a change in the PCV2d capsid accompanies the emergence of this genotype, we determined the cryo-electron microscopy image reconstruction of PCV2d VLP at 3.3 [A] resolution and compared it to the previously reported PCV2a and PCV2b structures. Differences between the CD and GH loops identify structural changes that accompany the emergence of PCV2b from PCV2a, and PCV2d from PCV2b. We also model additional amino acids for the N-terminus near the icosahedral 3-fold axes of symmetry and a tetranucleotide between the 5- and 2-fold axes of symmetry. To interpret the sequence diversity that defines the PCV2 genotypes on a structural platform we have performed structure-based sequence comparison. Our analysis demonstrates that each genotype possesses a unique set of amino acids located on the surface of the capsid that experience a high degree of substitution. These substitutions may be a response to the PCV2 vaccination program. The structural difference between PCV2a, b and d genotypes indicate that it is important to determine the PCV2 capsid structure as the virus evolves into different genotypes.\n\nImportancePCV2 is a significant epidemic agricultural pathogen that is the causative agent of a variety of swine illnesses. PCV2 infections have significant economic impact in the swine industry and must be controlled by vaccination. Outbreaks in farms vaccinated for PCV2 suggest that improvements to the current vaccination programs are needed. Better understanding of the assembly, structure, replication and evolution of these viruses is necessary for production of improved vaccines. The ability of PCV2 to rapidly shift genotypes suggests that expression systems capable of rapidly producing large quantities of virus-like particles should be pursued. To these ends we have established a mammalian cell-based virus-like particle expression system and performed high resolution structural studies of a new PCV2 genotype. Differences between the structure of this genotype and earlier genotypes demonstrate that it is important to study the PCV2 structure as it shifts genotypes.

biophysics↗

bioRxiv2018

Porcine circovirus 2 particles use distinct binding modes for cellular attachment

Porcine circovirus 2 is the smallest pathogenic virus capable of autonomous replication within its host. Infections result in immunosuppression and subsequent death of the host, and are initiated via the attachment of the PCV2 icosahedral capsid to heparan sulfate and chondroitin sulfate B glycosaminoglycans on the cell surface. However, the underlying mechanism of structural recognition remains to be explored. Using heparin, a routinely used analog of heparan sulfate, we demonstrate that increasing lengths of heparin exhibit greater affinity towards PCV2. Our competition assays indicate that dextra sulfate (8kDa) has higher affinity than heparin (12kDa), chondroitin sulfate B (41kDa) hyaluronic acid (1.6MDa), and dextran (6kDa) for PCV2. This suggests that polymers high in sulfate content are capable of competing with the PCV2-heparan sulfate interaction, and thus have the potential to inhibit PCV2 infection. Finally, we visualize the interaction between heparin and the PCV2 capsid using cryo-electron microscopy single particle analysis, symmetry expansion, and focused classification. The image reconstructions provide the first example of an asymmetric distribution of heparin on the surface of an icosahedral virus capsid. We demonstrate that each of the 60 capsid subunits that generate the T=1 capsid can bind heparin via one of five binding sites. However, not all of the binding sites are occupied by heparin and only one-to two-thirds of the binding sites are occupied. The binding sites are defined by arginine, lysine, and polar amino acids. Mutating the arginine, lysine, and polar amino acids to alanine diminishes the binding capacity of PCV2 to heparin.\n\nImportanceIt has been demonstrated that porcine circovirus 2 (PCV2) attaches to cells via heparan sulfate (HS) and chondroitin sulfate B (CSB) glycosaminoglycans; however, the underlying structural mechanism describing the HS/CSB recognition by PCV2 remains to be explored. We use cryo-electron microscopy with single particle analysis, symmetry expansion, and focused classification to visualize the interaction between the PCV2 capsid and heparin, an analog of heparan sulfate, to better than 3.6[A] resolution. We observe that the interaction between the PCV2 and heparin does not adhere to the icosahedral symmetry of the capsid. To the best of our knowledge, this is the first example where the interaction between heparin and an icosahedral capsid does not follow the symmetry elements of the capsid. Our findings also suggest that anionic polymers such as dextran sulfate may act to inhibit PCV2 infection.

biophysics↗