Biology subjects

Geller, R.

Publications and source records attributed to Geller, R..

10 recordsLinked to original sources

bioRxiv2024

The sequence and structural integrity of the SARS-CoV-2 Spike protein transmembrane domain is crucial for viral entry

The Spike (S) protein of SARS-CoV-2 is a type I membrane protein that mediates target cell recognition and membrane fusion. While its transmembrane domain (TMD) is traditionally viewed as a passive anchor to the viral envelope, emerging evidence suggests that TMDs often play active roles in the biogenesis and function of membrane proteins. Here, we investigated the functional role of the SARS-CoV-2 S protein TMD during viral entry. To this end, we introduced a series of amino acid substitutions and insertions within the hydrophobic core of the TMD and assessed their impact on S protein activity. Our findings reveal that the SARS-CoV-2 S protein is susceptible to alterations in its TMD. Functional determinants, including sequence features and structural parameters critical for viral entry, are distributed throughout the TMD, with a more pronounced contribution from its N-terminal region. We also demonstrate that the relative orientation of the regions flanking the TMD influences viral entry. Finally, our data suggest that the TMD mediates homo-oligomerization through a motif enriched in small residues, underscoring its functional importance beyond membrane anchoring.

molecular biology↗

bioRxiv2024

Mapping the mutational landscape of a full viral proteome reveals distinct profiles of mutation tolerability

RNA viruses have notoriously high mutation rates due to error-prone replication by their RNA polymerase. However, natural selection concentrates variability in a few key viral proteins. To test whether this stems from different mutation tolerance profiles among viral proteins, we measured the effect of >40,000 non-synonymous mutations across the full proteome of coxsackievirus B3 as well as >97% of all codon deletions in the non-structural proteins. We find significant variation in mutational tolerance within and between individual viral proteins, which correlated with both general and protein-specific structural and functional attributes. Further, mutational fitness effects remained largely constant across cell lines, highlighting conserved selection pressures. In addition to providing a rich dataset for understanding virus biology and evolution, our results illustrate that incorporation of mutational tolerance data into druggable pocket discovery can aid in selecting targets with high barriers to drug resistance.

microbiology↗

bioRxiv2024

Adaptive advantage of deletion repair in the N terminal domain of the SARS-CoV-2 spike protein in variants of concern

Mutations within the N-terminal domain (NTD) of the spike (S) protein are critical for the emergence of successful SARS-CoV-2 viral lineages. The NTD has been repeatedly impacted by deletions, often exhibiting complex and dynamic patterns, such as the recurrent emergence and disappearance of deletions in dominant variants. This study investigates the influence of repair of NTD lineage-defining deletions found in the BA.1 lineage (Omicron variant) on viral success. We performed comparative genomic analyses of more than 10 million SARS-CoV-2 genomes from GISAID to decipher the transmission success of viruses lacking S:{Delta}H69/V70, S:{Delta}V143/Y145, or both. These findings were contrasted against a screening of publicly available raw sequencing data, revealing substantial discrepancies between data repositories, suggesting that spurious deletion repair observations in GISAID may result from systematic artifacts. Specifically, deletion repair events were approximately an order of magnitude less frequent in the read-run survey. Our results suggest that deletion repair events are rare, isolated events with limited direct influence on SARS-CoV-2 evolution or transmission. Nevertheless, such events could facilitate the emergence of fitness-enhancing mutations. To explore potential drivers of NTD deletion repair patterns, we characterized the viral phenotype of such markers in a surrogate in vitro system. Repair of the S:{Delta}H69/V70 deletion reduced viral infectivity, while simultaneous repair with S:{Delta}V143/Y145 led to lower fusogenicity. In contrast, individual S:{Delta}V143/Y145 repair enhanced both fusogenicity and susceptibility to neutralization by sera from vaccinated individuals. This work underscores the complex genotype-phenotype landscape of the spike NTD in SARS-CoV-2, which impacts viral biology, transmission efficiency, and immune escape potential, offering insights with direct relevance to public health, viral surveillance, and the adaptive mechanisms driving emerging variants.

evolutionary biology↗

bioRxiv2023

Cell type-specific adaptation of the SARS-CoV-2 spike

SARS-CoV-2 can infect various human tissues and cell types, principally via interaction with its cognate receptor ACE2. However, how the virus evolves in different cellular environments is poorly understood. Here, we used experimental evolution to study the adaptation of the SARS-CoV-2 spike to four human cell lines expressing different levels of key entry factors. After 20 passages, cell type-specific phenotypic changes were observed. Selected spike mutations were identified and functionally characterized in terms of entry efficiency, ACE2 affinity, spike processing, TMPRSS2 usage, entry pathway and syncytia formation. We found that the effects of these mutations varied across cell types. Interestingly, two spike mutations (L48S and A372T) that emerged in cells expressing low ACE2 levels increased receptor affinity, syncytia induction, and entry efficiency under low-ACE2 conditions. Our results demonstrate specific adaptation of the SARS-CoV-2 spike to different cell types and have implications for understanding SARS-CoV-2 tissue tropism and evolution.

microbiology↗

bioRxiv2023

An efficient plasmid-based system for the recovery of recombinant vesicular stomatitis virus encoding foreign glycoproteins

Viral glycoproteins mediate entry into host cells, thereby dictating host range and pathogenesis. In addition, they constitute the principal target of neutralizing antibody responses, making them important antigens in vaccine development. Recombinant vesicular stomatitis virus (VSV) encoding foreign glycoproteins can provide a convenient and safe surrogate system to interrogate the function, evolution, and antigenicity of viral glycoproteins from viruses that are difficult to manipulate or those requiring high biosafety levels containment. However, the production of recombinant VSV can be technically challenging. In this work, we present an efficient and robust plasmid-based system for the production of recombinant VSV encoding foreign glycoproteins. We validate the system using glycoproteins from different viral families, including arenaviruses, coronaviruses, and hantaviruses, as well as highlight their utility for studying the effects of mutations on viral fitness. Overall, the methods described herein can facilitate the study of both native and recombinant VSV encoding foreign glycoproteins and can serve as the basis for the production of VSV-based vaccines.

microbiology↗

bioRxiv2022

Comprehensive profiling of polyclonal sera targeting a non-enveloped viral capsid

Despite their fundamental role in resolving viral infections, our understanding of how polyclonal neutralizing antibody responses target non-enveloped viruses remains limited. To define these responses, we obtained the full antigenic profile of multiple human and mouse polyclonal sera targeting the capsid of a prototypical picornavirus. Our results uncover significant variation in the breadth and strength of neutralization sites targeted by individual human polyclonal responses, which contrasted with homogenous responses observed in experimentally infected mice. We further use these comprehensive antigenic profiles to define key structural and evolutionary parameters that are predictive of escape, assess epitope dominance at the population level, and reveal a need for at least two mutations to achieve significant escape from multiple sera. Overall, our data provide the first comprehensive analysis of how polyclonal sera target a non-enveloped viral capsid and help define both immune dominance and escape at the population level.

microbiology↗

bioRxiv2022

Identification of cytoplasmic chaperone networks relevant for respiratory syncytial virus replication

RNA viruses have limited coding capacity and must therefore successfully subvert cellular processes to facilitate their replication. A fundamental challenge faced by both viruses and their hosts is the ability to achieve the correct folding and assembly of their proteome while avoiding misfolding and aggregation. In cells, this process is facilitated by numerous chaperone systems together with a large number of co-chaperones. In this work, we set out to define the chaperones and co-chaperones involved in the replication of respiratory syncytial virus (RSV). Using an RNAi screen, we identify multiple members of cellular protein folding networks whose knockdown alters RSV replication. The reduced number of chaperones and co-chaperones identified in this work can facilitate the unmasking of specific chaperone subnetworks required for distinct steps of the RSV life cycle and identifies new potential targets for antiviral therapy. Indeed, we show that the pharmacological inhibition of one of the genes identified in the RNAi screen, valosin-containing protein (VCP/p97), can impede the replication of RSV by interfering with the infection cycle at multiple steps.

microbiology↗

bioRxiv2021

The structural role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: the case of the spike A222V mutation

The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has now reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness.

molecular biology↗

bioRxiv2020

Globally defining the effects of mutations in a picornavirus capsid

The capsids of non-enveloped viruses are highly multimeric and multifunctional protein assemblies that protect the viral genome between infection cycles, dictate host and cell tropism, and mediate evasion of humoral immune responses. As such, capsids play key roles in viral biology and pathogenesis. Despite their importance, a comprehensive understanding of how mutations affect viral fitness across different structural and functional attributes of the capsid is lacking. To address this limitation, we globally define the effects of mutations in the capsid of a human picornavirus, generating a comprehensive dataset encompassing >90% of all possible single amino acid mutations. Moreover, we use this information to identify structural and sequence determinants that accurately predict mutational fitness effects, refine evolutionary analyses, and define the sequence specificity of key capsid encoded motifs. Finally, capitalizing on the sequence requirements identified in our dataset for capsid encoded protease cleavage sites, we implement and validate a bioinformatic approach for identifying novel host proteins targeted by viral proteases. Our findings present the most comprehensive investigation of mutational fitness effects in a picornavirus capsid to date and illuminate important aspects of viral biology, evolution, and host interactions.

microbiology↗

bioRxiv2017

Hsp90 dictates viral sequence space by balancing the evolutionary tradeoffs between protein stability, aggregation and translation rate

Acquisition of mutations is central to evolution but the detrimental effects of most mutations on protein folding and stability limit protein evolvability. Molecular chaperones, which suppress aggregation and facilitate polypeptide folding, are proposed to promote sequence diversification by buffering destabilizing mutations. However, whether and how chaperones directly control protein evolution remains poorly understood. Here, we examine the effect of reducing the activity of the key eukaryotic chaperone Hsp90 on poliovirus evolution. Contrary to predictions of a buffering model, inhibiting Hsp90 increases population sequence diversity and promotes accumulation of mutations reducing protein stability. Explaining this counterintuitive observation, we find that Hsp90 offsets the evolutionary tradeoff between protein stability and aggregation. Lower chaperone levels favor sequence variants of reduced hydrophobicity, thus decreasing protein aggregation propensity but at a cost to protein stability. Notably, reducing Hsp90 activity also promotes clusters of codon-deoptimized synonymous mutations at inter-domain boundaries, likely to promote local ribosomal slowdown to facilitate cotranslational domain folding. Our results reveal how a chaperone can shape the sequence landscape at both the protein and RNA levels to harmonize the competing constraints posed by protein stability, aggregation propensity and translation rate on successful protein biogenesis.

evolutionary biology↗