Cleavage of poly(A)-binding protein by enterovirus proteases concurrent with inhibition of translation in vitro

Abstract

Many enteroviruses, members of the family Picornaviridae, cause a rapid and drastic inhibition of host cell protein synthesis during infection, a process referred to as host cell shutoff. Poliovirus, one of the best-studied enteroviruses, causes marked inhibition of host cell translation while preferentially allowing translation of its own genomic mRNA. An abundance of experimental evidence has accumulated to indicate that cleavage of an essential translation initiation factor, eIF4G, during infection is responsible at least in part for this shutoff. However, evidence from inhibitors of viral replication suggests that an additional event is necessary for the complete translational shutoff observed during productive infection. This report examines the effect of poliovirus infection on a recently characterized 3' end translational stimulatory protein, poly(A)-binding protein (PABP). PABP is involved in stimulating translation initiation in lower eukaryotes by its interaction with the poly(A) tail on mRNAs and has been proposed to facilitate 5'-end-3'-end interactions in the context of the closed-loop translational model. Here, we show that PABP is specifically degraded during poliovirus infection and that it is cleaved in vitro by both poliovirus 2A and 3C proteases and coxsackievirus B3 2A protease. Further, PABP cleavage by 2A protease is accompanied by concurrent loss of translational activity in an in vitro-translation assay. Similar loss of translational activity also occurs simultaneously with partial 3C protease-mediated cleavage of PABP in translation assays. Further, PABP is not degraded during infections in the presence of guanidine-HCl, which blocks the complete development of host translation shutoff. These results provide preliminary evidence that cleavage of PABP may contribute to inhibition of host translation in infected HeLa cells, and they are consistent with the hypothesis that PABP plays a role in facilitating translation initiation in higher eukaryotes.

FIG. 1

PABP is degraded during poliovirus infection. (A) HeLa spinner cells were mock infected (Mock) or infected with poliovirus, and samples were harvested at the indicated times p.i. (hpi). Aliquots of these samples were subjected to SDS-PAGE and immunoblotted with a monoclonal antibody to PABP (10E10) (upper left gel) or a polyclonal antibody to eIF4G (lower left gel). cp, cleavage product. (B) Densitometric images of three immunoblots prepared as for panel A were quantitated with an Alpha Innotech imager; the average of the data is shown. The data are represented as percentages of intact PABP levels relative to the amount in mock-infected cells. (C) Immunoprecipitations of PABP were done as described in Materials and Methods on extracts prepared from cells that were pulse labeled for 2 h and then harvested (Control) or subsequently chased for the indicated length of time with either complete medium (mock) or complete medium containing 10 μg of cycloheximide/ml (CHI) or infected with poliovirus for the indicated length of time. The number below each lane corresponds to the densitometric quantitation of the PABP band relative to the control lane (% Ctrl.).

FIG. 2

PABP is not degraded during poliovirus infection in the presence of guanidine. (A) HeLa cells were mock-infected (Mock) or infected with poliovirus in the presence of 2 mM guanidine-HCl, and samples were harvested at the indicated times p.i. for immunoblot analysis with PABP antibody. (B) HeLa cells infected with poliovirus in the presence (Polio/GN) or absence (Polio) of guanidine were pulse labeled with [³⁵S]Met at the indicated times p.i., and aliquots were analyzed by SDS-PAGE for radiolabeled proteins. The positions of the poliovirus proteins are indicated.

FIG. 3

Poliovirus 2A^pro degrades PABP in vitro. In vitro-cleavage reaction mixtures were assembled as described in Materials and Methods, using 4 μl of RSW with 0 (lanes 1), 2 (lanes 2), 4 (lanes 3), or 10 μl (lanes 4) of recombinant poliovirus 2A^pro bacterial extract. Reaction mixtures were incubated at 37°C for 3 h and then analyzed by gel electrophoresis and immunoblotting. The blots shown are from the same gel analyzed with two different antibodies, PABP (left) and eIF4G (right). The positions of eIF4G cleavage products (cp) are indicated.

FIG. 4

Purified 2A^pro and 3C^pro cleave PABP in vitro. Cleavage reactions with RSW and purified recombinant 2A^pro from CVB3 (left) or purified recombinant poliovirus 3C^pro (right) with the indicated quantities of protease were incubated for 3 h at 37°C and analyzed by gel electrophoresis and immunoblotting with PABP antibody.

FIG. 5

PABP cleavage products from in vitro cleavages correspond to the cleavage detected during poliovirus infection in vivo. (A) Reticulocyte lysate-translated PABP was used as a substrate for PABP cleavage reactions, as detailed in Materials and Methods. The cleavage reaction mixtures contained 1.0 μg of CVB3 2A^pro, 1.5 μg of poliovirus 3C^pro, or a combination of both proteases and were incubated for 3 h at 37°C. The positions of the PABP cleavage products (cp) generated by each protease are indicated, as well as molecular mass marker positions. (B) In vivo cleavage product (PABP cp) detected by immunoblot analysis by PABP antibody in lysate derived from a poliovirus infection and harvested at 4 h. (C) Poliovirus 2A^pro and CVB3 2A^pro generate similar PABP cleavage products. Reticulocyte lysate-translated PABP was incubated alone or with 1.0 μg of CVB3 2A^pro or 10 μl of poliovirus 2A^pro extract and analyzed by gel electrophoresis and autoradiography. The position of the 57-kDa cleavage product (2A cp) is indicated.

FIG. 6

Cleavage of purified PABP by purified 2A^pro and 3C^pro. Purified recombinant histidine-tagged PABP (His-PABP) (left) or unmodified PABP (right) was incubated with purified CVB3 2A^pro or poliovirus 3C^pro or both. (A) Lane 1, unincubated His-PABP; lane 2, His-PABP incubated alone; lane 3, His-PABP plus 1.0 μg of 2A^pro; lane 4, His-PABP plus 1.5 μg of 3C^pro; lane 5, His-PABP plus 1.0 μg of 2A^pro–1.5 μg of 3C^pro. (B) Lane 1, PABP incubated alone; lane 2, PABP plus 1.0 μg of 2A^pro (37°C incubation); lane 3, PABP plus 1.0 μg of 2A^pro (30°C incubation); lane 4, PABP plus 1.0 μg of 3C^pro (37°C incubation). The reactions shown in panel A were incubated for 16 h at 30°C, and those shown in panel B were incubated at the indicated temperature for 16 h and then analyzed by SDS-PAGE and Coomassie blue staining. ∗∗, 2A^pro; ∗, 3C^pro.

FIG. 7

PABP is cleaved in its carboxyl-terminal region by both 2A^pro and 3C^pro. (A) Affinity-purified His-PABP was incubated with the indicated quantities of either CVB3 2A^pro or poliovirus 3C^pro for 3 h at 37°C and then analyzed by immunoblotting with antibody to hexahistidine tag. The positions of the large PABP cleavage products (cp) generated by each protease are indicated. (B) Schematic diagram of PABP structure showing known functional domains and localization of the protease cleavage sites on PABP. (C) Sequence of the primary cleavage site (∗) for CVB3 2A^pro on PABP.

FIG. 8

Inhibition of translation in vitro correlates with degradation of PABP by 2A^pro and 3C^pro. (A) Extracts generated from mock-infected HeLa cells (Mock) or HeLa cells infected with poliovirus with (PV/Gn) or without (Polio) guanidine were incubated in translation reactions as described in Materials and Methods. The reactions were assembled with or without protease (1.0 μg/reaction) and preincubated for 60 min at 30°C. Radiolabel and cocktail were then added, and incubation was continued for 60 additional minutes at 37°C. Half of the sample was used for SDS-PAGE and autoradiography (top), while the other half is shown analyzed by immunoblotting with antibody to PABP (bottom). Lane 7 shows translation derived from poliovirus extracts without added protease, while the lane labeled Mock, NE, designates translation in a mock-infected extract reaction without an added energy source (creatine phosphate-creatine kinase). (B) Extracts from mock-infected HeLa cells (Mock) or poliovirus-guanidine-infected cells (PV/Gn) were assembled into reactions as described in Materials and Methods. All components except radiolabel were added initially and incubated at 30°C to allow endogenous translation. At the indicated time, radiolabel (20 μCi) was added to each reaction, and translation was allowed to continue for 60 min more at 37°C. The samples were processed as described for panel A. The Mock, NE, lane was prepared the same way as the Mock, 0 min, lane without creatine phosphate-creatine kinase. Below each lane, the percent translation rate compared to that of mock-infected lysate is shown as an average of two separate experiments (quantitated with National Institutes of Health image software).