Biology subjects

King, P. D.

Publications and source records attributed to King, P. D..

6 recordsLinked to original sources

bioRxiv2026

The gp120 Envelope Glycoprotein of HIV-1 Triggers Macropinocytosis in Primary CD4+ T Cells to Promote HIV-1 Infection

Macropinocytosis is a large-scale, actin-driven, fluid-phase form of endocytosis that can be exploited by HIV-1 for entry into primary CD4+ T cells. Herein, we show that HIV-1 actively induces macropinocytosis in resting and activated primary CD4+ T cells. HIV-1-induced T cell macropinocytosis is triggered by the interaction of soluble gp120 Env with cell surface CD4, is clathrin-independent and actin-dependent, and does not require CXCR4 or any other known HIV-1 coreceptors. Notably, Env-induced macropinocytosis requires calcium-independent phospholipase A2 (iPLA2) activity, identifying a role for a lipid-signaling axis downstream of Env-CD4 engagement. Across a gp120 panel, multiple HIV-1 clade B gp120 Envs can induce CD4+ T cell macropinocytosis. However, the magnitude of macropinocytosis does not correlate with CD4-binding strength alone and instead aligns with Env-driven changes in CD4 epitope exposure in CD4 domains distal to the Env-binding domain. Importantly, inhibitors of Env-induced macropinocytosis block HIV-1 infection of resting CD4+ T cells. Therefore, Env-induced macropinocytosis is a functionally important mechanism that promotes HIV-1 infection of this cell type.

microbiology↗

bioRxiv2025

Macropinosomes are a site of HIV-1 entry into primary CD4+ T cells

HIV-1 has been observed to enter target cells at both the plasma membrane and endosomes. However, which pathways mediate its entry into primary CD4+ T cells, the major targets of this virus, remains unclear. Here, we show that HIV-1 can enter primary CD4+ T cells through macropinocytosis, a form of endocytosis. We found that HIV-1 can enter primary CD4+ T cells at both the plasma membrane and internal compartments, while entry into common T cell lines occurred primarily at the plasma membrane. Inhibition of macropinocytosis suppressed HIV-1 internalization into and subsequent fusion with primary CD4+ T cells regardless of the viral coreceptor usage. Microscopic analysis of viral contents exposed to the cytosol confirmed that HIV-1 fusion occurs at the macropinosomal membrane. Finally, the inhibition of macropinocytosis blocked HIV-1 infection of primary CD4+ T cells. Altogether, this study identifies macropinocytosis as one pathway for HIV-1 entry into primary CD4+ T cells. Significance statementHIV-1 entry is an important therapeutic target. However, the exact subcellular location of HIV-1 entry into primary CD4+ T cells, a major in vivo host for HIV-1, remains undetermined. The current study shows that macropinosomes serve as a site for productive HIV-1 entry into the cytoplasm of primary CD4+ T cells. Supporting the role for macropinosomes, inhibition of macropinocytosis prevents HIV-1 internalization into, fusion with, and infection of primary CD4+ T cells. By contrast, HIV-1 infection of a CD4+ T cell line commonly used in HIV-1 research is insensitive to macropinocytosis inhibition. Altogether, this study highlights the primary-T-cell-specific dependence of HIV-1 on macropinocytosis for productive entry and therefore suggests the macropinosome-mediated HIV-1 entry as a potential target for antiviral strategies.

microbiology↗

bioRxiv2024

The C2 domain augments Ras GTPase Activating Protein catalytic activity

Regulation of Ras GTPases by GTPase activating proteins (GAP) is essential for their normal signaling. Nine of the ten GAPs for Ras contain a C2 domain immediately proximal to their canonical GAP domain, and in RasGAP (p120GAP, p120RasGAP; RASA1) mutation of this domain is associated with vascular malformations in humans. Here, we show that the C2 domain of RasGAP is required for full catalytic activity towards Ras. Analysis of the RasGAP C2-GAP crystal structure, AlphaFold models, and sequence conservation reveal direct C2 domain interaction with the Ras allosteric lobe. This is achieved by an evolutionarily conserved surface centered around RasGAP residue R707, point mutation of which impairs the catalytic advantage conferred by the C2 domain in vitro. In mice, R707C mutation phenocopies the vascular and signaling defects resulting from constitutive disruption of the RASA1 gene. In SynGAP, mutation of the equivalent conserved C2 domain surface impairs catalytic activity. Our results indicate that the C2 domain is required to achieve full catalytic activity of Ras GTPase activating proteins.

biochemistry↗

bioRxiv2023

An EPHB4-RASA1 signaling complex inhibits shear stress-induced Ras-MAPK activation in lymphatic endothelial cells to promote the development of lymphatic vessel valves

EPHB4 is a receptor protein tyrosine kinase that is required for the development of lymphatic vessel (LV) valves. We show here that EPHB4 is necessary for the specification of LV valves, their continued development after specification, and the maintenance of LV valves in adult mice. EPHB4 promotes LV valve development by inhibiting the activation of the Ras-MAPK pathway in LV endothelial cells (LEC). For LV specification, this role for EPHB4 depends on its ability to interact physically with the p120 Ras-GTPase-activating protein (RASA1) that acts as a negative regulator of Ras. Through physical interaction, EPHB4 and RASA1 dampen oscillatory shear stress (OSS)-induced Ras-MAPK activation in LEC, which is required for LV specification. We identify the Piezo1 OSS sensor as a focus of EPHB4-RASA1 regulation of OSS-induced Ras-MAPK signaling mediated through physical interaction. These findings contribute to an understanding of the mechanism by which EPHB4, RASA1 and Ras regulate lymphatic valvulogenesis.

developmental biology↗

bioRxiv2023

The role of ion dissolution in metal and metal oxide surface inactivation of SARS-CoV-2

Antiviral surface coatings are under development to prevent viral fomite transmission from high-traffic touch surfaces in public spaces. Coppers antiviral properties have been widely documented; but the antiviral mechanism of copper surfaces is not fully understood. We screened a series of metal and metal oxide surfaces for antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease (COVID-19). Copper and copper oxide surfaces exhibited superior anti-SARS-CoV-2 activity; however, level of antiviral activity was dependent upon the composition of the carrier solution used to deliver virus inoculum. We demonstrate that copper ions released into solution from test surfaces can mediate virus inactivation, indicating a copper ion dissolution-dependent antiviral mechanism. Level of antiviral activity is, however, not dependent on the amount of copper ions released into solution per se. Instead, our findings suggest that degree of virus inactivation is dependent upon copper ion complexation with other biomolecules (e.g., proteins/metabolites) in the virus carrier solution that compete with viral components. Although using tissue culture-derived virus inoculum is experimentally convenient to evaluate the antiviral activity of copper-derived test surfaces, we propose that the high organic content of tissue culture medium reduces the availability of "uncomplexed" copper ions to interact with the virus, negatively affecting virus inactivation and hence surface antiviral performance. We propose that laboratory antiviral surface testing should include virus delivered in a physiologically relevant carrier solution (saliva or nasal secretions when testing respiratory viruses) to accurately predict real-life surface antiviral performance when deployed in public spaces. ImportanceThe purpose of evaluating antiviral activity of test surfaces in the laboratory is to identify surfaces that will perform efficiently in preventing fomite transmission when deployed on high-traffic touch surfaces in public spaces. The conventional method in laboratory testing is to use tissue culture-derived virus inoculum, however this study demonstrates that antiviral performance of test copper-containing surfaces is dependent on the composition of the carrier solution in which the virus inoculum is delivered to test surfaces. Therefore, we recommend that laboratory surface testing should include virus delivered in a physiologically relevant carrier solution, to accurately predict real-life test surface performance in public spaces. Understanding the mechanism of virus inactivation is key to future rational design of improved antiviral surfaces. Here, we demonstrate that copper ions released from copper surfaces into small liquid droplets containing SARS-CoV-2, is a mechanism by which the virus that causes COVID-19 can be inactivated.

microbiology↗

bioRxiv2020

RASA1-driven cellular export of collagen IV is required for the development of lymphovenous and venous valves in mice

RASA1, a negative regulator of the Ras-mitogen-activated protein kinase (MAPK) signaling pathway, is essential for the development and maintenance of lymphatic vessel (LV) valves. However, whether RASA1 is required for the development and maintenance of lymphovenous valves (LVV) and venous valves (VV) is unknown. In this study we show that induced endothelial cell (EC)-specific disruption of Rasa1 in mid-gestation mouse embryos did not affect initial specification of LVV or central VV but did affect their continued development. Similarly, switch to expression of a catalytically inactive form of RASA1 resulted in impaired LVV and VV development. Blocked development of LVV in RASA1-deficient embryos was associated with accumulation of the basement membrane protein, collagen IV, in LVV-forming EC and could be partially or completely rescued by MAPK inhibitors and drugs that promote collagen IV folding. Disruption of Rasa1 in adult mice resulted in venous hypertension and impaired VV function that was associated with loss of EC from VV leaflets. In conclusion, RASA1 functions as a negative regulator of Ras signaling in EC that is necessary for EC export of collagen IV, thus permitting the development of LVV and the development and maintenance of VV.

developmental biology↗