Biology subjects

Prasad, B. V. V.

Publications and source records attributed to Prasad, B. V. V..

5 recordsLinked to original sources

bioRxiv2025

Mutational Analysis of Human Norovirus VP2 Elucidates Critical Molecular Interactions for Virus Assembly

Human noroviruses (HuNoVs) are the leading cause of viral gastroenteritis with [≥]80% of infections caused by the GII genogroup. HuNoVs are non-enveloped, with an icosahedral capsid composed of 90 dimers of the major capsid protein VP1, which encloses a minor structural protein, VP2, and a VPg-linked positive sense ssRNA genome. Although the atomic structure of the icosahedral capsid formed by VP1 is well characterized using crystallography and cryo-electron microscopy analyses of HuNoV virus-like particles (VLPs), the structures and the localization of VP2 and VPg inside the capsid, how they are incorporated into the capsid, and whether this process requires interactions between them remain unresolved. Herein, we show VP2 is the molecular bridge for assembly of particles containing VP1, VP2 and VPg. We used deletion constructs and mutational analyses, guided by bioinformatic analyses, to determine the interaction site on VP2 for VP1 of the pandemic-causing GII.4 Sydney HuNoV. GII.4 HuNoV VP2 contains a unique insertion site at amino acids (AAs) 43-53, relative to VP2s of other GII HuNoV genotypes. We identified AA residues 40-43 on VP2 are required for interaction with VP1; mutation of VP2 AA 40-43 abrogates VP2 encapsidation. Computational analyses predicted VP2 has a highly conserved N-terminal -helical domain and an intrinsically disordered C-terminal domain that exhibits significant sequence diversity. We identified VP2, not VP1, uniquely binds VPg; the VP2 C-terminal domain is sufficient to interact with VPg. These findings reveal domain-specific functions of VP2 that are essential for coordinating capsid protein interactions for HuNoV assembly. ImportanceHuman noroviruses (HuNoVs) are the leading cause of epidemic and sporadic gastroenteritis in all age groups worldwide. Yet, we currently lack vaccines or therapeutics for these pathogens. Knowledge about HuNoV biology is limited, including the fundamental mechanisms governing particle assembly. Modern structural techniques have not resolved the complete structure of pandemic GII.4 norovirus that includes the localization of the interior capsid proteins VP2 and VPg. Furthermore, VP2s functional role(s) during infection remains obscure. Studies of feline and murine caliciviruses show VP2 may be involved in delivering the viral genome into cells, suggesting it synergizes with VP1 and VPg. We identify a motif on the N-terminal -helical domain of VP2, adjacent to a unique insertion site, that is essential for interaction with the major capsid protein VP1. We show VP2 uniquely binds the translation initiation protein, VPg, via its disordered C-terminus. These findings reveal principles of HuNoV capsid protein interactions and highlight VP2 as a bridge facilitating capsid assembly.

microbiology↗

bioRxiv2025

Functional Diversity in GII.4 Norovirus Entry: HBGA Binding and Capsid Clustering Dynamics

Human noroviruses (HuNoVs), especially GII.4 strains, are the leading cause of acute viral gastroenteritis worldwide, yet no approved vaccines or antivirals exist. The pandemic GII.4 Sydney 2012 strain enters cells via membrane wounding and clathrin-independent carrier (CLIC)-mediated endocytosis, but it is unclear whether this entry mechanism is conserved across GII.4 variants. We compared early binding and entry of multiple GII.4 variants using wildtype and mutant GII.4 virus-like particles (VLPs) and modified human intestinal enteroid (HIE) cultures. Only a subset of GII.4 variants, including GII.4 Sydney, form distinct, HBGA-dependent capsid clusters on the cell surface. Clustering strains display significantly enhanced membrane wounding and endocytosis compared to non-clustering strains and outcompete non-clustering strains in replication assays as shown by complete inhibition of GII.4 Sydney replication. Using mutant VLPs and a HBGA non-binding mutant (R345A), we identified two residues, V333 and R339, in the VP1 protruding domain as critical mediators of clustering and entry. Mutations of these residues disrupt clustering and endocytosis without affecting HBGA binding, suggesting a role in post-attachment processes. While clustering and endocytosis are contingent upon VLP binding to HBGAs, inhibitor studies show they are independent of host protein glycosylation and are driven by lipid raft remodeling regulated by cholesterol and ceramides. Quantitative analyses across multiple GII.4 variants reveal an apparent dichotomy between clustering and non-clustering phenotypes, with clustering variants exhibiting higher entry competence. This distinction offers insight into strain-specific cell entry mechanisms and may aid in identifying the elusive proteinaceous HuNoV cellular receptor(s) supporting targeted therapeutic development.

microbiology↗

bioRxiv2025

The Orai channel inhibitor BTP2 restricts Tulane virus and human norovirus replication independent of store-operated calcium entry

Human norovirus is the leading cause of viral gastroenteritis across all age groups. While there is a need for human norovirus antivirals, therapeutic development has been hindered by a lack of cell culture systems and animal models of infection. Surrogate viruses, such as Tulane virus (TV), have provided tractable systems to screen potential antiviral compounds. Our previous work demonstrated that Tulane virus encodes a viral ion channel, which dysregulates cytosolic calcium signaling. We set out to investigate whether host pathways triggered by viral ion channel activity, including store-operated calcium entry (SOCE), play a role in virus replication. Using pharmacologic inhibitors and genetically engineered cell lines, we establish that the SOCE inhibitor, BTP2, reduces TV replication in an SOCE-independent manner. We observed a significant reduction in TV replication, protein expression, and RNA synthesis in cells with both pre- and post-infection BTP2 treatment. By serial passage and plaque isolation, we demonstrate that TV quasi-species have mixed susceptibility and resistance to BTP2. Sequence comparison of the quasi-species revealed that amino acid changes in the structural proteins were associated with drug resistance. We utilized reverse genetics to generate TV with the resistance-associated VP1 and VP2 amino acid changes and found that a single amino acid change in VP1 (I380M) conferred BTP2 resistance. Further, expression of resistant VP2 alone was sufficient to partially rescue the replication of susceptible virus. Together, this supports that TV structural proteins are the targets of BTP2. Finally, using human intestinal organoids, we demonstrate that BTP2 significantly reduces human norovirus replication. ImportanceOur work identifies BTP2 as a potential human norovirus antiviral pharmacophore and highlights the utility of targeting calicivirus structural proteins to restrict viral replication. Further, we establish a system whereby Tulane virus can be used to screen novel antiviral candidates and establish their mechanism of action. Together, this will facilitate rapid preclinical validation of other novel human norovirus therapeutics.

microbiology↗

bioRxiv2024

Conformational flexibility is a critical factor in designing broad-spectrum human norovirus protease inhibitors

Human norovirus (HuNoV) infection is a global health and economic burden. Currently, there are no licensed HuNoV vaccines or antiviral drugs available. The protease encoded by the HuNoV genome plays a critical role in virus replication by cleaving the polyprotein and is, therefore, an excellent target for developing small molecule inhibitors. While rupintrivir, a potent small-molecule inhibitor of several picornavirus proteases, effectively inhibits GI.1 protease, it is an order of magnitude less effective against GII protease. Other GI.1 protease inhibitors also tend to be less effective against GII proteases. To understand the structural basis for the potency difference, we determined the crystal structures of proteases of GI.1, pandemic GII.4 (Houston and Sydney), and GII.3 in complex with rupintrivir. These structures show that the open substrate pocket in GI protease binds rupintrivir without requiring significant conformational changes, whereas, in GII proteases, the closed pocket flexibly extends, reorienting arginine-112 in the BII-CII loop to accommodate rupintrivir. Structures of R112A protease mutants with rupintrivir, coupled with enzymatic and inhibition studies, suggest R112 is involved in displacing both substrate and ligands from the active site, implying a role in the release of cleaved products during polyprotein processing. Thus, the primary determinant for differential inhibitor potency between the GI and GII proteases is the increased flexibility in the BII-CII loop of the GII proteases caused by H-G mutation in this loop. Therefore, the inherent flexibility of the BII-CII loop in GII proteases is a critical factor to consider when developing broad-spectrum inhibitors for HuNoV proteases. IMPORTANCEHuman noroviruses are a significant cause of sporadic and epidemic gastroenteritis worldwide. There are no vaccines or antiviral drugs currently available to treat infections. Our work elucidates the structural differences between GI.1 and GII proteases in response to inhibitor binding and will inform the future development of broad-spectrum norovirus protease inhibitors.

biochemistry↗

bioRxiv2020

Structure and mechanism of human diacylglycerol acyltransferase 1

Human diacylglycerol O-acyltransferase-1 (hDGAT1) synthesizes triacylglycerides and is required for dietary fat absorption and fat storage. The lack of 3-dimensional structure has limited our understanding of substrate recognition and mechanism of catalysis, and hampers rational targeting of hDGAT1 for therapeutic purposes. Here we present the structure of hDGAT1 in complex with a substrate oleoyl Coenzyme A at 3.1 [A] resolution. hDGAT1 forms a homodimer and each protomer has nine transmembrane helices that carve out a hollow chamber in the lipid bilayer. The chamber encloses highly conserved catalytic residues and has separate entrances for the two substrates fatty acyl Coenzyme A and diacylglycerol. The N-terminus of hDGAT1 makes extensive interactions with the neighboring protomer, and is required for enzymatic activity.

biochemistry↗