Bypass Suppression of Small-Plaque Phenotypes by a Mutation in Poliovirus 2A That Enhances Apoptosis

Abstract

The rate of protein secretion in host cells is inhibited during infection with several different picornaviruses, with consequences likely to have significant effects on viral growth, spread, and pathogenesis. This Sin ؉ (secretion inhibition) phenotype has been documented for poliovirus, foot-and-mouth disease virus, and coxsackievirus B3 and can lead to reduced cell surface expression of major histocompatibility complex class I and tumor necrosis factor receptor as well as reduced extracellular secretion of induced cytokines such as interleukin-6 (IL-6), IL-8, and beta interferon. The inhibition of protein secretion is global, affecting the movement of all tested cargo proteins through the cellular secretion apparatus. To test the physiological significance of the Sin ؊ and Sin ؉ phenotypes in animal models, Sin ؊ mutant viruses are needed that fail to inhibit host protein secretion and also exhibit robust growth properties. To identify such Sin ؊ mutant polioviruses, we devised a fluorescence-activated cell sorter-based screen to select virus-infected cells that nevertheless expressed newly synthesized surface proteins. After multiple rounds of selection, candidate Sin ؊ mutant viruses were screened for genetic stability, increased secretion of cargo molecules and wild-type translation and growth properties. A newly identified Sin ؊ mutant poliovirus that contained coding changes in nonstructural proteins 2A (N32D) and 2C (E253G) was characterized. In this virus, the 2C mutation is responsible for the Sin ؊ phenotype and the 2A mutation suppresses a resulting growth defect by increasing the rate of cell death and therefore the rate of viral spread. The 2A-N32D suppressor mutation was not allele specific and, by increasing the rate of cellular apoptosis, affected a completely different pathway than the 2C-E253G Sin ؊ mutation. Therefore, the 2A mutation suppresses the 2C-E253G mutant phenotype by a bypass suppression mechanism.

Global RNA Structure Analysis of Poliovirus Identifies a Conserved RNA Structure Involved in Viral Replication and Infectivity

“…These amino acid residues are thought to be involved in 3D pol oligomeric interfaces, and in vitro biochemical work with the polymerase mutants supports that view (52,54). However, the viral phenotypes described include an RNA synthesis defect (52) and small plaques (52,54), with the latter phenotype exhibiting a marked temperature sensitivity (53,54). All of these phenotypes are also exhibited by our synonymous 3D-7000 mutants.…”

2A Protease Is Not a Prerequisite for Poliovirus Replication

“…Calandria et al reported that independently expressed 2A pro and 3C pro induced apoptosis by mechanisms involving caspase activation (10). On the other hand, Burgon et al reported that a 2A N32D mutation independently caused cells to die by apoptosis much earlier than wild-type-infected cells (9). This is consistent with the hypothesis that a wild-type function of the 2A pro protein is to inhibit apoptosis and cause a canonical necrotic CPE late in infection, perhaps directly or indirectly leading to the aberrant cleavage of procaspase-9, and that this activity is abrogated by the 2A N32D mutation.…”

Valosin-Containing Protein (VCP/p97) Is Required for Poliovirus Replication and Is Involved in Cellular Protein Secretion Pathway in Poliovirus Infection

“…6C, right panel). This clone had only one amino acid change in the viral proteins, a 2C-E253G mutation, which is known as the determinant of a PV mutant for a secretion inhibition-negative phenotype (19). Sequences of 2C around aa 253 are not conserved in picornaviruses but are conserved among PV strains.…”

“…Knockdown of VCP did not suppress the replication of coxsackievirus B3 (CVB3), which is a member of another EV species, Human enterovirus B, or of Aichi virus (AV), which is a member of another genus, Kobuvirus, of the family Picornaviridae. A PV-resistant mutant that showed improved growth in VCP-knockdown cells contained the mutation A4881G (E253G in 2C [2C-E253G]), which is known as the determinant of a secretion inhibition-negative phenotype of a PV mutant (19). However, knockdown of VCP did not suppress the inhibition of cellular protein secretion caused by overexpression of viral proteins 2B, 2BC, 3A, and 3AB.…”

“…The inhibitory effect of 3A on cellular protein secretion is not generally conserved in picornavirus (23) but might be compensated by other viral proteins (e.g., 2BC protein for foot-and-mouth disease virus infection) (62). Cellular protein secretion is inhibited during PV infection (34); however, PV mutants that did not inhibit cellular protein secretion have been isolated and did not show a significant growth defect (15,19,32). Therefore, the importance of cellular protein secretion in picornavirus infection as well as the responsible host factors remained to be elucidated.…”