Specific Cleavage of the Nuclear Pore Complex Protein Nup62 by a Viral Protease

Nogi Park,Tim Skern,Kurt E Gustin

doi:10.1074/jbc.m110.143404

Abstract

Previous work has shown that several nucleoporins, including Nup62 are degraded in cells infected with human rhinovirus (HRV) and poliovirus (PV) and that this contributes to the disruption of certain nuclear transport pathways. In this study, the mechanisms underlying proteolysis of Nup62 have been investigated. Analysis of Nup62 in lysates from HRV-infected cells revealed that Nup62 was cleaved at multiple sites during viral infection. The addition of purified HRV2 2A protease (2A(pro)) to uninfected HeLa whole cell lysates resulted in the cleavage of Nup62, suggesting that 2A(pro) is a major contributor to Nup62 processing. The ability of purified 2A(pro) to cleave bacterially expressed and purified Nup62 demonstrated that 2A(pro) directly cleaves Nup62 in vitro. Site-directed mutagenesis of putative cleavage sites in Nup62 identified six different positions that are cleaved by 2A(pro) in vitro. This analysis revealed that 2A(pro) cleavage sites were located between amino acids 103 and 298 in Nup62 and suggested that the N-terminal FG-rich region of Nup62 was released from the nuclear pore complex in infected cells. Analysis of HRV- and PV-infected cells using domain-specific antibodies confirmed that this was indeed the case. These results are consistent with a model whereby PV and HRV disrupt nucleo-cytoplasmic trafficking by selectively removing FG repeat domains from a subset of nuclear pore complex proteins.

Highlights

Nup62 is a glycosylated FG-Nup located in the central channel of the nuclear pore complex (NPC)
To determine whether this occurred with group A rhinoviruses, the status of Nup62 in cells infected with human rhinovirus type 2 (HRV2) was examined
To determine whether 2A protease (2Apro) could degradation was seen in the absence of protease, and cleavage cause the degradation of Nup62 in the absence of HeLa cell could be completely prevented by the addition of N-(methoxyfactors, we examined the ability of HRV2 2Apro to cleave Nup62 succinyl)-Ala-Ala-Pro-Val-chloromethyl ketone, a known that had been translated and radiolabeled in rabbit reticulocyte inhibitor of 2Apro (Fig. 3B, lanes 1 and 5, respectively) [33]

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Virus—HeLa cells were maintained in a monolayer in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and penicillin/streptomycin at 37 °C in 5% CO2. The indicated amount of HRV2 2Apro was incubated with 25 ␮g of uninfected HeLa whole cell lysates, in vitro translated Nup, or the indicated amount of purified Nup at 30 °C for the indicated amount of time in 2A reaction buffer (50 mM Tris-Cl (pH 8.0), 50 mM NaCl, 5 mM DTT, and 1 mM EDTA) [29]. 0.5 ␮g of plasmid containing wild-type or mutant Nup open reading frame was incubated with 1 ␮l of TNT reaction buffer (Promega), 12.5 ␮l of rabbit reticulocyte lysate, 0.5 ␮l of T7 RNA polymerase, 0.5 ␮l of 1 mM amino acid mixture without methionine, and 10 ␮Ci of [35S]methionine (Ͼ1,000 Ci/mmol; GE Healthcare) for 90 min at 30 °C. Cells were observed using a Nikon Eclipse E1000 fluorescent microscope with a ϫ60 objective, and images were acquired using a Hamamatsu Orca digital camera and Metamorph software

RESULTS

2Apro cleavage sites identified by

DISCUSSION