Biology subjects

Thorne, L. G.

Publications and source records attributed to Thorne, L. G..

8 recordsLinked to original sources

bioRxiv2026

Allosteric capsid inhibitors and their escape mutants drive HIV-1 sensing

Small-molecule capsid inhibitors suppress HIV-1 infectivity by binding to capsid at the same site as FG motif-bearing host cofactors Sec24C, NUP153, CPSF6 and disordered nucleoporins residing in the nuclear pore complex central channel. We have used rational design to develop inhibitors called "allosteres" that target this pocket and inhibit HIV-1 infectivity. X-ray crystal structures of capsid/inhibitor complexes, reveal allosteric shifts upon inhibitor binding in the capsid C-terminal domain which impact the capsid lattice three-fold symmetry axis. Consistent with an uncoating mechanism, we find that allosteres cause HIV-1 to trigger innate immune response dependent on viral DNA and DNA sensor cGAS. Allosteres exhibit a similar loss of potency against clinically induced Lenacapavir resistance mutants but, strikingly, we find that HIV-1 bearing key resistance mutations induces innate immune activation in the absence of inhibitor. We hypothesise that resistant mutant sensitivity to cGAS contributes to reduction of HIV-1 transmission during Lenacapavir use in prophylaxis. Our work expands the physicochemical space and scaffold range for HIV capsid targeting inhibitors, provides mechanistic details of inhibition and facilitates improved inhibitor design.

microbiology↗

bioRxiv2025

A modified cyclosporine enhances lentivector transduction ex vivo and in vivo by degrading IFITM3

Intrinsic innate immune barriers have evolved to suppress viral infection and can reduce effective gene delivery in gene therapy. We have developed BG147, a novel cyclosporine A analogue, optimised via structure-guided design to prevent inhibition of HIV cofactor Cyclophilin A and to specifically inhibit interferon-induced transmembrane proteins (IFITM1-3). BG147 enhances VSV-G pseudotyped lentiviral vector transduction ex vivo in hematopoietic stem and progenitor cells (HSPCs) and in in vivo ocular gene therapy of photoreceptor cells in mice. Upon BG147 treatment, IFITM proteins are mislocalised and degraded through lysosomal acidification-dependent pathways. IFITM3 levels functionally return in cells 96 h after BG147 washout. BG147 promises to transform ex vivo and in vivo eye gene therapies by transiently inhibiting intrinsic immune barriers mediated by IFITM proteins to enhance a wide range of protocols. One Sentence SummaryModified cyclosporine, BG147, enhances lentivector gene therapy transduction, ex vivo in HSPC and in vivo in mouse photoreceptors, by degrading IFITM3.

cell biology↗

bioRxiv2022

Global landscape of the host response to SARS-CoV-2 variants reveals viral evolutionary trajectories

A series of SARS-CoV-2 variants of concern (VOCs) have evolved in humans during the COVID-19 pandemic--Alpha, Beta, Gamma, Delta, and Omicron. Here, we used global proteomic and genomic analyses during infection to understand the molecular responses driving VOC evolution. We discovered VOC-specific differences in viral RNA and protein expression levels, including for N, Orf6, and Orf9b, and pinpointed several viral mutations responsible. An analysis of the host response to VOC infection and comprehensive interrogation of altered virus-host protein-protein interactions revealed conserved and divergent regulation of biological pathways. For example, regulation of host translation was highly conserved, consistent with suppression of VOC replication in mice using the translation inhibitor plitidepsin. Conversely, modulation of the host inflammatory response was most divergent, where we found Alpha and Beta, but not Omicron BA.1, antagonized interferon stimulated genes (ISGs), a phenotype that correlated with differing levels of Orf6. Additionally, Delta more strongly upregulated proinflammatory genes compared to other VOCs. Systematic comparison of Omicron subvariants revealed BA.5 to have evolved enhanced ISG and proinflammatory gene suppression that similarly correlated with Orf6 expression, effects not seen in BA.4 due to a mutation that disrupts the Orf6-nuclear pore interaction. Our findings describe how VOCs have evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans. One sentence summarySystematic proteomic and genomic analyses of SARS-CoV-2 variants of concern reveal how variant-specific mutations alter viral gene expression, virus-host protein complexes, and the host response to infection with applications to therapy and future pandemic preparedness.

systems biology↗

bioRxiv2022

Enhanced innate immune suppression by SARS-CoV-2 Omicron subvariants BA.4 and BA.5

SARS-CoV-2 adaptation to humans is evidenced by the emergence of variants of concern (VOCs) with distinct genotypes and phenotypes that facilitate immune escape and enhance transmission frequency. Most recently Omicron subvariants have emerged with heavily mutated spike proteins which facilitate re-infection of immune populations through extensive antibody escape driving replacement of previously-dominant VOCs Alpha and Delta. Interestingly, Omicron is the first VOC to produce distinct subvariants. Here, we demonstrate that later Omicron subvariants, particularly BA.4 and BA.5, have evolved an enhanced capacity to suppress human innate immunity when compared to earliest subvariants BA.1 and BA.2. We find that, like previously dominant VOCs, later Omicron subvariants tend to increase expression of viral innate immune antagonists Orf6 and nucleocapsid. We show Orf6 to be a key contributor to enhanced innate immune suppression during epithelial replication by BA.5 and Alpha, reducing innate immune signaling through IRF3 and STAT1. Convergent VOC evolution of enhanced innate immune antagonist expression suggests common pathways of adaptation to humans and links VOC, and in particular Omicron subvariant, dominance to improved innate immune evasion.

microbiology↗

bioRxiv2022

SARS-CoV-2 Spike evolution influences GBP and IFITM sensitivity

SARS-CoV-2 spike requires proteolytic processing for viral entry. The presence of a polybasic furin-cleavage site (FCS) in spike, and evolution towards an optimised FCS by dominant variants of concern (VOCs), are linked to enhanced infectivity and transmission. Here we show that interferon-inducible antiviral restriction factors Guanylate binding proteins (GBP) 2 and 5 interfere with furin-mediated cleavage of SARS-CoV-2 spike and inhibit the infectivity of early-lineage Wuhan-Hu-1, while VOCs Alpha and Delta have evolved to escape restriction. Strikingly, we find Omicron is unique amongst VOCs, being restricted by GBP2/5, and also IFITM1, 2 and 3. Replacing the spike S2 domain in Omicron with Delta shows S2 is the determinant of entry route and IFITM sensitivity. We conclude that VOC evolution under different selective pressures has influenced sensitivity to spike-targeting restriction factors, with Omicron selecting spike changes that not only mediate antibody escape, and altered tropism, but also sensitivity to innate immunity.

microbiology↗

bioRxiv2021

Evolution of enhanced innate immune evasion by the SARS-CoV-2 B.1.1.7 UK variant

Emergence of SARS-CoV-2 variants, including the globally successful B.1.1.7 lineage, suggests viral adaptations to host selective pressures resulting in more efficient transmission. Although much effort has focused on Spike adaptation for viral entry and adaptive immune escape, B.1.1.7 mutations outside Spike likely contribute to enhance transmission. Here we used unbiased abundance proteomics, phosphoproteomics, mRNA sequencing and viral replication assays to show that B.1.1.7 isolates more effectively suppress host innate immune responses in airway epithelial cells. We found that B.1.1.7 isolates have dramatically increased subgenomic RNA and protein levels of Orf9b and Orf6, both known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein required for RNA sensing adaptor MAVS activation, and Orf9b binding and activity was regulated via phosphorylation. We conclude that B.1.1.7 has evolved beyond the Spike coding region to more effectively antagonise host innate immune responses through upregulation of specific subgenomic RNA synthesis and increased protein expression of key innate immune antagonists. We propose that more effective innate immune antagonism increases the likelihood of successful B.1.1.7 transmission, and may increase in vivo replication and duration of infection.

microbiology↗

bioRxiv2021

Characterisation of B.1.1.7 and Pangolin coronavirus spike provides insights on the evolutionary trajectory of SARS-CoV-2

The recent emergence of SARS-CoV-2 variants with increased transmission, pathogenesis and immune resistance has jeopardised the global response to the COVID-19 pandemic. Determining the fundamental biology of viral variants and understanding their evolutionary trajectories will guide current mitigation measures, future genetic surveillance and vaccination strategies. Here we examine virus entry by the B.1.1.7 lineage, commonly referred to as the UK/Kent variant. Pseudovirus infection of model cell lines demonstrate that B.1.1.7 entry is enhanced relative to the Wuhan-Hu-1 reference strain, particularly under low expression of receptor ACE2. Moreover, the entry characteristics of B.1.1.7 were distinct from that of its predecessor strain containing the D614G mutation. These data suggest evolutionary tuning of spike protein function. Additionally, we found that amino acid deletions within the N-terminal domain (NTD) of spike were important for efficient entry by B.1.1.7. The NTD is a hotspot of diversity across sarbecoviruses, therefore, we further investigated this region by examining the entry of closely related CoVs. Surprisingly, Pangolin CoV spike entry was 50-100 fold enhanced relative to SARS-CoV-2; suggesting there may be evolutionary pathways by which SARS-CoV-2 may further optimise entry. Swapping the NTD between Pangolin CoV and SARS-CoV-2 demonstrates that changes in this region alone have the capacity to enhance virus entry. Thus, the NTD plays a hitherto unrecognised role in modulating spike activity, warranting further investigation and surveillance of NTD mutations.

microbiology↗

bioRxiv2020

SARS-CoV-2 sensing by RIG-I and MDA5 links epithelial infection to macrophage inflammation

SARS-CoV-2 infection causes broad-spectrum immunopathological disease, exacerbated by inflammatory co-morbidities. A better understanding of mechanisms underpinning virus-associated inflammation is required to develop effective therapeutics. Here we discover that SARS-CoV-2 replicates rapidly in lung epithelial cells despite triggering a robust innate immune response through activation of cytoplasmic RNA-ensors RIG-I and MDA5. The inflammatory mediators produced during epithelial cell infection can stimulate primary human macrophages to enhance cytokine production and drive cellular activation. Critically, this can be limited by abrogating RNA sensing, or by inhibiting downstream signalling pathways. SARS-CoV-2 further exacerbates the local inflammatory environment when macrophages or epithelial cells are primed with exogenous inflammatory stimuli. We propose that RNA sensing of SARS-CoV-2 in lung epithelium is a key driver of inflammation, the extent of which is influenced by the inflammatory state of the local environment, and that specific inhibition of innate immune pathways may beneficially mitigate inflammation-associated COVID-19. HighlightsO_LISARS-CoV-2 activates RNA sensors and consequent inflammatory responses in lung epithelial cells C_LIO_LIEpithelial RNA sensing responses drive pro-inflammatory macrophage activation C_LIO_LIExogenous inflammatory stimuli exacerbate responses to SARS-CoV-2 in both eplithelial cells and macrophages C_LIO_LIImmunomodulators inhibit RNA sensing responses and consequent macrophage inflammation C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/424169v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@b07adborg.highwire.dtl.DTLVardef@51ddf7org.highwire.dtl.DTLVardef@c38f9aorg.highwire.dtl.DTLVardef@108db57_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗