Biology subjects

Yennawar, N. H.

Publications and source records attributed to Yennawar, N. H..

4 recordsLinked to original sources

bioRxiv2024

Non-lytic spread of poliovirus requires the nonstructural protein 3CD

Non-enveloped viruses like poliovirus (PV) have evolved the capacity to spread by non-lytic mechanisms. For PV, this mechanism exploits the host secretory autophagy pathway. Virions are selectively incorporated into autophagosomes, double-membrane vesicles that travel to the plasma membrane, fuse, and release single-membrane vesicles containing virions. Loading of cellular cargo into autophagosomes relies on direct or indirect interactions with microtubule-associated protein 1B-light chain 3 (LC3) that are mediated by motifs referred to as LC3-interaction regions (LIRs). We have identified a PV mutant with a severe defect in non-lytic spread. An F-to-Y substitution in a putative LIR of the nonstructural protein 3CD prevented virion incorporation into LC3-positive autophagosomes and virion trafficking to the plasma membrane for release. Using high-angle annular dark-field scanning transmission electron microscopy to monitor PV-induced autophagosome biogenesis, for the first time, we show that virus-induced autophagic signals yield normal autophagosomes, even in the absence of virions. The F-to-Y derivative of PV 3CD was unable to support normal autophagosome biogenesis. Together, these studies make a compelling case for a direct role of a viral nonstructural protein in the formation and loading of the vesicular carriers used for non-lytic spread that may depend on the proper structure, accessibility, and/or dynamics of its LIR. The studies of PV 3CD protein reported here will hopefully provoke a more deliberate look at the presence and function of LIR motifs in viral proteins of viruses known to use autophagy as the basis for non-lytic spread. IMPORTANCEPV and other enteroviruses hijack the cellular secretory autophagy pathway for non-lytic virus transmission. While much is known about the cellular factors required for non-lytic transmission, much less is known about viral factors contributing to transmission. We have discovered a PV nonstructural protein required for multiple steps of the pathway leading to vesicle-enclosed virions. This discovery should facilitate identification of the specific steps of the cellular secretory autophagy pathway and corresponding factors commandeered by the virus and may uncover novel targets for antiviral therapy.

microbiology↗

bioRxiv2024

Characterization of Recombinant Human Lactoferrin Expressed in Komagataella Phaffii

We performed a thorough analysis and characterization of multiple batches of Helaina recombinant human lactoferrin (rhLF, Effera) expressed at an industrial scale in a yeast system. Bottom-up LC-MS/MS-based proteomics analysis detected the full sequence of Helaina rhLF protein and confirmed that its amino acid sequence is identical to that of native human LF (Uniprot i.d. P02788). Helaina rhLF had a protein purity of 98% or higher as determined by three orthogonal methods; reversed-phase HPLC, SDS-PAGE, and LC-MS proteomics analysis. N-linked glycans were detected at three known glycosylation sites, namely, Asparagines-156, -497, and -642. The identified N-glycans of Helaina rhLF were predominantly oligomannose structures with five to nine mannoses (M5-M9), which we also report to be present in both the native human and bovine LF. human milk LF (hmLF) possessed lower levels of oligomannose structures and were mainly M5 and M6. Helaina rhLF protein secondary structure was nearly identical to that of hmLF, as revealed by microfluidic modulation spectroscopy. Results of small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analyses confirmed that, like hmLF, Helaina rhLF displayed well-folded globular structures in solution. Reconstructed solvent envelopes of Helaina rhLF, obtained through the SAXS analysis, demonstrated a remarkable fit with the reported crystalline structure of iron-bound native hmLF. Differential scanning calorimetry investigations into the thermal stability of Helaina rhLF revealed two distinct denaturation temperatures at 68.7{+/-}0.9 {degrees}C and 91.9{+/-}0.5 {degrees}C, consistently mirroring denaturation temperatures observed for apo-and holo-hmLF. Overall, the characterization analysis results affirmed that Helaina rhLF was of high purity and exhibited globular structures closely akin to that of hmLF.

biophysics↗

bioRxiv2022

Enteroviral 2C protein is an RNA-stimulated ATPase and uses a two-step mechanism for binding to RNA and ATP

The enteroviral 2C protein is a therapeutic target, but the absence of a mechanistic framework for this enzyme limits our understanding of inhibitor mechanisms. Here we use poliovirus 2C and a derivative thereof to elucidate the first biochemical mechanism for this enzyme and confirm the applicability of this mechanism to other members of the enterovirus genus. Our biochemical data are consistent with a dimer forming in solution, binding to RNA, which stimulates ATPase activity by increasing the rate of hydrolysis without impacting affinity for ATP substantially. Both RNA and DNA bind to the same or overlapping site on 2C, driven by the phosphodiester backbone, but only RNA stimulates ATP hydrolysis. We propose that RNA binds to 2C driven by the backbone, with reorientation of the ribose hydroxyls occurring in a second step to form the catalytically competent state. 2C also uses a two-step mechanism for binding to ATP. Initial binding is driven by the and {beta} phosphates of ATP. In the second step, the adenine base and other substituents of ATP are used to organize the active site for catalysis. These studies provide the first biochemical description of determinants driving specificity and catalytic efficiency of a picornaviral 2C ATPase.

biochemistry↗

bioRxiv2022

Adaptation-proof SARS-CoV-2 vaccine design

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface spike glycoprotein - a major antibody target - is critical for virus entry via engagement of human angiotensin-converting enzyme 2 (ACE2) receptor. Despite successes with existing vaccines and therapies that primarily target the receptor binding domain (RBD) of the spike protein, the susceptibility of RBD to mutations provides escape routes for the SARS-CoV-2 from neutralizing antibodies. On the other hand, structural conservation in the spike protein can be targeted to reduce escape mutations and achieve broad protection. Here, we designed candidate stable immunogens that mimic surface features of selected conserved regions of spike protein through epitope grafting, in which we present the target epitope topology on diverse heterologous scaffolds that can structurally accommodate the spike epitopes. Structural characterization of the epitope-scaffolds showed stark agreement with our computational models and target epitopes. The sera from mice immunized with engineered designs display epitope-scaffolds and spike binding activity. We also demonstrated the utility of the designed epitope-scaffolds in diagnostic applications. Taken all together, our study provides important methodology for targeting the conserved, non-RBD structural motifs of spike protein for SARS-CoV-2 epitope vaccine design and demonstrates the potential utility of epitope grafting in rational vaccine design.

bioengineering↗