skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: The Process of Filopodia Induction during HPV Infection
Human Papillomavirus 16 (HPV16) infects mucosal and epithelial cells and has been identified as a high-risk HPV type that is an etiologic agent of human cancers. The initial infectious process, i.e., the binding of the virus particle and its entry into the host cell, has been studied extensively, although it is not fully understood. There is still a gap in understanding the steps by which the virus is able to cross the plasma membrane after receptor binding. In this study, we demonstrate that after HPV16 comes into contact with a plasma membrane receptor, there are cytoskeletal changes resulting in an increase of filopodia numbers. This increase in filopodia numbers was transient and was maintained during the first two hours after virus addition. Our data show that there is a statistically significant increase in infection when filopodia numbers are increased by the addition of drug and virus simultaneously, and a decrease in virus infection when filopodia formation is inhibited. We describe that HPV16 binding results in the activation of Cdc42 GTPase that in turn results in an increase in filopodia. siRNA directed at Cdc42 GTPase resulted in a statistically significant reduction of infection and a corresponding lack of filopodia induction.  more » « less
Award ID(s):
2117625
PAR ID:
10450615
Author(s) / Creator(s):
;
Date Published:
Journal Name:
Viruses
Volume:
14
Issue:
6
ISSN:
1999-4915
Page Range / eLocation ID:
1150
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; (, Cells)
    Rho-GTPases are central regulators within a complex signaling network that controls cytoskeletal organization and cell movement. The network includes multiple GTPases, such as the most studied Rac1, Cdc42, and RhoA, along with their numerous effectors that provide mutual regulation through feedback loops. Here we investigate the temporal and spatial relationship between Rac1 and Cdc42 during membrane ruffling, using a simulation model that couples GTPase signaling with cell morphodynamics and captures the GTPase behavior observed with FRET-based biosensors. We show that membrane velocity is regulated by the kinetic rate of GTPase activation rather than the concentration of active GTPase. Our model captures both uniform and polarized ruffling. We also show that cell-type specific time delays between Rac1 and Cdc42 activation can be reproduced with a single signaling motif, in which the delay is controlled by feedback from Cdc42 to Rac1. The resolution of our simulation output matches those of time-lapsed recordings of cell dynamics and GTPase activity. Our data-driven modeling approach allows us to validate simulation results with quantitative precision using the same pipeline for the analysis of simulated and experimental data. 
    more » « less
  2. ; ; (, bioRxiv)
    Abstract PurposeReceptor tyrosine kinase (RTK) concentrations on the plasma membrane correlate with angiogenic functions in vitro and in rodent models. The intracellular RTK pool also regulates plasma membrane receptor availability and signaling pathways. Organs have specialized angiogenic functions essential to their distinct roles, supporting the hypothesis that plasma membrane and intracellular RTK concentrations vary across endothelial cells (ECs) from different organs. MethodsUsing quantitative flow cytometry on human ECs derived from dermis, umbilical vein, kidney, liver, and brain, we measured and statistically analyzed the concentrations of selected RTKs within ECs and on their plasma membranes. ResultsVEGFR1 exhibited the lowest concentrations on the plasma membrane (300–900 VEGFR1/cell) among VEGFRs. HDMECs (dermis) showed the lowest VEGFR1 level among the examined EC types. Whole-cell VEGFR1 concentrations were 2500–7500 VEGFR1/cell, with 12–26% located on the plasma membrane. The proportion of VEGFR2 located on the plasma membrane was higher at > 30%, except in HGMECs (kidney) where it was 24%. Plasma membrane VEGFR2 was significantly lower in HDMECs and HGMECs compared with HBMECs (brain), whereas whole-cell VEGFR2 levels were consistently in the range of 14,100–22,500 molecules/cell. VEGFR3 was the least localized to the plasma membrane, from 2% in HGMECs to 14% in HDMECs at the highest level of 4400 VEGFR3/cell. Whole-cell VEGFR3 concentrations ranged from 32,400 in HDMECs to 62,000 VEGFR3/cell in HLiSMECs (liver), with no significant differences among EC types. NRP1 was most abundant on the plasma membrane of HUVECs (umbilical vein) at 39,700 NRP1/cell; other ECs displayed 26,000–29,900 NRP1/cell, approximately 5-fold higher than the numbers of VEGFRs. Across EC types, Axl was present on the plasma membrane at levels (6900–12,200 Axl/cell) similar to those of VEGFR2. ConclusionsWe quantified and statistically analyzed plasma membrane and whole-cell expression of angiogenic RTKs across cultured human ECs from five different organs. Our findings suggest that RTK protein distribution might not fully reflect the differential angiogenic capacities in cultured ECs. In vitro monoculture conditions might reduce EC organ-specific features essential for refining vascular models. 
    more » « less
  3. ; ; ; ; ; ; ; ; ; (, Viruses)
    The efficiency of virus internalization into target cells is a major determinant of infectivity. SARS-CoV-2 internalization occurs via S-protein-mediated cell binding followed either by direct fusion with the plasma membrane or endocytosis and subsequent fusion with the endosomal membrane. Despite the crucial role of virus internalization, the precise kinetics of the processes involved remains elusive. We developed a pipeline, which combines live-cell microscopy and advanced image analysis, for measuring the rates of multiple internalization-associated molecular events of single SARS-CoV-2-virus-like particles (VLPs), including endosome ingression and pH change. Our live-cell imaging experiments demonstrate that only a few minutes after binding to the plasma membrane, VLPs ingress into RAP5-negative endosomes via dynamin-dependent scission. Less than two minutes later, VLP speed increases in parallel with a pH drop below 5, yet these two events are not interrelated. By co-imaging fluorescently labeled nucleocapsid proteins, we show that nucleocapsid release occurs with similar kinetics to VLP acidification. Neither Omicron mutations nor abrogation of the S protein polybasic cleavage site affected the rate of VLP internalization, indicating that they do not confer any significant advantages or disadvantages during this process. Finally, we observe that VLP internalization occurs two to three times faster in VeroE6 than in A549 cells, which may contribute to the greater susceptibility of the former cell line to SARS-CoV-2 infection. Taken together, our precise measurements of the kinetics of VLP internalization-associated processes shed light on their contribution to the effectiveness of SARS-CoV-2 propagation in cells. 
    more » « less
  4. ; ; ; ; ; ; (, Journal of Cell Science)
    ABSTRACT During cytokinesis, a series of coordinated events partition a dividing cell. Accurate regulation of cytokinesis is essential for proliferation and genome integrity. In fission yeast, these coordinated events ensure that the actomyosin ring and septum start ingressing only after chromosome segregation. How cytokinetic events are coordinated remains unclear. The GTPase Cdc42 promotes recruitment of certain cell wall-building enzymes whereas the GTPase Rho1 activates these enzymes. We show that Cdc42 prevents early Rho1 activation during fission yeast cytokinesis. Using an active Rho probe, we find that although the Rho1 activators Rgf1 and Rgf3 localize to the division site in early anaphase, Rho1 is not activated until late anaphase, just before the onset of ring constriction. We find that loss of Cdc42 activation enables precocious Rho1 activation in early anaphase. Furthermore, we provide functional and genetic evidence that Cdc42-dependent Rho1 inhibition is mediated by the Cdc42 target Pak1 kinase. Our work proposes a mechanism of Rho1 regulation by active Cdc42 to coordinate timely septum formation and cytokinesis fidelity. 
    more » « less
  5. ; ; ; ; ; ; ; ; ; ; et al (, Journal of Virology)
    Longnecker, Richard M. (Ed.)
    ABSTRACT A cascade of protein-protein interactions between four herpes simplex virus (HSV) glycoproteins (gD, gH/gL, and gB) drive fusion between the HSV envelope and host membrane, thereby allowing for virus entry and infection. Specifically, binding of gD to one of its receptors induces a conformational change that allows gD to bind to the regulatory complex gH/gL, which then activates the fusogen gB, resulting in membrane fusion. Using surface plasmon resonance and a panel of anti-gD monoclonal antibodies (MAbs) that sterically blocked the interaction, we previously showed that gH/gL binds directly to gD at sites distinct from the gD receptor binding site. Here, using an analogous strategy, we first evaluated the ability of a panel of uncharacterized anti-gH/gL MAbs to block binding to gD and/or inhibit fusion. We found that the epitopes of four gD-gH/gL-blocking MAbs were located within flexible regions of the gH N terminus and the gL C terminus, while the fifth was placed around gL residue 77. Taken together, our data localized the gD binding region on gH/gL to a group of gH and gL residues at the membrane distal region of the heterodimer. Surprisingly, a second set of MAbs did not block gD-gH/gL binding but instead stabilized the complex by altering the kinetic binding. However, despite this prolonged gD-gH/gL interaction, “stabilizing” MAbs also inhibited cell-cell fusion, suggesting a unique mechanism by which the fusion process is halted. Our findings support targeting the gD-gH/gL interaction to prevent fusion in both therapeutic and vaccine strategies against HSV. IMPORTANCE Key to developing a human HSV vaccine is an understanding of the virion glycoproteins involved in entry. HSV employs multiple glycoproteins for attachment, receptor interaction, and membrane fusion. Determining how these proteins function was resolved, in part, by structural biology coupled with immunological and biologic evidence. After binding, virion gD interacts with a receptor to activate the regulator gH/gL complex, triggering gB to drive fusion. Multiple questions remain, one being the physical location of each glycoprotein interaction site. Using protective antibodies with known epitopes, we documented the long-sought interaction between gD and gH/gL, detailing the region on gD important to create the gD-gH/gL triplex. Now, we have identified the corresponding gD contact sites on gH/gL. Concurrently we discovered a novel mechanism whereby gH/gL antibodies stabilize the complex and inhibit fusion progression. Our model for the gD-gH/gL triplex provides a new framework for studying fusion, which identifies targets for vaccine development. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email