Differential Targeting of Nuclear Pore Complex Proteins in Poliovirus-Infected Cells

Abstract

Poliovirus disrupts nucleocytoplasmic trafficking and results in the cleavage of two nuclear pore complex (NPC) proteins, Nup153 and Nup62. The NPC is a 125-MDa complex composed of multiple copies of 30 different proteins. Here we have extended the analysis of the NPC in infected cells by examining the status of Nup98, an interferon-induced NPC protein with a major role in mRNA export. Our results indicate that Nup98 is targeted for cleavage after infection but that this occurs much more rapidly than it does for Nup153 and Nup62. In addition, we find that cleavage of these NPC proteins displays differential sensitivity to the viral RNA synthesis inhibitor guanidine hydrochloride. Inhibition of nuclear import and relocalization of host nuclear proteins to the cytoplasm were only apparent at later times after infection when all three nucleoporins (Nups) were cleaved. Surprisingly, analysis of the distribution of mRNA in infected cells revealed that proteolysis of Nup98 did not result in an inhibition of mRNA export. Cleavage of Nup98 could be reconstituted by the addition of purified rhinovirus type 2 2Apro to whole-cell lysates prepared from uninfected cells, suggesting that the 2A protease has a role in this process in vivo. These results indicate that poliovirus differentially targets subsets of NPC proteins at early and late times postinfection. In addition, targeting of interferon-inducible NPC proteins, such as Nup98, may be an additional weapon in the arsenal of poliovirus and perhaps other picornaviruses to overcome host defense mechanisms.Poliovirus is a positive-strand RNA virus belonging to the family Picornaviridae. Poliovirus, like other members of this family, encodes a single large polyprotein that is co-and posttranslationally processed by virus-encoded proteases to produce the individual viral gene products (reviewed in reference 40). After the production of viral proteins, RNA synthesis ensues on virus-induced vesicles in the cell cytoplasm. Despite the fact that viral translation, RNA synthesis, and assembly occur in the cytoplasm, a number of host nuclear proteins have been attributed roles in the viral life cycle (3,6,27,32,33,51). Consistent with their having a role in the replication of poliovirus in the cytoplasm, several host nuclear factors have been shown to redistribute to the cytoplasm after infection (3,5,32,33,51).For nuclear factors to move from the nucleus to the cytoplasm they must transit the nuclear pore complex (NPC), a large protein channel found embedded in the nuclear envelope. The vertebrate NPC has a molecular mass of 125 MDa and is composed of multiple copies of roughly 30 different proteins that are collectively called nucleoporins (Nups; reviewed in reference 18). A number of findings have suggested that the redistribution of host nuclear factors to the cytoplasm in poliovirus-infected cells is due to alterations of the NPC and a disruption in nucleocytoplasmic trafficking. Previous work has shown that two NPC proteins, Nup153 and Nup62, are targeted for degradation in ...

Polypyrimidine tract-binding protein is relocated to the cytoplasm and is required during dengue virus infection in Vero cells

“…As other proteins such as La also translocate from the nucleus to the cytoplasm of infected cells (Yocupicio-Monroy et al, 2007), it is likely that a group of nuclear proteins is translocated to the cytoplasm in response to DENV infection. This phenomenon has been observed with other viruses such as poliovirus (Park et al, 2008). It will be interesting to dissect the mechanisms that induce translocation of these nuclear proteins.…”

Stable Formation of Compositionally Unique Stress Granules in Virus-Infected Cells

“…HeLa cell monolayers were cultured as described previously (38). The African Green monkey kidney cell line, Cos-7, was purchased from the American Type Culture Collection (ATCC) and grown in monolayers in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 2 mM L-glutamine, and penicillin-streptomycin.…”

“…The African Green monkey kidney cell line, Cos-7, was purchased from the American Type Culture Collection (ATCC) and grown in monolayers in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 2 mM L-glutamine, and penicillin-streptomycin. Mahoney type 1 poliovirus stocks were prepared as described previously (38). HeLa cells were infected at a multiplicity of infection (MOI) of 50 by adding virus in phosphate-buffered saline (PBS) supplemented with 1 mM MgCl 2 and 1 mM CaCl 2 for 30 min at 37°C.…”

“…For detection of polyadenylated mRNA, cells were plated on 12-mm-diameter coverslips and grown for 48 h before exposure to poliovirus, heat shock, or oxidative stress. Cells were then processed as previously described using an oligo(dT) probe (38) conjugated to fluorescein isothiocyanate, except that the hybridization temperature was increased to 46°C. For subsequent IFAs cells were incubated for 15 min at 25°C with blocking solution followed by the addition of the indicated antibody and processed as described above for IFAs.…”

“…In addition to reducing translation of cellular mRNAs, cleavage of host factors by 2A pro and 3C pro is also responsible for disrupting a number of cellular processes. For example, cleavage of nuclear pore complex proteins by 2A pro is thought to be responsible for inhibition of nucleo-cytoplasmic transport (2,38), while cleavage of TFIIIC and TBP by 3C pro contributes to the inhibition of transcription that occurs in infected cells (7,8,51). Numerous other targets for these proteases have been identified, including PABP (21), poly(rC) binding proteins 1 and 2 (41), the La autoantigen (46), the p65 subunit of NF-B (35), and polypyrimidine tract binding protein (1).…”