Biology subjects

Jangra, R. K.

Publications and source records attributed to Jangra, R. K..

15 recordsLinked to original sources

bioRxiv2026

Differential PCDH1 usage by Laguna Negra and Black Creek Canal hantaviruses

Laguna Negra virus and Black Creek Canal virus are hantaviruses associated with hantavirus cardiopulmonary syndrome (HCPS), but their cellular entry receptors have not been defined. We used replication-competent recombinant vesicular stomatitis viruses (rVSVs) bearing hantavirus Gn/Gc glycoprotein complexes to compare entry by these viruses, the Old World rodent-borne Dabieshan virus and Tigray virus, and the shrew-borne Cao Bang virus and Amga virus. All six Gn/Gc complexes supported infection of primary human pulmonary microvascular endothelial cells, with virus-specific efficiencies and kinetics. Genetic ablation of protocadherin-1 (PCDH1) reduced infection mediated by Laguna Negra virus and Black Creek Canal virus Gn/Gc in human osteosarcoma and endothelial cells, whereas PCDH1 re-expression restored susceptibility. Both viruses were captured by a soluble PCDH1 protein comprising the first two extracellular cadherin domains. Infection was also inhibited by a soluble decoy containing the first cadherin domain, and by an antibody specific to that domain, indicating that the first PCDH1 cadherin domain mediates viral engagement. Black Creek Canal Gn/Gc-bearing rVSVs incorporated less Gn/Gc and showed lower apparent capture by PCDH1 than Laguna Negra virus and Andes virus Gn/Gc-bearing particles, even after normalization for glycoprotein abundance. In contrast, PCDH1 depletion did not detectably alter infection mediated by the other four hantavirus Gn/Gc complexes. Thus, Laguna Negra virus and Black Creek Canal virus use PCDH1 for efficient entry but differ in apparent receptor engagement and responses to PCDH1-directed inhibitors, whereas all six viruses are potently neutralized by the broadly reactive Gn/Gc-directed antibody ADI-42898.

microbiology↗

bioRxiv2026

Expanded protocadherin-1 usage reveals a broader hantavirus entry landscape

Mammalian hantaviruses are RNA viruses that cause hantavirus cardiopulmonary syndrome in the Americas and hemorrhagic fever with renal syndrome in Eurasia. The cellular entry mechanisms of most hantaviruses remain poorly defined. To examine entry by phylogenetically distinct hantaviruses, we generated replication-competent recombinant vesicular stomatitis viruses (rVSVs) bearing Gn/Gc proteins from Necocli, Sangassou, Thottapalayam, Kenkeme, Nova, Oxbow, and Tula viruses. All these Gn/Gc proteins except Kenkeme supported infection of primary human endothelial cells, indicating that endothelial cell entry is permissive for a broader range of hantaviruses than previously appreciated. Except for rVSV-Kenkeme, these rVSVs did not acquire additional mutations beyond pre-engineered rescue-enhancing changes during rescue and passaging. Genetic studies in human cells lacking protocadherin-1 (PCDH1) showed that Necocli, Tula, and Nova viruses use PCDH1 for efficient infection, although the Nova phenotype was weaker. These three Gn/Gc proteins bound soluble PCDH1 with different apparent avidities, and infection by the corresponding rVSVs was inhibited by soluble PCDH1; Necocli and Tula, but not Nova, were also blocked by a PCDH1-targeting monoclonal antibody. Authentic Tula virus infection was similarly reduced in PCDH1 knockout endothelial cells. Finally, the broadly reactive anti-Gn/Gc human monoclonal antibody ADI-42898 efficiently neutralized Necocli, Nova, Sangassou, and Kenkeme rVSVs but showed weak or undetectable activity against Oxbow, Tula, and Thottapalayam rVSVs. Together, these findings expand the range of hantavirus glycoproteins capable of mediating infection of human endothelial cells, broaden the phylogenetic scope of PCDH1-dependent entry, and identify receptor-targeted and viral glycoprotein-targeted strategies with differential activity. ImportanceMany newly discovered hantaviruses are known only from sequence data, leaving their ability to enter human cells and their receptor usage unresolved. Using a BSL2-compatible rVSV system, we show that glycoproteins from several divergent hantaviruses can mediate infection of primary human endothelial cells, indicating that endothelial cell entry is permissive for a broader range of hantaviruses than previously appreciated. We also show that protocadherin-1 (PCDH1), previously linked mainly to New World hantaviruses, is used by Necocli, Tula, and Nova viruses but not universally across the panel, revealing broader but heterogeneous receptor usage. An authentic Tula virus experiment supports this conclusion beyond the surrogate system. Finally, a broadly reactive anti-Gn/Gc antibody neutralizes several, but not all, of these viruses, highlighting both the promise and the limits of broadly protective countermeasures and the utility of these rVSVs for evaluating entry inhibitors.

microbiology↗

bioRxiv2025

HKU5 bat merbecoviruses use divergent mechanisms to engage bat and mink ACE2 as entry receptors

Identifying receptors for bat coronaviruses is critical for spillover risk assessment, countermeasure development, and pandemic preparedness. While Middle East respiratory syndrome coronavirus (MERS-CoV) uses DPP4 for entry, the receptors of many MERS-related betacoronaviruses remain unknown. The bat merbecovirus HKU5 was previously shown to have an entry restriction in human cells. Using both pseudotyped and full-length virus, we show that HKU5 uses Pipistrellus abramus bat ACE2 but not human ACE2 or DPP4 as a receptor. Cryo-electron microscopy (cryo-EM) analysis of the virus-receptor complex and structure-guided mutagenesis reveal a spike and ACE2 interaction that is distinct from other ACE2-using coronaviruses. MERS-CoV vaccine sera poorly neutralize HKU5 informing pan-merbecovirus vaccine design. Notably, HKU5 can also engage American mink and stoat ACE2, revealing mustelids as potential intermediate hosts. These findings highlight the versatility of merbecovirus receptor use and underscore the need for continued surveillance of bat and mustelid species.

microbiology↗

bioRxiv2024

Viral susceptibility and innate immune competency of Carollia perspicillata bat cells produced for virological studies

Multiple viruses that are highly pathogenic in humans are known to have evolved in bats. How bats tolerate infection with these viruses, however, is poorly understood. As viruses engage in a wide range of interactions with their hosts, it is essential to study bat viruses in a system that resembles their natural environment like bat-derived in vitro cellular models. However, stable and accessible bat cell lines are not widely available for the broader scientific community. Here, we generated in vitro reagents for the Sebas short-tailed bat (Carollia perspicillata), tested multiple methods of immortalization, and characterized their susceptibility to virus infection and response to immune stimulation. Using a pseudotyped virus library and authentic virus infections, we show that these C. perspicillata cell lines derived from a diverse array of tissues are susceptible to viruses bearing the glycoprotein of numerous orthohantaviruses, including Andes and Hantaan virus and are also susceptible to live hantavirus infection. Furthermore, stimulation with synthetic double-stranded RNA prior to infection with VSV and MERS-CoV induced a protective antiviral response, demonstrating the suitability of our cell lines to study the bat antiviral immune response. Taken together, the approaches outlined here will inform future efforts to develop in vitro tools for virology from non-model organisms and these C. perspicillata cell lines will enable studies on virus-host interactions in bats.

microbiology↗

bioRxiv2024

Epistasis between N-terminal and receptor-binding domains drives cell entry in a bat coronavirus spike

Understanding the zoonotic risks posed by bat coronaviruses (CoVs) is critical for pandemic preparedness. Herein, we generated recombinant vesicular stomatitis viruses (rVSVs) bearing spikes from divergent bat CoVs to investigate their cell entry mechanisms. Unexpectedly, the successful recovery of rVSVs bearing the spike from SHC014, a SARS-like bat CoV, was associated with the acquisition of a novel substitution in the S2 fusion peptide-proximal region (FPPR). This substitution enhanced viral entry in both VSV and coronavirus contexts by increasing the availability of the spike receptor-binding domain to recognize its cellular receptor, ACE2. A second substitution in the spike N-terminal domain, uncovered through forward-genetic selection, interacted epistatically with the FPPR substitution to synergistically enhance spike:ACE2 interaction and viral entry. Our findings identify genetic pathways for adaptation by bat CoVs during spillover and host-to-host transmission, fitness trade-offs inherent to these pathways, and potential Achilles heels that could be targeted with countermeasures.

microbiology↗

bioRxiv2022

Two point mutations in protocadherin-1 disrupt Andes hantavirus recognition and afford protection against lethal infection

Andes virus and Sin Nombre virus are the etiologic agents of severe hantavirus cardiopulmonary syndrome (HCPS) in the Americas for which no FDA-approved countermeasures are available. Protocadherin-1 (PCDH1), a cadherin-superfamily protein recently identified as a critical host factor for ANDV and SNV, represents a new antiviral target; however, its precise role remains to be elucidated. Here, we used computational and experimental approaches to delineate the binding surface of the ANDV glycoprotein complex on PCDH1s first extracellular cadherin repeat domain. Strikingly, a single amino acid residue in this PCDH1 surface influenced the host species-specificity of SNV glycoprotein-PCDH1 interaction and cell entry. Mutation of this, and a neighboring residue, substantially protected Syrian hamsters from pulmonary disease and death caused by ANDV. We conclude that PCDH1 is a bona fide entry receptor for ANDV and SNV whose direct interaction with hantavirus glycoproteins could be targeted to develop new interventions against HCPS.

microbiology↗

bioRxiv2021

A combination of RBD and NTD neutralizing antibodies limits the generation of SARS-CoV-2 spike neutralization-escape mutants

Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150 and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent sera were reduced by 4-16-fold against rVSV-SARS2 bearing Y145D, K150E or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs modestly enhanced their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC. ImportanceThe US FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (mAb) therapies for the treatment of mild to moderate COVID-19. These mAb therapeutics are solely targeting the receptor binding domain of the SARS-CoV-2 spike protein. However, the N-terminal domain of the spike protein also carries crucial neutralizing epitopes. Here, we show that key mutations in the N-terminal domain can reduce the neutralizing capacity of convalescent COVID-19 sera. We report that a combination of two neutralizing antibodies targeting the receptor binding and N-terminal domains may have higher efficacy and is beneficial to combat the emergence of virus variants.

microbiology↗

bioRxiv2021

Genetic depletion studies define receptor usage by virulent hantaviruses in human endothelial cells

Hantaviruses are a large group of RNA viruses that include known epidemic threats and other agents poised for emergence. Several rodent-borne hantaviruses cause zoonoses accompanied by severe illness and death. However, assessments of zoonotic risk and the development of countermeasures alike are challenged by our limited knowledge of the molecular mechanisms of hantavirus infection, including the identities of cell entry receptors and their roles in influencing viral host range and virulence. Previous work has implicated several cell-surface molecules, most notably {beta}3- and {beta}1-containing integrin heterodimers, decay-accelerating factor (DAF), and the cadherin superfamily protein protocadherin-1 (PCDH1), in hantavirus entry in endothelial cells, the major targets of viral infection in humans. Despite the fact that {beta}3/{beta}1 integrins have been presumed to be the major hantavirus entry receptors for over two decades, rigorous genetic evidence supporting their requirement, and that of DAF as an entry cofactor, is lacking. Here, we used CRISPR/Cas9 engineering to knock out four candidate hantaviral receptors, singly and in combination, in a human endothelial cell line that recapitulates the properties of primary microvascular endothelial cells. PCDH1 loss substantially reduced entry and infection by a subset of hantaviruses endemic to the Americas. In contrast, the loss of {beta}3 integrin, {beta}1 integrin, and/or DAF had little or no effect on entry by any of a large panel of hantaviruses tested. We conclude that the major host molecules necessary for endothelial cell entry by PCDH1-independent hantaviruses remain to be discovered.

microbiology↗

bioRxiv2020

A glycoprotein mutation that emerged during the 2013-2016 Ebola virus epidemic alters proteolysis and accelerates membrane fusion

Genomic surveillance of viral isolates during the 2013-2016 Ebola virus epidemic in Western Africa--the largest and most devastating filovirus outbreak on record--revealed several novel mutations. The responsible strain, named Makona, carries an A to V substitution at position 82 in the glycoprotein (GP), which is associated with enhanced infectivity in vitro. Here, we investigated the mechanistic basis for this enhancement, as well as the interplay between A82V and a T to I substitution at residue 544 of GP, which also modulates infectivity in cell culture. We found that both 82V and 544I destabilize GP with the residue at 544 impacting overall stability, while 82V specifically destabilizes proteolytically cleaved GP. Both residues also promote faster kinetics of lipid mixing of the viral and host membranes in live cells, individually and in tandem, which correlates with faster times to fusion following co-localization with the viral receptor Niemann-Pick C1 (NPC1). Further, GPs bearing 82V are more sensitive to proteolysis by cathepsin L (CatL), a key host factor for viral entry. Intriguingly, CatL processed 82V variant GPs to a novel product of [~]12K size, which we hypothesize corresponds to a form of GP more fully primed for fusion than previously detected. We thus propose a model in which 82V promotes more efficient GP processing by CatL, leading to faster viral fusion kinetics and higher infectivity. ImportanceThe 2013-2016 outbreak of Ebola virus disease in West Africa demonstrated the potential for previously localized outbreaks to turn into regional, or even global, health emergencies. With over 28,000 cases and 11,000 confirmed deaths, this outbreak was over 50 times as large as any previously recorded. This outbreak also afforded the largest ever collection of Ebola virus genomic sequence data, allowing new insights into viral transmission and evolution. Viral mutants arising during the outbreak have attracted attention for their potentially altered patterns of infectivity in cell culture, with potential, if unclear, implications for increased viral spread and/or virulence. Here, we report on the properties of one such mutation in the viral glycoprotein, A82V, and its interplay with a previously described polymorphism at position 544. We show that mutations at both residues promote infection and fusion activation in cells, but that A82V additionally leads to increased infectivity under cathepsin-limited conditions, and the generation of a novel glycoprotein cleavage product.

microbiology↗

bioRxiv2020

A replication-competent vesicular stomatitis virus for studies of SARS-CoV-2 spike-mediated cell entry and its inhibition

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, and define correlates of immune protection, and to down-select candidate antivirals. Here, we describe a highly infectious recombinant vesicular stomatitis virus bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein that closely resembles the authentic agent in its entry-related properties. We show that the neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S and that neutralization of the rVSV and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific vaccines and therapeutics and for mechanistic studies of viral entry and its inhibition.

microbiology↗

bioRxiv2020

Broad sarbecovirus neutralizing antibodies define a key site of vulnerability on the SARS-CoV-2 spike protein

Broadly protective vaccines against known and pre-emergent coronaviruses are urgently needed. Critical to their development is a deeper understanding of cross-neutralizing antibody responses induced by natural human coronavirus (HCoV) infections. Here, we mined the memory B cell repertoire of a convalescent SARS donor and identified 200 SARS-CoV-2 binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of pre-existing memory B cells (MBCs) elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a new target for the rational design of pan-sarbecovirus vaccines.

immunology↗

bioRxiv2020

Direct intracellular visualization of Ebola virus-receptor interaction by in situ proximity ligation

Ebola virus (EBOV) entry into host cells comprises stepwise and extensive interactions of the sole viral surface glycoprotein GP with multiple host factors. During the intricate process, following virus uptake and trafficking to late endosomal/lysosomal compartments, GP is proteolytically processed to GPCL by the endosomal proteases cathepsin B and L unmasking GPs receptor-binding site. Engagement of GPCL with the universal filoviral intracellular receptor Niemann-Pick C1 (NPC1) eventually culminates in fusion between viral and cellular membranes, cytoplasmic escape of the viral nucleocapsid and subsequent infection. Mechanistic delineation of the indispensable GPCL:NPC1 binding step has been severely hampered by the unavailability of a robust cell-based assay assessing interaction of GPCL with full-length endosomal NPC1. Here, we describe a novel in situ assay to monitor GPCL:NPC1 engagement in intact, infected cells. Visualization of the subcellular localization of binding complexes is based on the principle of DNA-assisted, antibody-mediated proximity ligation. Virus-receptor binding monitored by proximity ligation was contingent on GPs proteolytic cleavage, and was sensitive to perturbations in the GPCL:NPC1 interface. Our assay also specifically decoupled detection of virus-receptor binding from steps post-receptor binding, such as membrane fusion and infection. Testing of multiple FDA-approved small molecule inhibitors revealed that drug treatments inhibited virus entry and GPCL:NPC1 recognition by distinctive mechanisms. Together, here we present a newly established proximity ligation assay, which will allow us to dissect cellular and viral requirements for filovirus-receptor binding, and to delineate the mechanisms of action of inhibitors on filovirus entry in a cell-based system. IMPORTANCEEbola virus causes episodic but increasingly frequent outbreaks of severe disease in Middle Africa, as shown by a currently ongoing outbreak in the Democratic Republic of Congo. Despite considerable effort, FDA-approved anti-filoviral therapeutics or targeted interventions are not available yet. Virus host-cell invasion represents an attractive target for antivirals; however our understanding of the inhibitory mechanisms of novel therapeutics is often hampered by fragmented knowledge of the filovirus-host molecular interactions required for viral infection. To help close this critical knowledge gap, here, we report an in situ assay to monitor binding of the EBOV glycoprotein to its receptor NPC1 in intact, infected cells. We demonstrate that our in situ assay based on proximity ligation represents a powerful tool to delineate receptor-viral glycoprotein interactions. Similar assays can be utilized to examine receptor interactions of diverse viral surface proteins whose studies have been hampered until now by the lack of robust in situ assays.

microbiology↗

bioRxiv2020

A virion-based assay for glycoprotein thermostability reveals key determinants of filovirus entry and its inhibition

Ebola virus (EBOV) entry into cells is mediated by its spike glycoprotein (GP). Following attachment and internalization, virions traffic to late endosomes where GP is cleaved by host cysteine proteases. Cleaved GP then binds its cellular receptor, Niemann-Pick C1. In response to an unknown cellular trigger, GP undergoes conformational rearrangements that drive fusion of viral and endosomal membranes . The temperature-dependent stability (thermostability) of the pre-fusion conformers of Class I viral fusion glycoproteins, including those of filovirus GPs, has provided insights into their propensity to undergo fusion-related rearrangements. However, previously described assays have relied on soluble glycoprotein ectodomains. Here, we developed a simple ELISA-based assay that uses the temperature-dependent loss of conformational epitopes to measure thermostability of GP embedded in viral membranes. The base and glycan cap subdomains of all filovirus GPs tested suffered a concerted loss of pre-fusion conformation at elevated temperatures, but did so at different temperature ranges, indicating virus-specific differences in thermostability. Despite these differences, all of these GPs displayed reduced thermostability upon cleavage to GPCL. Surprisingly, acid pH enhanced, rather than decreased, GP thermostability, suggesting it could enhance viral survival in hostile endo/lysosomal compartments. Finally, we confirmed and extended previous findings that some small-molecule inhibitors of filovirus entry destabilize EBOV GP and uncovered evidence that the most potent inhibitors act through multiple mechanisms. We establish the epitope-loss ELISA as a useful tool for studies of filovirus entry, engineering of GP variants with enhanced stability for use in vaccine development, and discovery of new stability-modulating antivirals. ImportanceThough a vaccine for Ebola virus has been approved by the FDA within the past year, no FDA-approved therapeutics are available to treat infections by Ebola virus or other filoviruses. The development of such countermeasures is challenged by our limited understanding of the mechanism by which Ebola virus enters cells, especially at the final step of membrane fusion. The sole surface-exposed viral protein, GP, mediates key steps in virus entry, including membrane fusion, and undergoes major structural rearrangements during this process. The stability of GP at elevated temperatures (thermostability) can provide insights into its capacity to undergo these structural rearrangements. Here, we describe a new assay that uses GP-specific antibodies to measure GP thermostability under a variety of conditions relevant to viral entry. We show that proteolytic cleavage and acid pH have significant effects on GP thermostability that shed light on their respective roles in viral entry. We also show that the assay can be used to study how small-molecule entry inhibitors affect GP stability. This work provides a simple and readily accessible assay to engineer forms of GP with enhanced stability that could be useful as part of an antiviral vaccine and to discover and improve drugs that act by modulating the stability of GP.

microbiology↗

bioRxiv2018

Human antibody cocktail deploys multiple functions to confer pan-ebolavirus protection

During the unprecedented 2013-2016 Ebola virus disease (EVD) epidemic in Western Africa and in its aftermath, the passive administration of monoclonal antibodies (mAbs) emerged as a promising treatment approach1-7. However, all antibody-based therapeutics currently in advanced development are specific for a single member of the Ebolavirus genus, Ebola virus (EBOV), and ineffective against divergent outbreak-causing ebolaviruses, including Bundibugyo virus (BDBV) and Sudan virus (SUDV)2,3,5,7. Here we advance MBP134, a cocktail of two broadly neutralizing human mAbs targeting the filovirus surface glycoprotein, GP, as a candidate pan-ebolavirus therapeutic. One component of this cocktail is a pan-ebolavirus neutralizing mAb, ADI-15878, isolated from a human EVD survivor8,9. The second, ADI-23774, was derived by affinity maturation of a human mAb8,9 via yeast display to enhance its potency against SUDV. MBP134 afforded exceptionally potent pan-ebolavirus neutralization in vitro and demonstrated greater protective efficacy than ADI-15878 alone in the guinea pig model of lethal EBOV challenge. A second-generation cocktail, MBP134AF, engineered to effectively harness natural killer (NK) cells afforded additional, unprecedented improvements in protective efficacy against EBOV and SUDV in guinea pigs relative to both its precursor and to any mAbs or mAb cocktails tested previously. MBP134AF is a best-in-class mAb cocktail suitable for evaluation as a pan-ebolavirus therapeutic in nonhuman primates.

microbiology↗

bioRxiv2018

Two point mutations in the Hantaan virus glycoproteins afford the generation of a highly infectious recombinant vesicular stomatitis virus vector

Rodent-to-human transmission of hantaviruses is associated with severe disease. Currently, no FDA-approved, specific antivirals or vaccines are available, and the requirement for high biocontainment (BSL3) laboratories limits hantavirus research. To study hantavirus entry in a BSL-2 laboratory, we set out to generate replication-competent, recombinant vesicular stomatitis viruses (rVSVs) bearing the Gn/Gc entry glycoproteins. As previously reported, rVSVs bearing New World hantavirus Gn/Gc were readily rescued from cDNAs, but their counterparts bearing Gn/Gc from the Old World hantavirus, Hantaan virus (HTNV), were refractory to rescue and only grew to low titers. However, serial passage of the rescued rVSV-HTNV Gn/Gc virus markedly increased its infectivity and capacity for cell-to-cell spread. This gain in viral fitness was associated with the acquisition of two point mutations; I532K in the cytoplasmic tail of Gn, and S1094L in the membrane-proximal stem of Gc. Follow-up experiments with rVSVs and single-cycle VSV pseudotypes confirmed these results. Mechanistic studies revealed that both mutations were determinative and contributed to viral infectivity in a synergistic manner. Our findings indicate that the primary mode of action of these mutations is to relocalize HTNV Gn/Gc from the Golgi complex to the cell surface, thereby affording significantly enhanced Gn/Gc incorporation into budding VSV particles. Our results suggest that enhancements in cell-surface expression of hantaviral glycoprotein(s) through incorporation of cognate mutations could afford the generation of rVSVs that are otherwise challenging to rescue. The robust replication-competent rVSV-HTNV Gn/Gc reported herein may also have utility as a vaccine.\n\nImportanceHuman hantavirus infections cause pulmonary syndrome in the Americas and hemorrhagic fever with renal syndrome (HFRS) in Eurasia. No FDA-approved vaccines and therapeutics exist for these deadly viruses, and their development is limited by the requirement for high biocontainment. In this study, we identified and characterized key amino acid changes in the surface glycoproteins of HFRS-causing Hantaan virus that enhance their incorporation into recombinant vesicular stomatitis virus (rVSV) particles. The replication-competent rVSV genetically encoding Hantaan virus glycoproteins described in this work provides a powerful and facile system to study hantavirus entry under lower biocontainment and may have utility as a hantavirus vaccine.

microbiology↗