The eukaryotic translation initiation factor 4GI is cleaved by different retroviral proteases

Abstract

The initiation factor eIF4G plays a central role in the regulation of translation. In picornaviruses, as well as in human immunodeficiency virus type 1 (HIV-1), cleavage of eIF4G by the viral protease leads to inhibition of protein synthesis directed by capped cellular mRNAs. In the present work, cleavage of both eIF4GI and eIF4GII has been analyzed by employing the proteases encoded within the genomes of several members of the family Retroviridae, e.g., Moloney murine leukemia virus (MoMLV), mouse mammary tumor virus, human T-cell leukemia virus type 1, HIV-2, and simian immunodeficiency virus. All of the retroviral proteases examined were able to cleave the initiation factor eIF4GI both in intact cells and in cell-free systems, albeit with different efficiencies. The eIF4GI hydrolysis patterns obtained with HIV-1 and HIV-2 proteases were very similar to each other but rather different from those obtained with MoMLV protease. Both eIF4GI and eIF4GII were cleaved very efficiently by the MoMLV protease. However, eIF4GII was a poor substrate for HIV proteases. Proteolytic cleavage of eIF4G led to a profound inhibition of cap-dependent translation, while protein synthesis driven by mRNAs containing internal ribosome entry site elements remained unaffected or was even stimulated in transfected cells.

FIG. 1.

Cleavage of eIF4GI in transfected cells. (A) COS-7 cells were transfected with the unmodified pTM1 vector or with pTM1 carrying inserts containing the PR-coding region of several retroviruses or encoding poliovirus 2A^pro. The cells were labeled with [³⁵S]Met-[³⁵S]Cys from 15 to 16 h posttransfection. Equal amounts of protein extract were loaded in a 17% polyacrylamide gel and analyzed by autoradiography. Estimates of protein synthesis inhibition in cell extracts obtained from transfections with different constructs are indicated. The data refer to control experiments carried out with the unmodified pTM1 vector, and determinations were obtained by densitometric scanning of the protein band of ∼45 kDa. (B) Detection of eIF4GI cleavage products by Western blotting using a mixture of antisera against its N- and C-terminal regions (top) or individual antisera against the N-terminal (middle) or C-terminal region of eIF4GI (bottom). c.p., cleavage fragments; Nt, N-terminal fragments of eIF4GI; Ct, C-terminal fragments of eIF4GI. The position of the intact eIF4GI is also indicated. The amount of hydrolyzed eIF4GI for each transfection experiment is indicated below the corresponding lane.

FIG. 2.

Effects of eIF4G cleavage by HIV-2 PR and MoMLV PR on cap-dependent and IRES-driven translation. (A) Schematic diagram of the three synthetic luciferase mRNAs used to transfect COS-7 cells. (B) Luciferase activity shown by COS-7 cells cotransfected with 2 μg of capped luciferase mRNAs (Cap-Luc) or mRNAs encoding the luciferase protein under the control of the IRES element from encephalomyocarditis virus (EMC-Luc) or capped luciferase mRNAs with the 5′ noncoding region from MoMLV (5′NCMo-Luc) and 2 μg of capped mRNAs encoding HIV-2 PR, MoMLV PR, poliovirus 2A^pro, or poliovirus 2C protein (used here as a control). Determinations were done 8 h posttransfection. Control experiments representing 100% of the luciferase activity in each experiment were performed by cotransfecting cells with the poliovirus 2C protein capped mRNA. The effects of the PRs on luciferase activity were calculated based on those values. Standard deviations determined from three independent experiments are indicated by the error bars. (C) Normalized translation stimulation in cotransfected cells. RNA was isolated from transfected cells at 8 h posttransfection, and the amount of luciferase mRNA was determined by real-time quantitative RT-PCR as described in Materials and Methods. The translation stimulation values were normalized to the relative amount of luciferase mRNA in each experiment and calculated based on values obtained for the corresponding controls: 2.3 × 10⁸ RNA molecules/10⁶ cells for Cap-Luc, 1.8 × 10⁹ RNA molecules/10⁶ cells for EMC-Luc, and 4.2 × 10⁸ RNA molecules/10⁶ cells for Cap-5′NCMo-Luc.

FIG. 3.

Cleavage of eIF4GI and eIF4GII by purified recombinant HIV-2 and MoMLV PRs. (A) Fifty micrograms of crude HeLa S10 extracts was incubated with 50 ng of each recombinant PR in a total volume of 20 μl for 90 min, and eIF4GI degradation products were detected by Western blotting using a mixture of antisera raised using eIF4GI peptides contained within the N-terminal and the C-terminal regions of the protein (left) or individual antisera recognizing the N-terminal (middle) or the C-terminal (right) region of eIF4GI. Nt1, Nt2, Ct1, Ct2, Ct3, Ct4, Ct5, and Ct6 are products generated after cleavage with retroviral PRs. SQ, saquinavir at 2 μM concentration; RTV, ritonavir at 20 μM concentration; −, without. (B) Detection of eIF4GII degradation products by Western blotting in crude HeLa S10 extracts, upon incubation with recombinant PRs, under the conditions described above. Western blots were obtained using a mixture of antisera against N-terminal and C-terminal regions of eIF4GII (left) or using the C-terminal antisera alone (right). Nt1, Nt2, Ct1, Ct2 and Ct3 are products generated after cleavage of eIF4GII with retroviral PRs.

FIG. 4.

Cleavage of radioactive eIF4GI and eIF4GII synthesized in vitro after incubation with recombinant HIV-2 and MoMLV PRs. mRNAs encoding eIF4GI or eIF4GII were translated in rabbit reticulocyte lysates (Promega) and labeled with [³⁵S]Met-[³⁵S]Cys. Then, the extracts were incubated for 90 min with 50 ng of each recombinant retroviral PR or MBP-2A^pro and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. Saquinavir (SQ) was used at a final concentration of 2 μM, and ritonavir (RTV) was used at 20 μM. −, without; +, with.

FIG. 5.

Identification of HIV-2 PR and MoMLV PR cleavage sites on eIF4GI. (A) Amino acid sequences of the eIF4GI cleavage sites identified using HIV-2 and MoMLV PRs. HIV-1 PR cleavage sites (51) are also shown. (B) Diagram showing the functional domains found in eIF4GI based on the available data, including the positions of mapped cleavage sites for HIV-2 and MoMLV PRs. The putative double-stranded-RNA binding domain (RRM) and the regions involved in binding to other translation factors are shown. The positions of peptides used to obtain antibodies are also indicated. PABP, poly(A)-binding protein. The sequence numbering refers to the eIF4GI-a isoform (9).