Biology subjects

Eyre, N. S.

Publications and source records attributed to Eyre, N. S..

4 recordsLinked to original sources

bioRxiv2026

Proteomic mapping of the dengue virus NS1 microenvironment in infected cells identifies novel host dependency factors including TM9SF3

Dengue virus (DENV) is endemic in over 100 countries and causes approximately 100 million symptomatic infections annually, with symptoms ranging from mild febrile illness to life-threatening severe vascular leakage and haemorrhagic fever. There are currently no approved antiviral therapies available to treat DENV infections. The DENV non-structural protein 1 (NS1) is essential for viral RNA replication and infectious virus particle production, while secreted NS1 contributes to immune evasion and pathogenicity. Towards the identification of novel NS1-host protein interactions that are critical to these functions, an APEX2 proximity labelling-coupled quantitative proteomics approach was employed to map the proteomic composition of the NS1 microenvironment in live infected cells. Our analysis identified a panel of 51 NS1-proximal host proteins, including established DENV host dependency factors (HDFs) involved in NS1 folding and N-glycosylation, as well as previously unrecognised host factors. Loss-of-function approaches were used to determine the importance of these NS1-proximal host proteins to DENV infection, identifying several novel HDFs, including transmembrane 9 superfamily member 3 (TM9SF3). Importantly, the knockout of TM9SF3 was shown to impair DENV infectious virion production and intracellular NS1 abundance and secretion, consistent with the recently described roles of TM9SF3 in Golgi integrity and glycosylation fidelity. Together, this study demonstrates the successful application of APEX2 proximity labelling-coupled quantitative proteomics to the identification of functionally relevant NS1-associated host proteins that may inform the development of future antiviral therapies. IMPORTANCEThe DENV NS1 protein is a non-enzymatic, multifunctional glycoprotein that plays multiple distinct roles in viral replication organelle formation, viral RNA replication and infectious virus particle production. It is also secreted from infected cells as an oligomeric lipoparticle that participates in immune evasion and vascular damage. Many of its enigmatic roles are thought to be mediated via its interactions with other viral proteins and host proteins. Here, we have employed an infectious NS1-tagged DENV reporter virus and proximity biotinylation-coupled mass spectrometry to characterise the protein microenvironment of NS1 during viral infection. We have then employed functional genomics approaches to identify NS1-proximal host factors that contribute to the viral replication cycle. Amongst the novel host factors that were identified was TM9SF3, which has recently emerged as a Golgi-resident Golgiphagy receptor that is important for maintenance of Golgi integrity and glycosylation fidelity and may represent a future DENV antiviral drug target.

microbiology↗

bioRxiv2026

Restraint of Powassan virus replication by TRIM5α facilitates viral avoidance of antiviral immunity

TRIpartite Motif (TRIM) protein 5 alpha (TRIM5) is a well characterized cellular inhibitor of lentivirus replication that limits transmission of related viruses between primates. We previously reported that TRIM5 derived from humans and rhesus macaques inhibits replication of orthoflaviviruses belonging to the tick-borne encephalitis virus (TBEV) serocomplex, including TBEV, Kyasanur forest disease virus and Langat virus (LGTV), but interestingly not the tick-borne Powassan virus (POWV). To further characterize the primate TRIM5 and orthoflavivirus interface, we screened TRIM5 variants from representative old- and new-world primates for restriction capacity. TRIM5 from old-world African green monkey, De Brazzas monkey and chimpanzee demonstrated virus-specific restriction of tick-borne orthoflaviviruses. Efforts to determine why TRIM5 fails to inhibit POWV revealed that our lab stock had acquired a non-synonymous mutation in NS3 that, when introduced into a POWV molecular clone, facilitated virus replication in the presence of all inhibitory primate TRIM5 proteins. Infection of human dendritic cells with TRIM5-resistant POWV resulted in high early replication and strong induction of interferon responses that limited replication compared with the wild-type virus. Thus, primate TRIM5 functions as a potent cellular barrier to infection with tick-borne orthoflaviviruses that restrains replication to a level that may help avoid early innate immune recognition.

microbiology↗

bioRxiv2024

Coatomer protein complex I is required for efficient secretion of dengue virus non-structural protein 1

Secreted non-structural protein 1 (sNS1) is an important orthoflavivirus pathogenic factor that can induce vascular leakage; a key symptom of severe dengue disease. Given the role of sNS1 in dengue pathogenesis, defining the molecular mechanisms of NS1 secretion may contribute towards development of NS1-targeting antiviral therapies. To this end, we performed a customised membrane-trafficking siRNA screen to identify human host factors involved in NS1 secretion. Our screen identified COPA, COPB2, and COPG1 as the top-ranking hits. These proteins are three of the seven subunits of the coatomer protein complex I (COPI) that coat transport vesicles that operate within the early secretory pathway, implicating COPI machinery as being involved in NS1 secretion. Validation studies employing host gene knockdown in dengue virus (DENV)-infected cells confirmed that COPI components are required for efficient NS1 secretion but are dispensable for infectious virus egress. Similar reductions in NS1 secretion were observed when COPI components were depleted in cells infected with West Nile virus Kunjin subtype (WNV/KUNV), indicating that the molecular mechanisms exploited to achieve NS1 secretion may be a conserved feature within the Orthoflavivirus genus. Heterologous expression of wildtype and pathogenic COPI variants in DENV NS1-NS5 polyprotein expressing cells resulted in altered NS1 secretion profiles, suggesting that allelic variants and altered expression levels of COPI components may indirectly influence the severity of dengue disease. The identification of COPI components as important determinants of NS1 secretion efficiency may aid in the identification of novel targets for anti-orthoflaviviral therapies. IMPORTANCEOver half of the worlds population is at risk of infection with mosquito-borne pathogenic orthoflaviviruses such as DENV. Although the secreted form of the viral NS1 protein has been identified as a major determinant of the pathogenic effects of DENV and related orthoflaviviruses, the exact mechanisms involved in NS1 secretion are poorly understood. Here we interrogated host factors involved in secretion of NS1 from infected cells using a customised membrane trafficking siRNA screen. This revealed 3 components of the COPI complex that regulates vesicular transport in the early secretory pathway as important factors in NS1 secretion. The involvement of COPI components in NS1 secretion was further validated using wildtype DENV and WNV/KUNV infection, overexpression approaches and chemical inhibition studies. Together, this study demonstrates the importance of COPI machinery in NS1 secretion and suggests that exploitation of this machinery in NS1 secretion may represent a future target of antiviral drug development.

cell biology↗

bioRxiv2022

Constitutive expression and distinct properties of IFN-epsilon protect the female reproductive tract from Zika virus infection

The immunological surveillance factors controlling vulnerability of the female reproductive tract (FRT) to sexually transmitted viral infections are not well understood. Interferon-epsilon (IFN{varepsilon}) is a distinct, immunoregulatory type-I IFN that is constitutively expressed by FRT epithelium and is not induced by pathogens like other antiviral IFNs , {beta} and {lambda}. We show the necessity of IFN{varepsilon} for Zika Virus (ZIKV) protection by: increased susceptibility of IFN{varepsilon}-/- mice; their "rescue" by intravaginal recombinant IFN{varepsilon} treatment and blockade of protective endogenous IFN{varepsilon} by neutralising antibody. Complementary studies in human FRT cell lines showed IFN{varepsilon} had potent anti-ZIKV activity, associated with transcriptome responses similar to IFN{lambda} but lacking the proinflammatory gene signature of IFN. IFN{varepsilon} activated STAT1/2 pathways similar to IFN and {lambda} that were inhibited by ZIKV-encoded non-structural (NS) proteins, but not if IFN{varepsilon} exposure preceded infection. This scenario is provided by the constitutive expression of endogenous IFN{varepsilon}. However, the IFN{varepsilon} expression was not inhibited by ZIKV NS proteins despite their ability to antagonise the expression of IFN{beta} or {lambda}. Thus, the constitutive expression of IFN{varepsilon} provides cellular resistance to viral strategies of antagonism and maximises the antiviral activity of the FRT. These results show that the unique spatiotemporal properties of IFN{varepsilon} provides an innate immune surveillance network in the FRT that is a significant barrier to viral infection with important implications for prevention and therapy. Author SummaryThe female reproductive tract (FRT) is vulnerable to sexually transmitted infections and therefore a well-tuned immune surveillance system is crucial for maintaining a healthy FRT. However, our understanding of the factors that impact viral infection of the FRT and the host response are not well understood. In this work we investigate the role of a hormonally regulated type I interferon, IFN epsilon (IFN{varepsilon}) in control of Zika virus (ZIKV) infection of the FRT. IFN{varepsilon} is unique compared to other canonical type-I IFNs in that it is constitutively expressed by epithelial cells of the FRT with expression levels controlled by progesterone and not in response to viral infection. We demonstrate that IFN{varepsilon} has anti-ZIKV properties using a combination of IFN{varepsilon} KO mice, blockade of endogenous IFN{varepsilon} by neutralising Abs and rescue of IFN{varepsilon} KO mice by recombinant IFN{varepsilon} administered directly to the FRT. Furthermore, we complemented our in vivo studies using human FRT derived cell lines. Importantly, ZIKV NS proteins did not block IFN{varepsilon} expression despite their ability to antagonise the expression of IFN{beta} or {lambda}. Collectively this work implicates IFN{varepsilon} as a key type-I IFN that provides a distinct homeostatic antiviral environment in the FRT.

immunology↗