Biology subjects

Huang, S.-W.

Publications and source records attributed to Huang, S.-W..

8 recordsLinked to original sources

bioRxiv2026

MX2 Mediates Collapse of the HIV-1 Capsid

The HIV-1 capsid core encapsulates the viral genome and mediates its delivery into the host cells nucleus. It is composed of multiple copies of the Capsid (CA, p24Gag) protein, assembled into hexamers and pentamers to create a lattice that forms a fullerene-like cone. Myxovirus resistance 2 (MX2) is an HIV-1 restriction factor that binds to the capsid core and blocks nuclear import of the viral genome. Here, we define a minimal region of MX2 required for HIV-1 restriction and produce a corresponding functional recombinant protein. We have used cryo-electron microscopy to determine the structure of this MX2 fragment bound to the tri-hexamer interface of the capsid lattice, revealing a large, buried interface combining electrostatic and hydrophobic interactions. This structure, together with assays that measure capsid core destabilisation, shows that MX2 binding induces conformational rearrangements in the capsid lattice that culminate in a loss of integrity. These results support a model whereby MX2 exerts its antiviral activity by disrupting the capsid lattice, inducing premature fragmentation and preventing HIV-1 nuclear import. By revealing the structural basis for MX2-mediated restriction, this work also provides the framework for the development of anti-HIV molecules that mimic MX2 restriction.

microbiology↗

bioRxiv2025

Targeted Computational Design of an Interleukin-7 Superkine with Enhanced Folding Efficiency and Immunotherapeutic Efficacy

Interleukin-7 (IL-7) plays a central role in maintaining T cell development and immune homeostasis, and enhancing the cytokines immune-stimulatory functionality has broad therapeutic implications against various oncological malignancies. Herein, we show a computationally designed IL7 superkine, Neo-7, which exhibits enhanced folding efficiency and superior binding affinity to its cognate receptors. To streamline the protein candidate prediction and validation process, the loop region of IL7 was strategically targeted for redesign while most of the receptor-interacting regions were preserved. Leveraging advanced computational tools such as AlphaFold2, we show loop remodeling to rectify structural irregularities that allows for iterative stabilization of protein backbone and leads to identification of beneficial mutations conducive to receptor engagement. Neo-7 superkine shows improved thermostability and production yield, and it exhibits heightened immune-stimulatory and anticancer effect in C57BL/6J mice. These findings underscore the utility of a targeted computational approach for de novo cytokine development.

bioengineering↗

bioRxiv2024

The primary mechanism for highly potent inhibition of HIV-1 maturation by lenacapavir

Lenacapavir (LEN) is a highly potent, long-acting antiretroviral medication for treating people infected with muti-drug-resistant HIV-1 phenotypes. The inhibitor targets multifaceted functions of the viral capsid protein (CA) during HIV-1 replication. Previous studies have mainly focused on elucidating LENs mode of action during viral ingress. Additionally, the inhibitor has been shown to interfere with mature capsid assembly during viral egress. However, the mechanism for how LEN affects HIV-1 maturation is unknown. Here, we show that pharmacologically relevant LEN concentrations do not impair proteolytic processing of Gag in virions. Instead, we have elucidated the primary mechanism for highly potent inhibition of HIV-1 maturation by sub-stoichiometric LEN:CA ratios. The inhibitor exerts opposing effects on formation of CA pentamers versus hexamers, the key capsomere intermediates in mature capsid assembly. LEN impairs formation of pentamers, whereas it induces assembly of hexameric lattices by imposing an opened CA conformation and stabilizing a dimeric form of CA. Consequently, LEN treatment results in morphologically atypical virus particles containing malformed, hyper-stable CA assemblies, which fail to infect target cells. Moreover, we have uncovered an inverse correlation between inhibitor potency and CA levels in cell culture assays, which accounts for LENs ability to potently (with pM EC50 values) inhibit HIV-1 maturation at clinically relevant drug concentrations. Author SummaryLenacapavir (LEN) is the first-in-class HIV-1 capsid targeting antiretroviral that exhibits multimodal modality to inhibit both early and late steps of viral replication. Our studies here have elucidated previously undescribed structural and mechanistic bases for a highly potent antiviral activity of LEN during viral egress. These findings will inform clinical applications of LEN as a potent HIV-1 maturation inhibitor and aid the development of second-generation inhibitors targeting assembly of the mature viral capsid.

microbiology↗

bioRxiv2024

Structural and Mechanistic Bases for Resistance of the M66I Capsid Variant to Lenacapavir

Lenacapavir (LEN) is the first in class viral capsid protein (CA) targeting antiretroviral for treating multi-drug-resistant HIV-1 infection. Clinical trials and cell culture experiments have identified resistance associated mutations (RAMs) in the vicinity of the hydrophobic CA pocket targeted by LEN. The M66I substitution conferred by far the highest level of resistance to the inhibitor compared to other RAMs. Here we investigated structural and mechanistic bases for how the M66I change affects LEN binding to CA and viral replication. The high-resolution X-ray structure of the CA(M66I) hexamer revealed that the {beta}-branched side chain of Ile66 induces steric hindrance specifically to LEN thereby markedly reducing the inhibitor binding affinity. By contrast, the M66I substitution did not affect binding of Phe-Gly (FG)-motif-containing cellular cofactors CPSF6, NUP153, or SEC24C, which engage the same hydrophobic pocket of CA. In cell culture the M66I variant did not acquire compensatory mutations or replicate in the presence of LEN. Analysis of viral replication intermediates revealed that HIV-1(M66I CA) predominantly formed correctly matured viral cores, which were more stable than their wildtype counterparts. The mutant cores stably bound to the nuclear envelope but failed to penetrate inside the nucleus. Furthermore, the M66I substitution markedly altered HIV-1 integration targeting. Taken together, our findings elucidate mechanistic insights for how the M66I change confers remarkable resistance to LEN and affects HIV-1 replication. Moreover, our structural findings provide powerful means for future medicinal chemistry efforts to rationally develop second generation inhibitors with a higher barrier to resistance. IMPORTANCELenacapavir (LEN) is a highly potent and long-acting antiretroviral that works by a unique mechanism of targeting the viral capsid protein. The inhibitor is used in combination with other antiretrovirals to treat multi-drug-resistant HIV-1 infection in heavily treatment-experienced adults. Furthermore, LEN is in clinical trials for preexposure prophylaxis (PrEP) with interim results indicating 100 % efficacy to prevent HIV-1 infections. However, one notable shortcoming is a relatively low barrier of viral resistance to LEN. Clinical trials and cell culture experiments identified emergent resistance mutations near the inhibitor binding site on capsid. The M66I variant was the most prevalent capsid substitution identified in patients receiving LEN to treat muti-drug resistant HIV-1 infections. The studies described here elucidate the underlying mechanism by which the M66I substitution confers a marked resistance to the inhibitor. Furthermore, our structural findings will aid future efforts to develop the next generation of capsid inhibitors with enhanced barriers to resistance.

microbiology↗

bioRxiv2024

Plant volatile-based fumigation improves mating competitiveness of males for population suppression of the global fruit pest Cydia pomonella

BACKGROUNDThe implementation of sterile insect technique (SIT) has proven effective in the area-wide suppression of several significant agricultural and sanitary pests by employing traditional cobalt-60 (60 Co-{gamma}) as a radiation source. Recently, X-ray has been validated as a feasible alternative to 60 Co-{gamma} radiation sources. Nonetheless, higher doses of X-ray irradiation lead to insect sterility but diminish mating competitiveness, thereby impacting the effectiveness of SIT applications. Thus, it is crucial to ascertain the optimal irradiation dose and develop strategies to enhance the mating competitiveness of sterile insects to enhance SIT efficacy. RESULTSIn this study, we determined the effect of various X-ray irradiation doses (ranging from 0 to 366 Gy) on the fecundity, fertility, and mating competitiveness of Cydia pomonella, a globally invasive fruit pest. Results demonstrated that the sterility rate of sterile males increased proportionally with irradiation dose up to 200 Gy, beyond which it plateaued. Notably, exposure to 200 Gy of irradiation notably decreased the mating competitiveness of male, as evidenced by a mating competitiveness index of 0.17 in laboratory and 0.096 in the orchard. This decline in mating competitiveness is likely linked to the down-regulation of genes associated with the recognition of sex pheromones, specifically CpomOR3a, CpomOR3b, and CpomOR5, following X-ray irradiation. Fumigation of the plant volatile, linalool at varying concentrations (70, 83, and 96 L/m 3) resulted in differential enhancements in male mating competitiveness, with the moderate concentration significantly improving the competitiveness of sterilized males, possibly by restoring their ability to recognize sex pheromones. Implementation of repeated releases of sterilized males on a pilot scale led to a notable reduction in the population of C. pomonella in the field. CONCLUSIONThese findings indicate that fumigation with plant volatiles has the potential to mitigate male sterility induced by X-ray irradiation, offering a promising approach to enhance the efficacy of SIT applications for the control of C. pomonella. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/604520v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@7ac9aeorg.highwire.dtl.DTLVardef@14ed866org.highwire.dtl.DTLVardef@1611e8borg.highwire.dtl.DTLVardef@11c6377_HPS_FORMAT_FIGEXP M_FIG C_FIG We determined the optimal X-ray irradiation dose and developed a linalool fumigation approach to improve the mating competitiveness of sterilized insects, thereby bolstering the efficacy of against Cydia pomonella.

zoology↗

bioRxiv2024

Syngeneic natural killer cell therapy activates dendritic and T cells in metastatic lungs and effectively treat low-burden metastases

Natural killer (NK) cells can control metastasis through cytotoxicity and IFN-{gamma} production independently of T cells in experimental metastasis mouse models. The inverse correlation between NK activity and metastasis incidence supports a critical role for NK cells in human metastatic surveillance. However, autologous NK cell therapy has shown limited benefit in treating patients with metastatic solid tumors. Using a spontaneous metastasis mouse model of MHC-I+ breast cancer, we found that transfer of IL-15/IL-12-conditioned syngeneic NK cells after primary tumor resection promoted long-term survival of mice with low metastatic burden and induced a tumor-specific protective T cell response that is essential for the therapeutic effect. Furthermore, NK cell transfer augments activation of conventional dendritic cells (cDCs), Foxp3-CD4+ T cells and stem cell-like CD8+ T cells in metastatic lungs, to which IFN-{gamma} of the transferred NK cells contributes significantly. These results imply direct interactions between transferred NK cells and endogenous cDCs to enhance T cell activation. We conducted an investigator-initiated clinical trial of autologous NK cell therapy in six patients with advanced cancer and observed that the NK cell therapy was safe and showed signs of effectiveness. These findings indicate that autologous NK cell therapy is effective in treating established low burden metastases of MHC-I+ tumor cells by activating the cDC-T cell axis at metastatic sites.

immunology↗

bioRxiv2023

Virus specificity and nucleoporin requirements for MX2 activity are affected by GTPase function and capsid-CypA interactions

Human myxovirus resistance 2 (MX2/MXB) is an interferon-induced GTPase that inhibits human immunodeficiency virus-1 (HIV-1) infection by preventing nuclear import of the viral preintegration complex. The HIV-1 capsid (CA) is the major viral determinant for sensitivity to MX2, and complex interactions between MX2, CA, nucleoporins (Nups), cyclophilin A (CypA), and other cellular proteins influence the outcome of viral infection. To explore the interactions between MX2, the viral CA, and CypA, we utilized a CRISPR-Cas9/AAV approach to generate CypA knock-out cell lines as well as cells that express CypA from its endogenous locus, but with specific point mutations that would abrogate CA binding but should not affect enzymatic activity or cellular function. We found that infection of CypA knock-out and point mutant cell lines with wild-type HIV-1 and CA mutants recapitulated the phenotypes observed upon cyclosporine A (CsA) addition, indicating that effects of CsA treatment are the direct result of blocking CA-CypA interactions and are therefore independent from potential interactions between CypA and MX2 or other cellular proteins. Notably, abrogation of GTP hydrolysis by MX2 conferred enhanced antiviral activity when CA-CypA interactions were abolished, and this effect was not mediated by the CA-binding residues in the GTPase domain, or by phosphorylation of MX2 at position T151. We additionally found that elimination of GTPase activity also altered the Nup requirements for MX2 activity. Our data demonstrate that the antiviral activity of MX2 is affected by CypA-CA interactions in a virus-specific and GTPase activity-dependent manner. These findings further highlight the importance of the GTPase domain of MX2 in regulation of substrate specificity and interaction with nucleocytoplasmic trafficking pathways. Author SummaryHIV-1 entry into the nucleus is an essential step in viral replication that involves complex interactions between the viral capsid and multiple cellular proteins, including the proline isomerase cyclophilin A. Nuclear entry of HIV-1 and other primate lentiviruses is inhibited by the antiviral protein MX2. Here, we show that direct interactions between capsid and cyclophilin A affect the antiviral activity and specificity of MX2, and that these interactions are altered when the enzymatic activity of MX2 is eliminated. We demonstrate that abolishing enzymatic activity of MX2 also alters the requirements for nuclear pore complex components for viral restriction. Our study provides new insights into how the enzymatic function of MX2 affects inhibition of lentiviral nuclear import.

microbiology↗

bioRxiv2023

Overcoming the nutritional immunity by engineering iron scavenging bacteria for cancer therapy

Certain bacteria demonstrate the ability to target and colonize the tumor microenvironment, a characteristic that positions them as innovative carriers for delivering various therapeutic agents in cancer therapy. Nevertheless, our understanding of how bacteria adapt their physiological condition to the tumor microenvironment remains elusive. In this work, we employed liquid chromatography-tandem mass spectrometry to examine the proteome of E. coli colonized in murine tumors. Comparing to E. coli cultivated in the rich medium, we found that E. coli colonized in tumors notably upregulated the processes related to ferric ions, including the enterobactin biosynthesis and iron homeostasis. This finding indicated that the tumor is an iron-deficient environment to E. coli. We also found that the colonization of E. coli in the tumor led to an increased expression of lipocalin 2 (LCN2), a hosts protein that can sequester the enterobactin. We therefore engineered E. coli in order to evade the nutritional immunity provided by LCN2. By introducing the IroA cluster, the E. coli synthesizes the glycosylated enterobactin, which creates steric hindrance to avoid the LCN2 sequestration. The IroA-E. coli showed enhanced resistance to LCN2 and significantly improved the anti-tumor activity in mice. Moreover, the mice cured by the IroA-E. coli treatment became resistant to the tumor re-challenge, indicating the establishment of immunological memory. Overall, our study underscores the crucial role of bacterias ability to acquire ferric ions within the tumor microenvironment for effective cancer therapy.

bioengineering↗