The Essential Role of Clathrin-mediated Endocytosis in the Infectious Entry of Human Enterovirus 71

Hussain, Khairunnisa’ Mohamed; Leong, Kim Lian Janet; Ng, Mary Mah-Lee; Chu, Justin Jang Hann

doi:10.1074/jbc.m110.168468

Cited by 127 publications

(130 citation statements)

References 70 publications

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“…To further understand the process of EV71 internalization, we performed double staining of EV71 (in the absence of neutralizing MAbs) with the early and late endosome markers EEA1 and LAMP1, respectively. Colocalization of EV71 with EEA1 was observed as early as 5 min postinfection, increased afterwards, and was sustained until 60 min postinfection (the last time point tested), whereas colocalization of EV71 with LAMP1 was clearly seen from 30 to 60 min postinfection (data not shown), indicating that internalization and subsequent transport of EV71 virions to the early and late endosomes occur mainly within 15 to 30 min after infection, in agreement with the similar findings reported previously (50). By comparing the localization pattern and timing of EV71 in the presence of D5 to those in the absence of the antibody, it was concluded that the internalization of EV71 is prohibited by the D5 antibody treatment.…”

Section: Discussionsupporting

confidence: 81%

“…For a given sample, the neutralization efficiency was calculated by normalization of its OD 490 value to that of the sample treated with virus only as follows: percent neutralization ϭ [(OD 490 of the given sample Ϫ OD 490 of the sample treated with virus only)/(OD 490 of the mock-treated sample Ϫ OD 490 of the sample treated with virus only)] ϫ 100. The 50% inhibitory concentration (IC 50 ) was defined as the MAb concentration that reduced cell death by 50%.…”

Section: Cells and Virusesmentioning

confidence: 99%

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Single Neutralizing Monoclonal Antibodies Targeting the VP1 GH Loop of Enterovirus 71 Inhibit both Virus Attachment and Internalization during Viral Entry

Shi

et al. 2015

J Virol

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Antibodies play a critical role in immunity against enterovirus 71 (EV71). However, how EV71-specific antibodies neutralize infections remains poorly understood. Here we report the working mechanism for a group of three monoclonal antibodies (MAbs) that potently neutralize EV71. We found that these three MAbs (termed D5, H7, and C4, respectively) recognize the same conserved neutralizing epitope within the VP1 GH loop of EV71. Single MAbs in this group, exemplified by D5, could inhibit EV71 infection in cell cultures at both the pre-and postattachment stages in a cell type-independent manner. Specifically, MAb treatment resulted in the blockade of multiple steps of EV71 entry, including virus attachment, internalization, and subsequent uncoating and RNA release. Furthermore, we show that the D5 and C4 antibodies can interfere with EV71 binding to its key receptors, including heparan sulfate, SCARB2, and PSGL-1, thus providing a possible explanation for the observed multi-inhibitory function of the MAbs. Collectively, our study unravels the mechanism of neutralization by a unique group of anti-EV71 MAbs targeting the conserved VP1 GH loop. The findings should enhance our understanding of MAb-mediated immunity against enterovirus infections and accelerate the development of MAb-based anti-EV71 therapeutic drugs. IMPORTANCEEnterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease (HFMD), which has caused significant morbidities and mortalities in young children. Neither a vaccine nor an antiviral drug is available. Neutralizing antibodies are major protective components in EV71 immunity. Here, we unraveled an unusual mechanism of EV71 neutralization by a group of three neutralizing monoclonal antibodies (MAbs). All of these MAbs bound the same conserved epitope located at the VP1 GH loop of EV71. Interestingly, mechanistic studies showed that single antibodies in this MAb group could block EV71 attachment and internalization during the viral entry process and interfere with EV71 binding to heparan sulfate, SCARB2, and PSGL-1 molecules, which are key receptors involved in different steps of EV71 entry. Our findings greatly enhance the understanding of the interplays among EV71, neutralizing antibodies, and host receptors, which in turn should facilitate the development of an MAbbased anti-EV71 therapy. E nterovirus 71 (EV71) is one of the major causative agents of hand, foot, and mouth disease (HFMD), which has been prevalent in Southeast Asia (1). Patients with severe HFMD cases manifest neurological complications, such as brainstem encephalitis and pulmonary edema, resulting in death, and such cases are often associated with EV71 infection (2). No prophylactic vaccine against EV71 infection or therapeutic drug for EV71 infection is available. Even though inactivated whole-virus-based EV71 vaccines have progressed into clinical trials (3, 4), their licensure faces serious challenges (5). Thus, the development of efficacious antiviral drugs for the treatment of EV71-infected patients ...

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Section: Discussionsupporting

confidence: 81%

Section: Cells and Virusesmentioning

confidence: 99%

Single Neutralizing Monoclonal Antibodies Targeting the VP1 GH Loop of Enterovirus 71 Inhibit both Virus Attachment and Internalization during Viral Entry

Shi

et al. 2015

J Virol

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“…The second receptor, reported by Yamayoshi et al (9), was human SCARB2, which is expressed on most types of cells (7). EV-71 binds to the SCARB2 receptor and is then internalized through clathrin-mediated endocytosis (47,48). However, blocking of these receptors did not abolish EV-71 infection, and this finding led to the discovery of the third receptor, annexin II.…”

Section: Discussionmentioning

confidence: 99%

Enterovirus 71 Uses Cell Surface Heparan Sulfate Glycosaminoglycan as an Attachment Receptor

Tan

Poh

Sam

et al. 2013

J Virol

169

183

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“…The EF loop region of VP1, which lines the wall of the canyon on the viral surface, was found to be important for binding to SCARB2 (24). EV71 infection via the SCARB2-dependent pathway was inhibited by a small interfering RNA (siRNA) treatment against the molecules that are involved in the clathrin-dependent endocytic pathway and by inhibitors of endosomal acidification (25,26). In addition to EV71, coxsackievirus A7 (CVA7), CVA14, and CVA16 have utilized SCARB2 as a receptor for infection (17,22).…”

mentioning

confidence: 99%

Functional Comparison of SCARB2 and PSGL1 as Receptors for Enterovirus 71

Yamayoshi

Ohka

Fujii

et al. 2013

J Virol

110

126

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Human scavenger receptor class B, member 2 (SCARB2), and P-selectin glycoprotein ligand-1 (PSGL1) have been identified to be the cellular receptors for enterovirus 71 (EV71). We compared the EV71 infection efficiencies of mouse L cells that expressed SCARB2 (L-SCARB2) and PSGL1 (L-PSGL1) and the abilities of SCARB2 and PSGL1 to bind to the virus. L-SCARB2 cells bound a reduced amount of EV71 compared to L-PSGL1 cells. However, EV71 could infect L-SCARB2 cells more efficiently than L-PSGL1 cells. The results suggested that the difference in the binding capacities of the two receptors was not the sole determinant of the infection efficiency and that SCARB2 plays an essential role after attaching to virions. Therefore, we examined the viral entry into L-SCARB2 cells and L-PSGL1 cells by immunofluorescence microscopy. In both cells, we detected internalized EV71 virions that colocalized with an early endosome marker. We then performed a sucrose density gradient centrifugation analysis to evaluate viral uncoating. After incubating the EV71 virion with L-SCARB2 cells or soluble SCARB2 under acidic conditions below pH 6.0, we observed that part of the native virion was converted into an empty capsid that lacked both genomic RNA and VP4 capsid proteins. The results suggested that the uncoating of EV71 requires both SCARB2 and an acidic environment and occurs after the internalization of the virus-receptor complex into endosomes. However, the empty capsid formation was not observed after incubation with L-PSGL1 cells or soluble PSGL1 under any of the tested pH conditions. These results indicated that SCARB2 is capable of viral binding, viral internalization, and viral uncoating and that the low infection efficiency of L-PSGL1 cells is due to the inability of PSGL1 to induce viral uncoating. The characterization of SCARB2 as an uncoating receptor greatly contributes to the understanding of the early steps of EV71 infection.

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The Essential Role of Clathrin-mediated Endocytosis in the Infectious Entry of Human Enterovirus 71

Cited by 127 publications

References 70 publications

Single Neutralizing Monoclonal Antibodies Targeting the VP1 GH Loop of Enterovirus 71 Inhibit both Virus Attachment and Internalization during Viral Entry

Single Neutralizing Monoclonal Antibodies Targeting the VP1 GH Loop of Enterovirus 71 Inhibit both Virus Attachment and Internalization during Viral Entry

Enterovirus 71 Uses Cell Surface Heparan Sulfate Glycosaminoglycan as an Attachment Receptor

Functional Comparison of SCARB2 and PSGL1 as Receptors for Enterovirus 71

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