Biology subjects

Hsieh, L. T.-H.

Publications and source records attributed to Hsieh, L. T.-H..

3 recordsLinked to original sources

bioRxiv2026

Structure and functional analyses of vaccinia virus J5 protein reveal distinct determinants for entry-fusion complex assembly and activation

Vaccinia virus enters host cells through a multi-component entry fusion complex (EFC) that is structurally distinct from canonical viral fusion systems. Understanding how vaccinia virus EFC mediates membrane fusion is crucial for elucidating poxvirus entry and identifying potential antiviral targets. Here, we report the solution NMR structure of a truncated ectodomain of vaccinia J5 protein, residues 2-68. Using recombinant vaccinia viruses expressing J5 mutants, we analyzed substitutions in conserved and surface-exposed residues, as well as chimeric constructs between vaccinia J5 and its entomopoxvirus ortholog AMV232. Functional analyses revealed that the conserved P38YYCWY43 motif is dispensable for EFC assembly but required for membrane fusion activity whereas the flexible region spanning residues 90-110 mediates interactions required for stable incorporation of J5 into the EFC. ImportanceVaccinia virus enters host cells through membrane fusion mediated by a unique multi-component entry fusion complex (EFC) that is distinct from classical viral fusion proteins. Although J5 has been identified as a central component of the pre-fusion EFC, the structural regions of J5 required for membrane fusion remain unclear. Here, we determined the solution NMR structure of the J5 ectodomain and identified two regions, the conserved P38YYCWY43 motif and residues 90-110, as key determinants of EFC function during vaccinia virus entry.

microbiology↗

bioRxiv2025

Structure and functional analyses of Vaccinia virus entry- fusion complex component J5 protein

Vaccinia virus enters host cells through an 11-protein entry fusion complex (EFC) that operates by a mechanism distinct from those of canonical viral fusion systems. Understanding how this multiprotein complex mediates membrane fusion is crucial for elucidating poxvirus entry and identifying potential antiviral targets. Here, we determined the NMR structure of a truncated J5 protein, (J5 2-68). To further dissect the functional determinants of J5, we generated recombinant vaccinia viruses expressing various J5 mutants, including substitutions in conserved residues, alterations of exposed charged residues, and chimeric constructs between vaccinia J5 and its orthologous AMV232 gene from an entomopoxvirus. Functional analyses revealed that residues 90-110 and the conserved P38YYCWY43 motif are indispensable for maintaining EFC integrity and promoting membrane fusion. Together, we define the structural and functional elements of J5 that are essential for poxvirus entry and advance our understanding of the unique membrane fusion mechanism employed by poxviruses. ImportanceVaccinia virus enters host cells through an eleven-protein entry fusion complex (EFC) that is mechanistically distinct from canonical viral fusion systems. Understanding how this multiprotein machinery mediates membrane fusion is essential for elucidating poxvirus entry mechanisms and for developing antiviral strategies. Here, we determined the NMR structure of the J5 ectodomain and generated a series of recombinant vaccinia viruses carrying J5 mutations. Functional analyses identified two regions, residues 90-110 and the conserved P38YYCWY43 motif, as essential for EFC stability and membrane fusion. These findings provide the first structure-function framework for J5, a core component of the EFC, and reveal key determinants required for complex integrity and viral entry.

microbiology↗

bioRxiv2023

Mycolactone causes catastrophic Sec61-dependent loss of the endothelial glycocalyx and basement membrane: a new indirect mechanism driving tissue necrosis in Mycobacterium ulcerans infection

The drivers of tissue necrosis in Mycobacterium ulcerans infection (Buruli ulcer disease) have historically been ascribed solely to the directly cytotoxic action of the diffusible exotoxin, mycolactone. However, its role in the clinically-evident vascular component of disease aetiology remains poorly explained. We have now dissected mycolactones effects on primary vascular endothelial cells in vitro and in vivo. We show that mycolactone-induced changes in endothelial morphology, adhesion, migration, and permeability are dependent on its action at the Sec61 translocon. Unbiased quantitative proteomics identified a profound effect on proteoglycans, driven by rapid loss of type II transmembrane proteins of the Golgi, including enzymes required for glycosaminoglycan (GAG) synthesis, combined with a reduction in the core proteins themselves. Loss of the glycocalyx is likely to be of particular mechanistic importance, since knockdown of galactosyltransferase II (beta-1,3-galactotransferase 6; B3GALT6), the GAG linker-building enzyme, phenocopied the permeability and phenotypic changes induced by mycolactone. Additionally, mycolactone depleted many secreted basement membrane components and microvascular basement membranes were disrupted in vivo. Remarkably, exogenous addition of laminin-511 reduced endothelial cell rounding, restored cell attachment and reversed the defective migration caused by mycolactone. Hence supplementing mycolactone-depleted extracellular matrix may be a future therapeutic avenue, to improve wound healing rates.

microbiology↗