Cell Surface Vimentin Is an Attachment Receptor for Enterovirus 71

Enterovirus 71 (EV71), a positive-stranded RNA virus, is the major cause of hand, foot, and mouth disease (HFMD) in children, which can cause severe central nervous system disease and death. The capsids of EV71 consist of 60 copies of each of four viral structural proteins (VP1 to VP4), with VP1, VP2, and VP3 exposed on the surface and VP4 arranged internally. VP1 plays a central role in particle assembly and cell entry. To gain insight into the role of positively charged residues in VP1 function in these processes, a charged-to-alanine scanning analysis was performed using an infectious cDNA clone of EV71. Twenty-seven mutants containing single charged-to-alanine changes were tested. Sixteen of them were not viable, seven mutants were replication defective, and the remaining four mutants were replication competent. By selecting revertants, second-site mutations which could at least partially restore viral infectivity were identified within VP1 for four defective mutations and two lethal mutations. The resulting residue pairs represent a network of intra-and intermolecular interactions of the VP1 protein which could serve as a potential novel drug target. Interestingly, mutation K215A in the VP1 GH loop led to a significant increase in thermal stability, demonstrating that conditional thermostable mutants can be generated by altering the charge characteristics of VP1. Moreover, all mutants were sensitive to the EV71 entry inhibitor suramin, which binds to the virus particle via the negatively charged naphthalenetrisulfonic acid group, suggesting that single charged-to-alanine mutation is not sufficient for suramin resistance. Taken together, these data highlight the importance of positively charged residues in VP1 for production of infectious particles. IMPORTANCEInfection with EV71 is more often associated with neurological complications in children and is responsible for the majority of fatalities. No licensed vaccines or antiviral therapies are currently available for the prevention or treatment of EV71 infection. Understanding the determinants of virion assembly and entry will facilitate vaccine development and drug discovery. Here, we identified 23 out of 27 positively charged residues in VP1 which impaired or blocked the production of infectious particles. The defect could be rescued by second-site mutations within the VP1 protein. Our findings highlight the importance of positively charged residues in VP1 during infectious particle production and reveal a potential strategy for blocking EV71 infections by inhibiting intra-or intermolecular interactions of the VP1 protein. Enterovirus 71 (EV71) is a nonenveloped icosahedral RNA virus belonging to genus Enterovirus within the family Picornaviridae. Since its first isolation from the stool of an infant with encephalitis in California in 1969 (1), EV71 has been recognized as a major cause of hand, foot, and mouth disease (HFMD), which is associated with severe neurological symptoms in a small proportion of cases (2). There has been a significant incre...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Identification of Positively Charged Residues in Enterovirus 71 Capsid Protein VP1 Essential for Production of Infectious Particles

Yuan

Wang

et al. 2016

“…P-selectin glycoprotein ligand 1 (PSGL-1) binds at the 5-fold vertex and enhances EV71 infection of cells that express low levels of SCARB2 (7,8). The binding sites for sialylated glycans, annexin II, heparin sulfate, and extracellular vimentin are unknown (9)(10)(11)(12)(13)(14). After receptor-mediated cell entry, the virus replicates in the cytoplasm.…”

Section: Importancementioning

confidence: 99%

The Enterovirus 71 Procapsid Binds Neutralizing Antibodies and Rescues Virus Infection In Vitro

Shingler

Cifuente

Ashley

et al. 2015

Enterovirus 71 (EV71) is responsible for seasonal outbreaks of hand, foot, and mouth disease in the Asia-Pacific region. The virus has the capability to cause severe disease and death, especially in young children. Although several vaccines are currently in clinical trials, no vaccines or therapeutics have been approved for use. Previous structural studies have revealed that two antigenically distinct capsid forms are produced in EV71-infected cells: an expanded empty capsid, sometimes called a procapsid, and the infectious virus. Specifically, an immunodominant epitope of EV71 that maps to the virus canyon is structurally different in the procapsid and virus. This structure-function study shows that the procapsid can sequester antibodies, thus enhancing EV71 infection in vitro. The results presented here suggest that, due to conformational differences between the EV71 procapsid and virus, the presence of the procapsid in natural virus infections should be considered in the future design of vaccines or therapeutics. IMPORTANCEIn a picornavirus infection, both an infectious and a noninfectious empty capsid, sometimes referred to as a procapsid, are produced. It was novel to discover that the procapsid form of EV71 was expanded and antigenically distinct from the infectious virus. Previously, it had been supposed that this empty capsid was an off-pathway dead end or at best served for storage of pentameric subunits, which was later shown to be unlikely. It remains unexplained why picornaviruses evolutionarily conserve the wasteful production of so much noninfectious capsid. Here, we demonstrate that the EV71 procapsid has different antigenic properties than the infectious virus. Thus, the procapsid has the capacity to sequester neutralizing antibody and protect the virus, promoting or restoring a successful infection in vitro. This important observation should be considered in the future design and development of vaccines and therapeutics.

“…Because virus receptors determine the host range, tissue tropism, and pathogenesis (4-6), the identification of factors that mediate the recognition and/or entry of EV71 to host cells is essential to decipher infection mechanisms. Several receptors or attachment molecules for EV71 have been identified, including scavenger receptor B2 (SCARB2), P-selectin glycoprotein ligand-1 (PSGL-1), sialylated glycoprotein, dendritic cell-specific ICAM 3-grabbing nonintegrin, annexin II, vimentin, and polysaccharide (heparin sulfate) (7)(8)(9)(10)(11)(12)(13). EV71 strains can be divided into different subgenotypes based on sequence homology (14).…”

mentioning

confidence: 99%

Cell Surface Nucleolin Facilitates Enterovirus 71 Binding and Infection

Wang

Huang

et al. 2015

Because the pathogenesis of enterovirus 71 (EV71) remains mostly ambiguous, identifying the factors that mediate viral binding and entry to host cells is indispensable to ultimately uncover the mechanisms that underlie virus infection and pathogenesis. Despite the identification of several receptors/attachment molecules for EV71, the binding, entry, and infection mechanisms of EV71 remain unclear. Herein, we employed glycoproteomic approaches to identify human nucleolin as a novel binding receptor for EV71. Glycoproteins purified by lectin chromatography from the membrane extraction of human cells were treated with sialidase, followed by immunoprecipitation with EV71 particles. Among the 16 proteins identified by tandem mass spectrometry analysis, cell surface nucleolin attracted our attention. We found that EV71 interacted directly with nucleolin via the VP1 capsid protein and that an antinucleolin antibody reduced the binding of EV71 to human cells. In addition, the knockdown of cell surface nucleolin decreased EV71 binding, infection, and production in human cells. Furthermore, the expression of human nucleolin on the cell surface of a mouse cell line increased EV71 binding and conferred EV71 infection and production in the cells. These results strongly indicate that human nucleolin can mediate EV71 binding to and infection of cells. Our findings also demonstrate that the use of glycoproteomic approaches is a reliable methodology to discover novel receptors for pathogens. IMPORTANCEOutbreaks of EV71 have been reported in Asia-Pacific countries and have caused thousands of deaths in young children during the last 2 decades. The discovery of new EV71-interacting molecules to understand the infection mechanism has become an emergent issue. Hence, this study uses glycoproteomic approaches to comprehensively investigate the EV71-interacting glycoproteins. Several EV71-interacting glycoproteins are identified, and the role of cell surface nucleolin in mediating the attachment and entry of EV71 is characterized and validated. Our findings not only indicate a novel target for uncovering the EV71 infection mechanism and anti-EV71 drug discovery but also provide a new strategy for virus receptor identification.