Biology subjects

Lundkvist, A.

Publications and source records attributed to Lundkvist, A..

8 recordsLinked to original sources

bioRxiv2025

Heparanase attenuates Zika virus infection by destabilizing the viral envelope protein

Heparanase (Hpa) is the only endoglycosidase enzyme in mammalian cells capable of cleaving heparan sulfate. In addition to its well-known functions in the regulation of glycosaminoglycans integrity, accumulating evidence indicates that Hpa plays vital roles in viral infection, while the mechanisms are not yet fully understood, especially in RNA virus infection. In this study, we report that Hpa functions as a restriction factor for Zika virus (ZIKV) infection. Our results demonstrated that Hpa, but not the enzymatic inactive mutant (Hpa-DM), resulted in degradation of the ZIKV envelope (E) protein, which could be rescued by treatment of the proteasome inhibitor (MG132) and the autophagy inhibitor (NH4Cl), separately. Additionally, the ubiquitination of ZIKV E did not show an significant change in the presence of Hpa. Overexpression of Hpa, but not Hpa-DM, dramatically decreased ZIKV infection in different cell models, evidenced by the reduction of viral proteins and a compromised production of infectious virions. This was further confirmed by the results in MEF cells, in which knockout Hpa enhanced ZIKV infection, while overexpression of Hpa suppressed the production of virions. In addition, ZIKV was found to downregulate the Hpa expression, which could counteract the inhibitory effects of Hpa. Altogether, our study discovers an unrecognized role of Hpa in virus infection and demonstrates that Hpa serves as a restriction factor for ZIKV infection. SummaryZika virus (ZIKV), primarily transmitted by mosquitoes, can cause a wide range of symptoms, including myalgia, fever, rash, and severe neurological complications such as microcephaly, epilepsy, and Guillain-Barre syndrome. A deep understanding of ZIKV-host cell interactions, particularly the roles of pro- and anti-viral factors, is essential for the development of effective therapeutic strategies, which are currently unavailable. In this study, we uncover that heparanase (Hpa), the only endoglycosidase enzyme in mammalian cells capable of cleaving heparan sulfate (HS), can degrade the ZIKV envelope (E) protein. Hpa exhibits an inhibitory role in ZIKV infection in different cell models. Additionally, we find that ZIKV downregulates the Hpa expression, which could be used by the virus to mitigate the inhibitory effects of Hpa. Taken together, our study demonstrates Hpa as a restriction factor in ZIKV infection and highlights complex interactions between ZIKV infection and HS machinery enzymes.

microbiology↗

bioRxiv2024

The evolutionary and molecular history of a chikungunya virus outbreak lineage

In 2018-2019, Thailand experienced a nationwide spread of chikungunya virus (CHIKV), with approximately 15,000 confirmed cases of disease reported. Here, we investigated the evolutionary and molecular history of the East/Central/South African (ECSA) genotype to determine the origins of the 2018-2019 CHIKV outbreak in Thailand. This was done using newly sequenced clinical samples from travellers returning to Sweden from Thailand in late 2018 and early 2019 and previously published genome sequences. Our phylogeographic analysis showed that before the outbreak in Thailand, the Indian Ocean lineage (IOL) found within the ESCA, had evolved and circulated in East Africa, South Asia, and Southeast Asia for about 15 years. In the first half of 2017, an introduction occurred into Thailand from another South Asian country, most likely Bangladesh, which subsequently developed into a large outbreak in Thailand with export to neighbouring countries. Based on comparative phylogenetic analyses of the complete CHIKV genome and protein modelling, we also identified amino acid substitutions that may be associated with immune evasion, increased spread, and virulence. We identified several mutations in the E1/E2 spike complex, such as E1 K211E and E2 V264A, which are highly relevant as they may lead to changes in vector competence, transmission efficiency and pathogenicity of the virus. A number of mutations (E2 G205S, Nsp3 D372E, Nsp2 V793A), that emerged shortly before the outbreak of the virus in Thailand in 2018 may have altered antibody binding and recognition due to their position. This study not only improves our understanding of the factors contributing to the epidemic in Southeast Asia, but also has implications for the development of effective response strategies and the potential development of new vaccines. Author SummaryWe investigated the evolutionary and molecular history of the East/Central/South African (ECSA) genotype to determine the origins of the 2018-2019 chikungunya virus (CHIKV) outbreak in Thailand. We used newly sequenced clinical samples from travellers returning to Sweden from Thailand in late 2018 and early 2019 together with previously published genome sequences. Our phylogeographic analysis shows that the Indian Ocean lineage (IOL), found within ECSA, evolved in Eastern Africa, Southern Asia, and Southeast Asia for about 15 years before the outbreak in Thailand in 2018. We have also identified amino acid substitutions that may be associated with immune evasion, increased spread, and higher virulence that occurred prior to the outbreak and may have played a critical role in the rapid spread of the virus. Our study concludes that monitoring and understanding CHIKV dynamics remains critical for an effective response to the previously unpredictable outbreaks of the virus.

microbiology↗

bioRxiv2024

Identification of unique and potent inhibitors of SARS-CoV-2 main protease from DNA-encoded chemical libraries

In vitro screening of large compounds libraries with automated high-throughput screening is expensive, time consuming and requires dedicated infrastructures. Conversely, the selection of DNA-encoded chemical libraries (DECL) can be rapidly performed with routine equipment available in most laboratories. In this study we identified novel inhibitors of SARS-CoV-2 main protease (Mpro) through the affinity-based selection of the DELopen library (open access for academics), containing 4.2 billion compounds. The identified inhibitors were peptide-like compounds containing an N-terminal electrophilic group able to form a covalent bond with the nucleophilic Cys145 of Mpro, as confirmed by x-ray crystallography. This DECL selection campaign enabled the discovery of the unoptimized compound SLL11 displaying an IC50 of 30 nM, proving that the rapid exploration of large chemical spaces enabled by DECL technology, allows for the direct identification of potent inhibitors avoiding several rounds of iterative medicinal chemistry. Compound MP1, a close analogue of SLL11, showed antiviral activity against SARS-CoV-2 in the low micromolar range when tested in Caco-2 and Calu-3 (EC50 = 2.3 {micro}M) cell lines. As peptide-like compounds can suffer from low cell permeability and metabolic stability, the cyclization of the compounds as well as the substitution of selected residues with D-enantiomers will be explored in the future to improve the antiviral activity of these novel compounds.

microbiology↗

bioRxiv2023

Validation of an antigenic site targeted by monoclonal antibodies against Puumala virus

Identification of B-cell epitopes facilitates the development of vaccines, therapeutic antibodies and diagnostic tools. Previously, the binding site of the bank vole monoclonal antibody (mAb) 4G2 against Puumala virus (PUUV, an orthohantavirus in the Hantaviridae family of the Bunyavirales order) was predicted using a combination of methods, including pepscan, phage-display, and site-directed mutagenesis of vesicular stomatitis virus (VSV) particles pseudotyped with Gn and Gc glycoproteins from PUUV. These techniques led to the identification of the neutralization escape mutation F915A. To our surprise, a recent crystal structure of PUUV Gc in complex with Fab 4G2 revealed that residue F915 is distal from epitope of mAb 4G2. To clarify this issue and explore potential explanations for the inconsistency, we designed a mutagenesis experiment to probe the 4G2 epitope, with three PUUV pseudoviruses carrying amino acid changes E725A, S944F, and S946F, located within the structure-based 4G2 epitope in the Gc. These amino acid changes were able to convey neutralization escape from 4G2, and S944F and S946F also conveyed escape from neutralization by human mAb 1C9. Furthemore, our mapping of all the known neutralization evasion sites from hantaviral Gcs onto PUUV Gc revealed that over 60% of these sites reside within or close to the epitope of mAb 4G2, indicating that this region represents a crucial area targeted by neutralizing antibodies against various hantaviruses. The identification of this site of vulnerability could guide the creation of subunit vaccines against PUUV and other hantaviruses in the future.

molecular biology↗

bioRxiv2022

Discovery of a novel coronavirus in Swedish bank voles (Myodes glareolus)

We identified a novel Betacoronavirus from bank voles (Myodes glareolus) in Grimso, Sweden. Repeated detection over three years and an overall prevalence of 3.4% suggests the virus commonly occurs in bank voles. Furthermore, phylogenetic analyses indicate the virus belongs to a highly divergent Embecovirus lineage predominantly associated with bank voles.

microbiology↗

bioRxiv2020

Revealing interspecies transmission barriers of avian influenza A viruses

Influenza A virus (IAV) pandemics result from interspecies transmission events within the avian reservoir and further to mammals including humans. Investigating molecular virus-host interactions dictating this process and the adaptations to the new hosts that follow is vital to understand zoonotic IAV spread. Receptor incompatibility has been suggested to limit zoonotic IAV transmission from the wild bird reservoir. Other barriers to interspecies transmission, particularly within the avian system, largely remain elusive. Through assessment of infection dynamics of mallard origin IAV in two different avian hosts, coupled with studies of receptor expression and host response we aimed to reveal the host-pathogen interactions in a cross-species transmission event. We found that shedding patterns and innate immune responses were highly dependent on viral genotypes, host species and inoculation routes, but less dependent on receptor expression. Further, in contrary to the prevailing dogma we demonstrate that birds can produce a wide range of different sialylated structures also found in mammals, e.g. extended N- and O-linked Neu5Ac2,6 terminated glycans. Overall, receptor incompatibility is not the sole transmission barrier for IAV between birds and to humans, but other host-pathogen factors deserve dedicated studies to achieve proper pandemic preparedness.

microbiology↗

bioRxiv2020

Molecular rationale for hantavirus neutralization by a reservoir host-derived monoclonal antibody

The intricate lattice of Gn and Gc glycoprotein spike complexes at the surface of hantaviruses facilitates host-cell entry and is the primary target of the neutralizing antibody-mediated immune response. Here, through study of a neutralizing monoclonal antibody (mAb 4G2) generated in a bank vole reservoir host following infection with Puumala virus (PUUV), we provide molecular-level insights into how antibody-mediated targeting of the hantaviral glycoprotein lattice effectively neutralizes the virus. Crystallographic analysis reveals that mAb 4G2 binds to a multi-domain site on Gc in the pre-fusion state, and that Fab binding is incompatible with the conformational changes of the Gc that are required for host cell entry. Cryo-electron microscopy of PUUV-like particles treated with Fab 4G2 demonstrates that the antibody binds to monomeric Gc at breaks in the Gn-Gc lattice, highlighting the immunological accessibility of Gc monomers on the mature hantavirus surface and the plastic nature of the higher-order lattice assembly. This work provides a structure-based blueprint for rationalizing antibody-mediated targeting of hantaviruses.

microbiology↗

bioRxiv2018

Defining of MAbs-neutralizing sites on the surface glycoproteins Gn and Gc of a hantavirus using vesicular stomatitis virus pseudotypes and site-directed mutagenesis

Earlier four Monoclonal antibodies (MAbs) against surface glycoproteins Gn and Gc of Puumala virus (PUUV, genus Orthohantavirus, family Hantaviridae, order Bunyavirales) were generated and for three MAbs with neutralizing capacity the localization of binding epitopes was predicted using pepscan and phage-display techniques. In this work, we produced vesicular stomatitis virus (VSV) particles pseudotyped with the Gn and Gc glycoproteins of PUUV and applied site-directed mutagenesis to dissect the structure of neutralizing epitopes. Replacement of cysteine amino acid (aa) residues with alanines resulted in pseudotype particles with diminished (16 to 18%) neut-titers; double Cys[->]Ala mutants, as well as mutants with bulky aromatic and charged residues replaced with alanines, have shown even stronger reduction in neut-titers (from 25% to the escape phenotype). In silico modelling of the neut-epitopes supported the hypothesis that these critical residues are located on the surface of viral glycoprotein molecules and thus can be recognized by the antibodies indeed. Similar pattern was observed in experiments with mutant pseudotypes and sera collected from patients suggesting that these neut-epitopes are utilized in a course of human PUUV infection.\n\nIMPORTANCENeutralization of viruses by antibodies is one of the key events in infection. We identified a set of mutations in the surface proteins of PUUV that reduced the virus-neutralizing activity of MAbs. Moreover, we found three mutants with escaped phenotype. These data will help understanding the mechanisms of hantavirus neutralization and assist construction of vaccine candidates.

microbiology↗