scholarly journals ATP1A1-Mediated Src Signaling Inhibits Coronavirus Entry into Host Cells

2015 ◽  
Vol 89 (8) ◽  
pp. 4434-4448 ◽  
Author(s):  
Christine Burkard ◽  
Monique H. Verheije ◽  
Bart L. Haagmans ◽  
Frank J. van Kuppeveld ◽  
Peter J. M. Rottier ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Crucial Role ◽  
Influenza A ◽  
Hepatitis Virus ◽  
Early Stage ◽  
Virus Entry ◽  
Host Cells ◽  
Α Subunit ◽  
Src Signaling ◽  
Recent Emergence

ABSTRACTIn addition to transporting ions, the multisubunit Na+,K+-ATPase also functions by relaying cardiotonic steroid (CTS)-binding-induced signals into cells. In this study, we analyzed the role of Na+,K+-ATPase and, in particular, of its ATP1A1 α subunit during coronavirus (CoV) infection. As controls, the vesicular stomatitis virus (VSV) and influenza A virus (IAV) were included. Using gene silencing, the ATP1A1 protein was shown to be critical for infection of cells with murine hepatitis virus (MHV), feline infectious peritonitis virus (FIPV), and VSV but not with IAV. Lack of ATP1A1 did not affect virus binding to host cells but resulted in inhibited entry of MHV and VSV. Consistently, nanomolar concentrations of the cardiotonic steroids ouabain and bufalin, which are known not to affect the transport function of Na+,K+-ATPase, inhibited infection of cells with MHV, FIPV, Middle East respiratory syndrome (MERS)-CoV, and VSV, but not IAV, when the compounds were present during virus inoculation. Cardiotonic steroids were shown to inhibit entry of MHV at an early stage, resulting in accumulation of virions close to the cell surface and, as a consequence, in reduced fusion. In agreement with an early block in infection, the inhibition of VSV by CTSs could be bypassed by low-pH shock. Viral RNA replication was not affected when these compounds were added after virus entry. The antiviral effect of ouabain could be relieved by the addition of different Src kinase inhibitors, indicating that Src signaling mediated via ATP1A1 plays a crucial role in the inhibition of CoV and VSV infections.IMPORTANCECoronaviruses (CoVs) are important pathogens of animals and humans, as demonstrated by the recent emergence of new human CoVs of zoonotic origin. Antiviral drugs targeting CoV infections are lacking. In the present study, we show that the ATP1A1 subunit of Na+,K+-ATPase, an ion transporter and signaling transducer, supports CoV infection. Targeting ATP1A1 either by gene silencing or by low concentrations of the ATP1A1-binding cardiotonic steroids ouabain and bufalin resulted in inhibition of infection with murine, feline, and MERS-CoVs at an early entry stage. Infection with the control virus VSV was also inhibited. Src signaling mediated by ATP1A1 was shown to play a crucial role in the inhibition of virus entry by ouabain and bufalin. These results suggest that targeting the Na+,K+-ATPase using cardiotonic steroids, several of which are FDA-approved compounds, may be an attractive therapeutic approach against CoV and VSV infections.

scholarly journals LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

2020 ◽  
Vol 94 (18) ◽  
Author(s):  
Xuesen Zhao ◽  
Shuangli Zheng ◽  
Danying Chen ◽  
Mei Zheng ◽  
Xinglin Li ◽  
...  
Keyword(s):  
Influenza A ◽  
Yellow Fever Virus ◽  
Virus Entry ◽  
Ectopic Expression ◽  
A549 Cells ◽  
Cellular Protein ◽  
Host Cells ◽  
Cellular Proteins ◽  
Human Coronavirus ◽  
Human Coronaviruses

ABSTRACT C3A is a subclone of the human hepatoblastoma HepG2 cell line with strong contact inhibition of growth. We fortuitously found that C3A was more susceptible to human coronavirus HCoV-OC43 infection than HepG2, which was attributed to the increased efficiency of virus entry into C3A cells. In an effort to search for the host cellular protein(s) mediating the differential susceptibility of the two cell lines to HCoV-OC43 infection, we found that ArfGAP with dual pleckstrin homology (PH) domains 2 (ADAP2), gamma-interferon-inducible lysosome/endosome-localized thiolreductase (GILT), and lymphocyte antigen 6 family member E (LY6E), the three cellular proteins identified to function in interference with virus entry, were expressed at significantly higher levels in HepG2 cells. Functional analyses revealed that ectopic expression of LY6E, but not GILT or ADAP2, in HEK 293 cells inhibited the entry of HCoV-O43. While overexpression of LY6E in C3A and A549 cells efficiently inhibited the infection of HCoV-OC43, knockdown of LY6E expression in HepG2 significantly increased its susceptibility to HCoV-OC43 infection. Moreover, we found that LY6E also efficiently restricted the entry mediated by the envelope spike proteins of other human coronaviruses, including the currently pandemic SARS-CoV-2. Interestingly, overexpression of serine protease TMPRSS2 or amphotericin treatment significantly neutralized the IFN-inducible transmembrane 3 (IFITM3) restriction of human coronavirus (CoV) entry, but did not compromise the effect of LY6E on the entry of human coronaviruses. The work reported herein thus demonstrates that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis via a mechanism distinct from other factors that modulate CoV entry. IMPORTANCE Virus entry into host cells is one of the key determinants of host range and cell tropism and is subjected to the control of host innate and adaptive immune responses. In the last decade, several interferon-inducible cellular proteins, including IFITMs, GILT, ADAP2, 25CH, and LY6E, had been identified to modulate the infectious entry of a variety of viruses. Particularly, LY6E was recently identified as a host factor that facilitates the entry of several human-pathogenic viruses, including human immunodeficiency virus, influenza A virus, and yellow fever virus. Identification of LY6E as a potent restriction factor of coronaviruses expands the biological function of LY6E and sheds new light on the immunopathogenesis of human coronavirus infection.

In situ labelling chemistry of respiratory syncytial viruses by employing the biotinylated host-cell membrane protein for tracking the early stage of virus entry

2014 ◽  
Vol 50 (99) ◽  
pp. 15776-15779 ◽  
Author(s):  
Lin Ling Zheng ◽  
Xiao Xi Yang ◽  
Yue Liu ◽  
Xiao Yan Wan ◽  
Wen Bi Wu ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Membrane Protein ◽  
Quantum Dot ◽  
Early Stage ◽  
Virus Entry ◽  
Host Cells ◽  
Host Cell Membrane ◽  
Respiratory Syncytial Viruses ◽  
Cell Membrane Protein

Anin situstrategy for producing quantum dot-labelled respiratory syncytial viruses by incorporating the biotinylated membrane protein of the host cells into mature virions is reported.

scholarly journals The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry

2019 ◽  
Vol 94 (2) ◽  
Author(s):  
Guangwen Wang ◽  
Li Jiang ◽  
Jinliang Wang ◽  
Jie Zhang ◽  
Fandi Kong ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Early Stage ◽  
A549 Cells ◽  
Replication Cycle ◽  
Host Factors ◽  
Host Cells ◽  
Signaling Cascade ◽  
G Protein Coupled

ABSTRACT Influenza A virus (IAV) coopts numerous host factors to complete its replication cycle. Here, we identify free fatty acid receptor 2 (FFAR2) as a cofactor for IAV entry into host cells. We found that downregulation of FFAR2 or Ffar2 expression significantly reduced the replication of IAV in A549 or RAW 264.7 cells. The treatment of A549 cells with small interfering RNA (siRNA) targeting FFAR2 or the FFAR2 pathway agonists 2-(4-chlorophenyl)-3-methyl-N-(thiazol-2-yl)butanamide (4-CMTB) and compound 58 (Cmp58) [(S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide] dramatically inhibited the nuclear accumulation of viral nucleoprotein (NP) at early time points postinfection, indicating that FFAR2 functions in the early stage of the IAV replication cycle. FFAR2 downregulation had no effect on the expression of sialic acid (SA) receptors on the cell membrane, the attachment of IAV to the SA receptors, or the activity of the viral ribonucleoprotein (vRNP) complex. Rather, the amount of internalized IAVs was significantly reduced in FFAR2-knocked-down or 4-CMTB- or Cmp58-treated A549 cells. Further studies showed that FFAR2 associated with β-arrestin1 and that β-arrestin1 interacted with the β2-subunit of the AP-2 complex (AP2B1), the essential adaptor of the clathrin-mediated endocytosis pathway. Notably, siRNA knockdown of either β-arrestin1 or AP2B1 dramatically impaired IAV replication, and AP2B1 knockdown or treatment with Barbadin, an inhibitor targeting the β-arrestin1/AP2B1 complex, remarkably decreased the amount of internalized IAVs. Moreover, we found that FFAR2 interacted with three G protein-coupled receptor (GPCR) kinases (i.e., GRK2, GRK5, and GRK6) whose downregulation inhibited IAV replication. Together, our findings demonstrate that the FFAR2 signaling cascade is important for the efficient endocytosis of IAV into host cells. IMPORTANCE To complete its replication cycle, IAV hijacks the host endocytosis machinery to invade cells. However, the underlying mechanisms of how IAV is internalized into host cells remain poorly understood, emphasizing the need to elucidate the role of host factors in IAV entry into cells. In this study, we identified FFAR2 as an important host factor for the efficient replication of both low-pathogenic and highly pathogenic IAV. We revealed that FFAR2 facilitates the internalization of IAV into target cells during the early stage of infection. Upon further characterization of the role of FFAR2-associated proteins in virus replication, we found that the FFAR2–β-arrestin1–AP2B1 signaling cascade is important for the efficient endocytosis of IAV. Our findings thus further our understanding of the biological details of IAV entry into host cells and establish FFAR2 as a potential target for antiviral drug development.

scholarly journals The Mechanism of Poly-Galloyl-Glucoses Preventing Influenza A Virus Entry into Host Cells

PLoS ONE ◽  
2014 ◽  
Vol 9 (4) ◽  
pp. e94392 ◽  
Author(s):  
Hu Ge ◽  
Ge Liu ◽  
Yang-Fei Xiang ◽  
Yu Wang ◽  
Chao-Wan Guo ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Virus Entry ◽  
Host Cells

scholarly journals Amantadine Variants Activity Against Multiple Influenza A Viruses

2021 ◽  
Author(s):  
Μarianna Stampolaki ◽  
Christina Tzitzoglaki ◽  
Christos Liolios ◽  
Anja Hoffmann ◽  
Brent Johnson ◽  
...  
Keyword(s):  
Influenza A ◽  
Early Stage ◽  
Biological Activities ◽  
Point Mutations ◽  
New Drugs ◽  
Host Cells ◽  
Wild Type ◽  
Influenza A Viruses ◽  
Proton Current ◽  
Antigenic Shift

Future pandemic influenza necessitates the development of new drugs against the current circulating, amantadine and rimantadine drugs resistant, influenza A M2 S31N viruses. The possibility of an antigenic shift to M2 S31 necessitates ranking the biological activities of amantadine variants. Several amantadine variants have been tested by different laboratories, but various M2 wild type influenza A strains have been used with different sensitivity against amantadine and the unambiguous comparison between potencies is not straightforward. Here, we compared the anti-influenza activities of 57 synthetic amantadine variants against influenza A WSN/33 viruses with amantadine-sensitive M2 WT, with a range of over three digits providing a reference set of potencies for structure-activity relationships, and amantadine-resistant M2 S31N proteins (and observed no potent compounds). 17 compounds were selected and tested against M2 L26F, V27A, A30T, G34E viruses. We tested few reference compounds using electrophysiology and explored point mutations which both showed that M2 is the target of potent antiviral potency against the M2 WT, L26F, V27A viruses. Major findings are: (a) Several amantadine variants from Kolocouris group block only M2 WT and M2 L26F-mediated proton current and the corresponding viruses replication. (b) A compound from Vazquez’s group is a triple blocker of M2 WT, L26F, V27A channels and viruses replication. (c) A compound from Vazquez’s group blocks only M2 L26 channel and virus replication. (d) Several compounds from Kolocouris group have potent activity against several influenza A M2 WT and three M2 S31N viruses, eg. the pandemic A/H1N1/California/07/2009 (H1N1pdm09) or A/H1N1/PuertoRico/08/1934 without blocking M2 S31N. The compounds and their cocktails while not to be more toxic than amantadine might be useful for re-purposing of amantadine class of drugs in the case (i) of the prevalence of M2 L26F and or M2 V27A strains (ii) of an antigenic shift of the virus to M2 WT and (iii) because they inhibited a broad panel of M2 WT and M2 S31N viruses including the H1N1pdm09). (d) We showed that the mechanism of antiviral activity against A/California/07/2009 or A/PR/08/1934 and possibly also M2 WT viruses compared to WSN/33 viruses is not due to inhibition of an early stage of virus infection or a late stage of M2 channel function during endocytosis or inhibition of HA binding to host cells or a different pH for HA fusion or a lysosomotropic effect.

scholarly journals The immunomodulatory CEA cell adhesion molecule 6 (CEACAM6/CD66c) is a candidate receptor for the influenza A virus

2017 ◽  
Author(s):  
Shah Kamranur Rahman ◽  
Mairaj Ahmed Ansari ◽  
Pratibha Gaur ◽  
Imtiyaz Ahmad ◽  
Chandrani Chakravarty ◽  
...  
Keyword(s):  
Cell Surface ◽  
Influenza A Virus ◽  
Influenza A ◽  
Virus Entry ◽  
Semipermeable Membrane ◽  
Host Cells ◽  
Lung Cells ◽  
Membrane Glycoprotein ◽  
Virus Binding ◽  
Host Membrane

AbstractTo establish a productive infection in host cells, viruses often use one or multiple host membrane glycoprotein as their receptors. For Influenza A virus (IAV) such a glycoprotein receptor has not been described, to date. Here we show that IAV is using the host membrane glycoprotein CD66c as a receptor for entry into human epithelial lung cells. Neuraminidase (NA), a viral spike protein binds to CD66c on the cell surface during IAV entry into the host cells. Lung cells overexpressing CD66c showed an increase in virus binding and subsequent entry into the cell. Upon comparison, CD66c demonstrated higher binding capacity than other membrane glycoproteins (EGFR and DC-SIGN) reported earlier to facilitate IAV entry into host cells. siRNA mediated knockdown of CD66c from lung cells inhibited virus binding on cell surface and entry into cells. Blocking CD66c by antibody on the cell surface resulted in decreased virus entry. We found CD66c is a specific glycoprotein receptor for influenza A virus that did not affect entry of non-IAV RNA virus (Hepatitis C virus). Finally, IAV pre-incubated with recombinant CD66c protein when administered intranasally in mice showed decreased cytopathic effects in mice lungs. This publication is the first to report CD66c (CEACAM6) as a glycoprotein receptor for Influenza A virus.Significance StatementCells are enclosed by a semipermeable membrane that allows selective exchange of biomolecules between cells and their surroundings. A set of specialized proteins in this semipermeable membrane, work like gatekeepers to the cell and regulate entry of these biomolecules. One class of such surface proteins is termed as receptors. Viruses bind to one or more of these receptors and manipulate gatekeepers for their own successful entry into host-cells. A membrane protein that influenza A virus (Flu virus) uses for entry into the cells was not discovered till date. This study reports for the first time, a receptor for influenza A virus, that was sought after by researchers for decades. The viral receptor is a promising target that can be used to inhibit virus entry into host cells.

scholarly journals LY6E Restricts the Entry of Human Coronaviruses, including the currently pandemic SARS-CoV-2

2020 ◽  
Author(s):  
Xuesen Zhao ◽  
Shuangli Zheng ◽  
Danying Chen ◽  
Mei Zheng ◽  
Xinglin Li ◽  
...  
Keyword(s):  
Influenza A ◽  
Yellow Fever Virus ◽  
Virus Entry ◽  
Ectopic Expression ◽  
A549 Cells ◽  
Cellular Protein ◽  
Host Cells ◽  
Cellular Proteins ◽  
Human Coronavirus ◽  
Human Coronaviruses

ABSTRACTC3A is a sub-clone of human hepatoblastoma HepG2 cell line with the strong contact inhibition of growth. We fortuitously found that C3A was more susceptible to human coronavirus HCoV-OC43 infection than HepG2, which was attributed to the increased efficiency of virus entry into C3A cells. In an effort to search for the host cellular protein(s) mediating the differential susceptibility of the two cell lines to HCoV-OC43 infection, we found that ADAP2, GILT and LY6E, three cellular proteins with known activity of interfering virus entry, expressed at significantly higher levels in HepG2 cells. Functional analyses revealed that ectopic expression of LY6E, but not GILT or ADAP2, in HEK 293 cells inhibited the entry of HCoV-OC43. While overexpression of LY6E in C3A and A549 cells efficiently inhibited the infection of HCoV-OC43, knockdown of LY6E expression in HepG2 significantly increased its susceptibility to HCoV-OC43 infection. Moreover, we found that LY6E also efficiently restricted the entry mediated by the envelope spike proteins of other human coronaviruses, including the currently pandemic SARS-CoV-2. Interestingly, overexpression of serine protease TMPRSS2 or amphotericin treatment significantly neutralized the IFITM3 restriction of human coronavirus entry, but did not compromise the effect of LY6E on the entry of human coronaviruses. The work reported herein thus demonstrates that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis via a distinct mechanism.ImportanceVirus entry into host cells is one of the key determinants of host range and cell tropism and is subjected to the control by host innate and adaptive immune responses. In the last decade, several interferon inducible cellular proteins, including IFITMs, GILT, ADAP2, 25CH and LY6E, had been identified to modulate the infectious entry of a variety of viruses. Particularly, LY6E was recently identified as host factors to facilitate the entry of several human pathogenic viruses, including human immunodeficiency virus, influenza A virus and yellow fever virus. Identification of LY6E as a potent restriction factor of coronaviruses expands the biological function of LY6E and sheds new light on the immunopathogenesis of human coronavirus infection.

scholarly journals Stress-Inducible Gene Atf3 Dictates a Dichotomous Macrophage Activity in Chemotherapy-Enhanced Lung Colonization

2021 ◽  
Vol 22 (14) ◽  
pp. 7356
Author(s):  
Justin D. Middleton ◽  
Jared Fehlman ◽  
Subhakeertana Sivakumar ◽  
Daniel G. Stover ◽  
Tsonwin Hai
Keyword(s):  
Cancer Cells ◽  
Cancer Cell ◽  
Early Stage ◽  
Mechanistic Explanation ◽  
Host Cells ◽  
Secondary Site ◽  
Initial Increase ◽  
Cell Retention ◽  
Lung Colonization ◽  
Inducible Gene

Previously, we showed that chemotherapy paradoxically exacerbated cancer cell colonization at the secondary site in a manner dependent on Atf3, a stress-inducible gene, in the non-cancer host cells. Here, we present evidence that this phenotype is established at an early stage of colonization within days of cancer cell arrival. Using mouse breast cancer models, we showed that, in the wild-type (WT) lung, cyclophosphamide (CTX) increased the ability of the lung to retain cancer cells in the vascular bed. Although CTX did not change the WT lung to affect cancer cell extravasation or proliferation, it changed the lung macrophage to be pro-cancer, protecting cancer cells from death. This, combined with the initial increase in cell retention, resulted in higher lung colonization in CTX-treated than control-treated mice. In the Atf3 knockout (KO) lung, CTX also increased the ability of lung to retain cancer cells. However, the CTX-treated KO macrophage was highly cytotoxic to cancer cells, resulting in no increase in lung colonization—despite the initial increase in cell retention. In summary, the status of Atf3 dictates the dichotomous activity of macrophage: pro-cancer for CTX-treated WT macrophage but anti-cancer for the KO counterpart. This dichotomy provides a mechanistic explanation for CTX to exacerbate lung colonization in the WT but not Atf3 KO lung.

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