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

Abstract

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...

A Single Mutation in the VP1 of Enterovirus 71 Is Responsible for Increased Virulence and Neurotropism in Adult Interferon-Deficient Mice

“…The importance of having positively charged residues at the 5-fold axis of VP1 has been shown to be crucial for production of infectious particles (10). However, we provide evidence that suggests K244E mutants can produce infectious virus and do in fact replicate in mice (Fig.…”

“…K244E did not produce virus whose titer could be determined on RD cells by TCID 50 . A similar phenomenon was observed when position 244 of VP1 was mutated from a lysine to another amino acid in previous EV71 studies (10,14). We concluded that the mutant virus was not viable and hypothesized that a positively charged residue at position 244 was needed to produce infectious virus.…”

“…Also, the virion has been shown to change drastically at the N terminus of VP1 during uncoating and moderately at the 5-fold axis (42). Also, previous work has shown that viable virus could only be recovered when positive charges at the 5-fold axis were mutated by introducing compensatory mutations hypothesized to enhance the posi-tive electrostatic potential, restoring interactions between VP1 monomers (10). The observation that K244E requires compensation for replication in tissue culture has also been reported (14).…”

“…This region is critical for receptor attachment and necessary for the stability of infectious virions. Several of these positively charged residues are conserved in sequences among all genogroups of EV71, suggesting their importance to the virus (10,11). These residues aid EV71 to bind to several receptors on different tissues as it disseminates throughout its host.…”

“…One critical domain documented as an important contributor to virulence includes a group of positively charged amino acids near the 5-fold vertices of the EV71 capsid (5,10). This region is critical for receptor attachment and necessary for the stability of infectious virions.…”