Viral Proteinase Requirements for the Nucleocytoplasmic Relocalization of Cellular Splicing Factor SRp20 during Picornavirus Infections

Abstract

Infection of mammalian cells by picornaviruses results in the nucleocytoplasmic redistribution of certain host cell proteins. These viruses interfere with import-export pathways, allowing for the cytoplasmic accumulation of nuclear proteins that are then available to function in viral processes. We recently described the cytoplasmic relocalization of cellular splicing factor SRp20 during poliovirus infection. SRp20 is an important internal ribosome entry site (IRES) trans-acting factor (ITAF) for poliovirus IRES-mediated translation; however, it is not known whether other picornaviruses utilize SRp20 as an ITAF and direct its cytoplasmic relocalization. Also, the mechanism by which poliovirus directs the accumulation of SRp20 in the cytoplasm of the infected cell is currently unknown. Work described in this report demonstrated that infection by another picornavirus (coxsackievirus B3) causes SRp20 to relocalize from the nucleus to the cytoplasm of HeLa cells, similar to poliovirus infection; however, SRp20 is relocalized to a somewhat lesser extent in the cytoplasm of HeLa cells during infection by yet another picornavirus (human rhinovirus 16). We show that expression of poliovirus 2A proteinase is sufficient to cause the nucleocytoplasmic redistribution of SRp20. Following expression of poliovirus 2A proteinase in HeLa cells, we detect cleavage of specific nuclear pore proteins known to be cleaved during poliovirus infection. We also find that expression of human rhinovirus 16 2A proteinase alone can cause efficient cytoplasmic relocalization of SRp20, despite the lower levels of SRp20 relocalization observed during rhinovirus infection compared to poliovirus. Taken together, these results further define the mechanism of SRp20 cellular redistribution during picornavirus infections, and they provide additional insight into some of the differences observed between human rhinovirus and other enterovirus infections. Members of the Picornaviridae family of viruses cause a range of diseases in humans, including poliomyelitis, myocarditis, and the common cold. Picornaviruses are small single-stranded, positive-sense RNA viruses that include the enteroviruses poliovirus and coxsackievirus, as well as human rhinoviruses (HRVs). They contain a ϳ7.0-kb-to-8.5-kb genome that consists of a single open reading frame, which is translated to generate a polyprotein that is proteolytically processed by viral proteinases into structural and nonstructural proteins. Instead of a 7-methyl guanosine cap structure, picornaviruses contain a small viral protein, VPg, covalently linked to the 5= end of the RNA. This unique protein-RNA linkage serves as a primer for viral RNA synthesis. Viral translation occurs via a cap-independent mechanism and is driven by an internal ribosome entry site (IRES) located in the long, highly structured 5= noncoding region (5= NCR).In addition to proteolytic processing of the viral polyprotein, the viral proteinases 2A and 3C/3CD cleave several cellular proteins, including eukaryotic initiation factor 4G ...

Selective Removal of FG Repeat Domains from the Nuclear Pore Complex by Enterovirus 2A pro

“…2D, lanes 1, 3, and 4) (44). Cumulatively, these results are in agreement with other reports and support the conclusion that 2A pro is the viral protease responsible for proteolysis of Nup98 (32,34).…”

“…pro from all three rhinovirus species targets Nup98 and Nup153 (34) and that expression of 2A pro alone is sufficient to bring about the proteolysis of these NPC proteins (32). The results presented here indicate that HRV2 and poliovirus 2A pro proteins proteolyze Nup98 at two locations, G374 and G552.…”

“…These results indicate differences in the mechanisms underlying proteolysis of Nup98 compared to other FG-Nups, although the significance of these differences is not understood. Despite the differences in the kinetics of cleavage and sensitivity to guanidine, the viral 2A protease (2A pro ) is the primary protease responsible for the proteolysis of Nup98, Nup153, and Nup62 (28,(30)(31)(32)(33)(34).…”

Picornavirus IRES elements: RNA structure and host protein interactions

“…Recent studies have shown the localization in the cytoplasm of infected cells of several ITAFs, as illustrated by far upstream element binding protein 1 (FBPI) (Huang et al, 2011) or hnRNP A (Lin et al, 2009b). Redistribution of SRp20 to the cytoplasm of infected cells (Fitzgerald et al, 2013;Fitzgerald and Semler, 2011) is consistent with their capacity to stimulate enterovirus 71 (EV71) or PV IRES activity (Bedard et al, 2007).…”

The nuclear pore complex: structure and function

“…Polioviruses or rhinoviruses, for example, express a proteinase enzyme that is causing the cleavage and degradation of Nup153, Nup98 and Nup62, which consequently inhibits nucleocytoplasmic transport (Belov et al, 2000) and increases the permeability of the NPC. This increased permeability promotes passive diffusion and the re-localization of host cell nuclear proteins that take part in viral replication (Meerovitch et al, 1993;McBride et al, 1996;Gustin and Sarnow, 2001;Gustin and Sarnow, 2002;Park at al., 2008;Fitzgerald et al, 2013). Inhibition of nucleocytoplasmic transport might reduce the export of host mRNAs to the cytoplasm, which in turn is reducing the competition for the translation machinery and which is promoting viral protein synthesis.…”