Biology subjects

Raghuvamsi, P. V.

Publications and source records attributed to Raghuvamsi, P. V..

5 recordsLinked to original sources

bioRxiv2022

GM1a functions as a coreceptor/ attachment factor for Dengue virus during infection in mammalian systems

Dengue virus (DENV) is a flavivirus causing an estimated 390 million infections per year around the world. Despite the immense global health and economic impact of this virus, its true receptor(s) for internalization into live cells has not yet been identified, and no successful antivirals or treatments have been isolated to this date. This study aims to improve our understanding of virus entry routs by exploring the sialic acid-based cell surface molecule GM1a and its role in DENV infection. The interaction of the virus with GM1a was studied using fluorescence correlation spectroscopy (FCS), fluorescence cross correlation spectroscopy (FCCS), imaging FCS (ImFCS) and amide hydrogen/deuterium exchange mass spectrometry (HDXMS), and the effect on infectivity and movement of the virus during infection was explored using plaque assays and fluorescence-based imaging and single particle tracking (SPT). GM1a was deemed to interact with DENV at domain I (DI) and domain II (DII) of the E protein of the protein coat at quaternary contacts of a fully assembled virus, leading to a ten-fold increase and seven-fold increase in infectivity for DENV1 and DENV2 in mammalian cell systems respectively. The interaction of virus with GM1a triggers a speeding up of virus movement on live cell surfaces, possibly resulting from a reduction in rigidity of cellular rafts during infection, and functions as a coreceptor/ attachment factor for DENV during infection in mammalian systems. Author SummaryDengue virus (DENV) is a flavivirus causing an estimated 390 million infections per year around the world. Despite the immense global health and economic impact of this virus, no successful antivirals or treatments have been isolated to this date. This may be due to the incomplete understanding of the virus infection mechanism, including a lack of an identified true receptor and entry related attachment factors or co-receptors responsible for internalization of the virus. This work focuses on the early infection stage of DENV1 and DENV2 strains, to identify how the virus moves on cell surfaces in its search for its receptors, and identifies the critical role of the sialic acid ganglioside GM1a during internalization of the virus.

biophysics↗

bioRxiv2021

Mapping the allosteric effects that define functional activity of SARS-CoV-2 specific antibodies

Previous studies on the structural relationship between human antibodies and SARS-CoV-2 have focused on generating static snapshots of antibody complexes with the Spike trimer. However, antibody-antigen interactions are dynamic, with significant binding-induced allosteric effects on conformations of antibody and its target antigen. In this study, we employ hydrogen-deuterium exchange mass spectrometry, in vitro assays, and molecular dynamics simulations to investigate the allosteric perturbations linked to binding events between a group of human antibodies with differential functional activities, and the Spike trimer from SARS-CoV-2. Our investigations have revealed key dynamic features that define weakly or moderately neutralizing antibodies versus those with strong neutralizing activity. These results provide mechanistic insights into the functional modes of human antibodies against COVID-19, and provide a rationale for effective antiviral strategies. TeaserDifferent neutralizing antibodies induce site-specific allosteric effects across SARS-CoV-2 Spike protein

biochemistry↗

bioRxiv2021

Viral genome-capsid core senses host environments to prime RNA release

Viruses are metastable macromolecular assemblies containing a nucleic acid core packaged by capsid proteins that are primed to disassemble in host-specific environments leading to genome release and replication. The mechanism of how viruses sense environmental changes associated with host entry to prime them for disassembly is unknown. We have applied a combination of mass spectrometry, cryo-EM, and simulation-assisted structure refinement to Turnip crinkle virus (TCV), which serves as a model non-enveloped icosahedral virus (Triangulation number = 3, 180 copies/icosahedron). Our results reveal genomic RNA tightly binds a subset of viral coat proteins to form a stable RNA-capsid core which undergoes conformational switching in response to host-specific environmental changes. These changes include: i) Depletion of Ca2+ which triggers viral particle expansion ii) Increase in osmolytes further disrupt interactions of outer coat proteins from the RNA-capsid core to promote complete viral disassembly. A cryo-EM structure of the expanded particle shows that RNA is asymmetrically extruded from a single 5-fold axis during disassembly. The genomic RNA:capsid protein interactions confer metastability to the TCV capsid and drive release of RNA from the disassembling virion within the plant host cell. AUTHOR SUMMARYRNA viruses including coronaviruses, dengue, influenza, and HIV are a significant threat to human health. These viral particles are finely tuned to undergo complex conformational changes that allow for response to varied environments. Turnip crinkle virus (TCV) serves as an excellent model for studying RNA virus dynamics. Since TCV is non-enveloped and has no post-translational modifications, we can specifically investigate the contributions of RNA to viral dynamics. Genomic RNA is not a passive entity but plays a crucial and previously uncharacterized role in viral disassembly. Our results reveal that the genomic RNA-capsid core serves as an environmental sensor and undergoes conformational switching in response to host cell conditions.

plant biology↗

bioRxiv2021

Uncovering cryptic pockets in the SARS-CoV-2 spike glycoprotein

The recent global COVID-19 pandemic has prompted a rapid response in terms of vaccine and drug development targeting the viral pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this work, we modelled a complete membrane-embedded SARS-CoV-2 spike (S) protein, the primary target of vaccine and therapeutics development, based on available structural data and known glycan content. We then used molecular dynamics (MD) simulations to study the system in the presence of benzene probes designed to enhance discovery of cryptic, potentially druggable pockets on the S protein surface. We uncovered a novel cryptic pocket with promising druggable properties located underneath the 617-628 loop, which was shown to be involved in the formation of S protein multimers on the viral surface. A marked multi-conformational behaviour of this loop in simulations was validated using hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments, supportive of opening and closing dynamics. Interestingly, the pocket is also the site of the D614G mutation, known to be important for SARS-CoV-2 fitness, and within close proximity to mutations in the novel SARS-CoV-2 strains B.1.1.7 and B.1.1.28, both of which are associated with increased transmissibility and severity of infection. The pocket was present in systems emulating both immature and mature glycosylation states, suggesting its druggability may not be dependent upon the stage of virus maturation. Overall, the predominantly hydrophobic nature of the cryptic pocket, its well conserved surface, and proximity to regions of functional relevance in viral assembly and fitness are all promising indicators of its potential for therapeutic targeting. Our method also successfully recapitulated hydrophobic pockets in the receptor binding domain and N-terminal domain associated with detergent or lipid binding in prior cryo-electron microscopy (cryo-EM) studies. Collectively, this work highlights the utility of the benzene mapping approach in uncovering potential druggable sites on the surface of SARS-CoV-2 targets.

biophysics↗

bioRxiv2021

Dengue virus 2 capsid protein chaperones strand displacement without altering the capsid-coding region hairpin element's structural functionality

By virtue of its chaperone activity, the capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements. However, the role of DENV2C during the interaction of RNA elements involved in stabilizing the 5-3 panhandle structure of DENV RNA is still unclear. Therefore, we determined how DENV2C affects structural functionality of the capsid-coding region hairpin element (cHP) during RNA rearrangement of the 9-nt conserved sequence (5CS) to its complementary 3CS counterpart. The cHP element has two distinct functions: a role in translation start codon selection and a role in RNA synthesis. Our results showed that the cHP hairpin impedes annealing between the 5CS and the 3CS elements. Although DENV2C does not modulate structural functionality of the cHP hairpin, it accelerates annealing and specifically promotes strand displacement of 3CS during 5-3 panhandle formation. Furthermore, DENV2C exerts its chaperone activity by favoring one of the active conformations of the cHP element. Based on our results, we propose mechanisms for annealing and strand displacement involving the cHP element. Thus, our results provide mechanistic insights on how DENV2C regulates RNA synthesis by modulating essential RNA elements in the capsid-coding region, that in turn allow for DENV replication.

molecular biology↗