Biology subjects

Manicassamy, B.

Publications and source records attributed to Manicassamy, B..

10 recordsLinked to original sources

bioRxiv2024

Conserved role of spike S2 domain N-glycosylation across beta-coronavirus family

Besides acting as an immunological shield, the N-glycans of SARS-CoV-2 are also critical for viral life cycle. As the S2 subunit of spike is highly conserved across beta-coronaviruses, we determined the functional significance of the five stem N-glycans located in S2 between N1098-N1194. Studies were performed with 31 Asn-to-Gln mutants, beta-coronavirus virus-like particles and single-cycle viral replicons. Deletions of stem N-glycans enhanced S1 shedding from trimeric spike, reduced ACE2 binding and abolished syncytia formation. When three or more N-glycans were deleted, spike expression on cell surface and incorporation into virions was both reduced. Viral entry function was progressively lost upon deleting the N1098 glycan in combination with additional glycosite modifications. In addition to SARS-CoV-2, deleting stem N-glycans in SARS-CoV and MERS-CoV spike also prevented viral entry into target cells. These data suggest multiple functional roles for the stem N-glycans, and evolutionarily conserved properties for these complex carbohydrates across human beta-coronaviruses. Author SummaryPrevious work shows that the N-linked glycans of SARS-CoV-2 are essential for viral life cycle. Few natural mutations have been observed in the S2-subunit of the viral spike glycoprotein in GISAID data, and mutations are absent in the five stem N-glycans located between N1098-N1194. In the post-fusion spike structure these glycans lie equidistant, ~4 nm apart, suggesting functional significance. Upon testing the hypothesis that these glycans are critical for SARS-CoV-2 function, we noted multiple roles for the complex carbohydrates including regulation of S1-subunit shedding, spike expression on cells and virions, syncytial formation/cell-cell fusion and viral entry. Besides SARS-CoV-2, these glycans were also critical for other human beta-coronaviruses. Thus, these carbohydrates represent targets for the development of countermeasures against future outbreaks.

microbiology↗

bioRxiv2024

A detailed protocol for generating spike trans-complemented SARS-CoV-2 replicons

Multiple approaches have been implemented for basic science studies that attempt to investigate SARS-CoV-2 biology or virology in biosafety level-2 setting. These include pseudotyped-virus based on lentivirus and vesicular stomatitis virus, virus-like particles that only contain the SARS-CoV-2 structural proteins, and single-cycle replicons. Among these, the single-cycle replicons most closely resemble the authentic virus as they essentially include the full viral genome, except for essential elements required for active virus multiplication. In this regard, we previously developed a SARS-CoV-2 replicon system where a Gaussia Dura luciferase-P2A-mNeonGreen reporter cassette replaced viral spike. In this short paper, we present an optimized protocol for the use of this reagent that overcomes previous technical limitations. We demonstrate that co-transfection of this bacmid along with spike plasmid, using the improved protocol, yields high-quality spike bearing SARS-CoV-2 virus particles with single-cycle infectivity. Due to the nature of bacmid construction, this approach is particularly useful for studying the impact of spike mutagenesis on virus evolution in BSL-2 setting.

microbiology↗

bioRxiv2022

ISG15-modification of the Arp2/3 complex restricts pathogen spread

The ubiquitin-like protein, ISG15, can act as a cytokine or can covalently modify host and pathogen-derived proteins. The consequences of ISG15 modification on substrate fate remain unknown. Here we reveal that ISGylation of the Arp2/3 complex slows actin filament formation and stabilizes Arp2/3 dependent structures including cortical actin and lamella. When properly controlled, this serves as an antibacterial and antiviral host defense strategy to directly restrict actin-mediated pathogen spread. However, Listeria monocytogenes takes advantage in models of dysregulated ISGylation, leading to increased mortality due to augmented spread. The underlying molecular mechanism responsible for the ISG15-dependent impact on actin-based motility is due to failed bacterial separation after division. This promotes spread by enabling the formation of multi-headed bacterial "bazookas" with stabilized comet tails that can disseminate deeper into tissues. A bacterial mutant that cannot recruit Arp2/3 or a non-ISGylatable mutant of Arp3 is sufficient to rescue slowed comet tail speed and restrict spread. Importantly, ISG15-deficient neonatal mice have aberrant epidermal epithelia characterized by keratinocytes with diffuse cortical actin, which could underlie observed defects in wound healing in human patients who lack ISG15. Ultimately, our discovery links host innate immune responses to cytoskeletal dynamics with therapeutic implications for viral infection and metastasis.

cell biology↗

bioRxiv2022

Avian and Human Influenza Viruses Exhibit Distinct Glycoconjugate Receptor Specificities in Human Lung Cells

IAV utilize sialic acid (Sia) containing cell surface glycoconjugates for host cell infection, and IAV strains from different host species show preferences for structurally distinct Sia at the termini of glycoconjugates. Various types of cell surface glycoconjugates (N-glycans, O-glycans, glycolipids) display significant diversity in both structure and carbohydrate composition. To define the types of glycoconjugates that facilitate IAV infection, we utilized the CRISPR/Cas9 technique to truncate different types of glycoconjugates, either individually or in combination, by targeting glycosyltransferases essential to glycan biosynthesis in a human lung epithelial cell line. Our studies show that both human and avian IAV strains do not display strict preferences for a specific type of glycoconjugate. Interestingly, truncation of all three types of glycoconjugates significantly decreased the replication of human IAV strains, yet did not impact the replication of avian IAV strains. Taken together, our studies demonstrate that avian IAV strains utilize a broader repertoire of glycoconjugates for host cell infection as compared to human IAV strains. Author SummaryIt is well known that influenza A viruses (IAV) initiate host cell infection by binding to sialic acid, a sugar molecule present at the ends of various sugar chains called glycoconjugates. These glycoconjugates can vary in chain length, structure, and composition. However, it remains unknown if IAV strains preferentially bind to sialic acid on specific glycoconjugates for host cell infection. Here, we utilized CRISPR gene editing to abolish sialic acid on different glycoconjugate types in human lung cells, and evaluated human versus avian IAV infections. Our studies show that both human and avian IAV strains can infect human lung cells by utilizing any of the three major sialic acid-containing glycoconjugate types, specifically N-glycans, O-glycans, and glycolipids. Interestingly, simultaneous elimination of sialic acid on all three glycoconjugate types in human lung cells dramatically decreased human IAV infection, yet had little effect on avian IAV infection. Our studies indicate that avian IAV strains can utilize a wide variety of glycoconjugates for infection, whereas human IAV strains display restrictions in glycoconjugate type usage. These novel studies show distinct differences in host glycoconjugate preferences between human and avian IAV strains.

microbiology↗

bioRxiv2021

Generation of potent cellular and humoral immunity against SARS-CoV-2 antigens via conjugation to a polymeric glyco-adjuvant

The SARS-CoV-2 virus has caused an unprecedented global crisis, and curtailing its spread requires an effective vaccine which elicits a diverse and robust immune response. We have previously shown that vaccines made of a polymeric glyco-adjuvant conjugated to an antigen were effective in triggering such a response in other disease models and hypothesized that the technology could be adapted to create an effective vaccine against SARS-CoV-2. The core of the vaccine platform is the copolymer p(Man-TLR7), composed of monomers with pendant mannose or a toll-like receptor 7 (TLR7) agonist. Thus, p(Man-TLR7) is designed to target relevant antigen-presenting cells (APCs) via mannose-binding receptors and then activate TLR7 upon endocytosis. The p(Man-TLR7) construct is amenable to conjugation to protein antigens such as the Spike protein of SARS-CoV-2, yielding Spike-p(Man-TLR7). Here, we demonstrate Spike-p(Man-TLR7) vaccination elicits robust antigen-specific cellular and humoral responses in mice. In adult and elderly wild-type mice, vaccination with Spike-p(Man-TLR7) generates high and long-lasting titers of anti-Spike IgGs, with neutralizing titers exceeding levels in convalescent human serum. Interestingly, adsorbing Spike-p(Man-TLR7) to the depot-forming adjuvant alum, amplified the broadly neutralizing humoral responses to levels matching those in mice vaccinated with formulations based off of clinically-approved adjuvants. Additionally, we observed an increase in germinal center B cells, antigen-specific antibody secreting cells, activated T follicular helper cells, and polyfunctional Th1-cytokine producing CD4+ and CD8+ T cells. We conclude that Spike-p(Man-TLR7) is an attractive, next-generation subunit vaccine candidate, capable of inducing durable and robust antibody and T cell responses.

bioengineering↗

bioRxiv2021

Polymersomes decorated with SARS-CoV-2 spike protein receptor binding domain elicit robust humoral and cellular immunity

A diverse portfolio of SARS-CoV-2 vaccine candidates is needed to combat the evolving COVID-19 pandemic. Here, we developed a subunit nanovaccine by conjugating SARS-CoV-2 Spike protein receptor binding domain (RBD) to the surface of oxidation-sensitive polymersomes. We evaluated the humoral and cellular responses of mice immunized with these surface-decorated polymersomes (RBDsurf) compared to RBD-encapsulated polymersomes (RBDencap) and unformulated RBD (RBDfree), using monophosphoryl lipid A-encapsulated polymersomes (MPLA PS) as an adjuvant. While all three groups produced high titers of RBD-specific IgG, only RBDsurf elicited a neutralizing antibody response to SARS-CoV-2 comparable to that of human convalescent plasma. Moreover, RBDsurf was the only group to significantly increase the proportion of RBD-specific germinal center B cells in the vaccination-site draining lymph nodes. Both RBDsurf and RBDencap drove similarly robust CD4+ and CD8+ T cell responses that produced multiple Th1-type cytokines. We conclude that multivalent surface display of Spike RBD on polymersomes promotes a potent neutralizing antibody response to SARS-CoV-2, while both antigen formulations promote robust T cell immunity.

bioengineering↗

bioRxiv2021

Host Factor Rab11a is Critical for Efficient Assembly of Influenza A Virus Genomic Segments

It is well documented that influenza A viruses selectively package 8 distinct viral ribonucleoprotein complexes (vRNPs) into each virion; however, the role of host factors in genome assembly is not completely understood. To evaluate the significance of cellular factors in genome assembly, we generated a reporter virus carrying a tetracysteine tag in the NP gene (NP-Tc virus) and assessed the dynamics of vRNP localization with cellular components by fluorescence microscopy. At early time points, vRNP complexes were preferentially exported to the MTOC; subsequently, vRNPs associated on vesicles positive for cellular factor Rab11a and formed distinct vRNP bundles that trafficked to the plasma membrane on microtubule networks. In Rab11a deficient cells, however, vRNP bundles were smaller in the cytoplasm with less co-localization between different vRNP segments. Furthermore, Rab11a deficiency increased the production of non-infectious particles with higher RNA copy number to PFU ratios, indicative of defects in specific genome assembly. These results indicate that Rab11a+ vesicles serve as hubs for the congregation of vRNP complexes and enable specific genome assembly through vRNP:vRNP interactions, revealing the importance of Rab11a as a critical host factor for influenza A virus genome assembly.

microbiology↗

bioRxiv2021

Rab11a mediates cell-cell spread and reassortment of influenza A virus genomes via tunneling nanotubes.

Influenza A virus (IAV) genomes comprise eight negative strand RNAs packaged into virions in the form of viral ribonucleoproteins (vRNPs). Rab11a plays a crucial role in the transport of vRNPs from the nucleus to the plasma membrane via microtubules, allowing assembly and virus production. Here, we identify a novel function for Rab11a in the inter-cellular transport of IAV vRNPs using tunneling nanotubes (TNTs) as molecular highways. TNTs are F-Actin rich tubules that link the cytoplasm of nearby cells. In IAV-infected cells, Rab11a was visualized together with vRNPs in these actin-rich intercellular connections. To better examine viral spread via TNTs, we devised an infection system in which conventional, virion-mediated, spread was not possible. Namely, we generated HA-deficient reporter viruses which are unable to produce progeny virions but whose genomes can be replicated and trafficked. In this system, vRNP transfer to neighboring cells was observed and this transfer was found to be dependent on both actin and Rab11a. Generation of infectious virus via TNT transfer was confirmed using donor cells infected with HA-deficient virus and recipient cells stably expressing HA protein. Mixing donor cells infected with genetically distinct IAVs furthermore revealed the potential for Rab11a and TNTs to serve as a conduit for genome mixing and reassortment in IAV infections. These data therefore reveal a novel role for Rab11a in the IAV life cycle, which could have significant implications for within-host spread, genome reassortment and immune evasion. Author SummaryInfluenza A viruses infect epithelial cells of the upper and lower respiratory tract in humans. Infection is propagated by the generation of viral particles from infected cells, which disseminate within the tissue. Disseminating particles can encounter obstacles in the extracellular environment, including mucus, ciliary movement, antibody neutralization and uptake by phagocytic immune cells. An alternative mode of spread, which avoids these hazards, involves direct transport of viral components between cells. This cell-cell spread of infection is not a well understood process. In this study we demonstrate that the host factor Rab11a mediates the transport of viral genomes in the cell-cell spread of infection. Rab11a is already known to play a pro-viral role in the transport of viral genomes to the plasma membrane for assembly into virus particles. Here, we see that this same transport mechanism is co-opted for direct cell-cell spread through cellular connections called tunneling nanotubes. We show that complexes of Rab11a and viral components can be trafficked across tunneling nanotubes, transmitting infection without the formation of virus particles. Importantly, this route of spread often seeds viral genomes from multiple donor cells into recipient cells, which in turn increases viral genetic diversity.

microbiology↗

bioRxiv2020

The p150 Isoform of ADAR1 Blocks Sustained RLR signaling and Apoptosis during Influenza Virus Infection

Signaling through retinoic acid inducible gene I (RIG-I) like receptors (RLRs) is tightly regulated, with activation occurring upon sensing of viral nucleic acids, and suppression mediated by negative regulators. Under homeostatic conditions aberrant activation of melanoma differentiation-associated protein-5 (MDA5) is prevented through editing of endogenous dsRNA by RNA editing enzyme Adenosine Deaminase Acting on RNA (ADAR1). In addition, ADAR1 is postulated to play proviral and antiviral roles during viral infections that are dependent or independent of RNA editing activity. Here, we investigated the importance of ADAR1 isoforms in modulating influenza A virus (IAV) replication and revealed the opposing roles for ADAR1 isoforms, with the nuclear p110 isoform restricting versus the cytoplasmic p150 isoform promoting IAV replication. Importantly, we demonstrate that p150 is critical for preventing sustained RIG-I signaling, as p150 deficient cells showed increased IFN-{beta} expression and apoptosis during IAV infection, independent of RNA editing activity. Taken together, the p150 isoform of ADAR1 is important for preventing sustained RIG-I induced IFN-{beta} expression and apoptosis during viral infection.

microbiology↗

bioRxiv2020

Suppression of Cytotoxic T Cell Functions and Decreased Levels of Tissue Resident Memory T cell During H5N1 infection

Seasonal influenza virus infections cause mild illness in healthy adults, as timely viral clearance is mediated by the functions of cytotoxic T cells. However, avian H5N1 influenza virus infections can result in prolonged and fatal illness across all age groups, which has been attributed to the overt and uncontrolled activation of host immune responses. Here we investigate how excessive innate immune responses to H5N1 impair subsequent adaptive T cell responses in the lungs. Using recombinant H1N1 and H5N1 strains sharing 6 internal genes, we demonstrate that H5N1 (2:6) infection in mice causes higher stimulation and increased migration of lung dendritic cells to the draining lymph nodes, resulting in higher numbers of virus specific T cells in the lungs. Despite robust T cell responses in the lungs, H5N1 (2:6) infected mice showed inefficient and delayed viral clearance as compared to H1N1 infected mice. In addition, we observed higher levels of inhibitory signals including increased PD1 and IL-10 expression by cytotoxic T cells in H5N1 (2:6) infected mice, suggesting that delayed viral clearance of H5N1 (2:6) was due to suppression of T cell functions in vivo. Importantly, H5N1 (2:6) infected mice displayed decreased numbers of tissue resident memory T cells as compared to H1N1 infected mice; however, despite decreased number of tissue resident memory T cells, H5N1 (2:6) were protected against a heterologous challenge from H3N2 virus (X31). Taken together, our study provides mechanistic insight for the prolonged viral replication and protracted illness observed in H5N1 infected patients. ImportanceInfluenza viruses cause upper respiratory tract infections in humans. In healthy adults, seasonal influenza virus infections result in mild disease. Occasionally, influenza viruses endemic in domestic birds can cause severe and fatal disease even in healthy individuals. In avian influenza virus infected patients, the host immune system is activated in an uncontrolled manner and is unable to control infection in a timely fashion. In this study, we investigated why the immune system fails to effectively control a modified form of avian influenza virus. Our studies show that T cell functions important for clearing virally infected cells are impaired by higher negative regulatory signals during modified avian influenza virus infection. In addition, memory T cell numbers were decreased in modified avian influenza virus infected mice. Our studies provide a possible mechanism for the severe and prolonged disease associated with avian influenza virus infections in humans.

microbiology↗