Activation of cellular Arf GTPases by poliovirus protein 3CD correlates with virus replication

Abstract

We have previously shown that synthesis of poliovirus protein 3CD in uninfected HeLa cell extracts induces an increased association with membranes of the cellular Arf GTPases, which are key players in cellular membrane traffic. Arfs cycle between an inactive, cytoplasmic, GDP-bound form and an active, membrane-associated, GTP-bound form. 3CD promotes binding of Arf to membranes by initiating recruitment to membranes of guanine nucleotide exchange factors (GEFs), BIG1 and BIG2. GEFs activate Arf by replacing GDP with GTP. In poliovirus-infected cells, there is a dramatic redistribution of cellular Arf pools that coincides with the reorganization of membranes used to form viral RNA replication complexes. Here we demonstrate that Arf translocation in vitro can be induced by purified recombinant 3CD protein; thus, concurrent translation of viral RNA is not required. Coexpression of 3C and 3D proteins was not sufficient to target Arf to membranes. 3CD expressed in HeLa cells was retained after treatment of the cells with digitonin, indicating that it may interact with a membrane-bound host factor. A F441S mutant of 3CD was shown previously to have lost Arf translocation activity and was also defective in attracting the corresponding GEFs to membranes. A series of other mutations were introduced at 3CD residue F441. Mutations that retained Arf translocation activity of 3CD also supported efficient growth of virus, regardless of their effects on 3D polymerase elongation activity. Those that abrogated Arf activation by 3CD generated quasi-infectious RNAs that produced some plaques from which revertants that always restored the Arf activation property of 3CD were rescued.

FIG. 1.

Translocation of Arf to membranes in vitro. (A) Recombinant proteins 3CD, 3AB, and 3D were added to HeLa S10 extracts and incubated for 3.5 h. The membranous fraction was collected by centrifugation and assessed by immunoblotting with anti-Arf antibodies. (B) RNAs coding for proteins 3C, 3D, and 3CD were translated in HeLa S10 extracts. After translation, the membranous fractions were collected by centrifugation and assessed by immunoblotting with anti-Arf antibodies (upper panel). The immunoblot membrane was stripped and probed with antibodies to GGA3, which bind the GTP-bound form of Arf (middle panel). The lower panel shows autoradiography of the gel showing [³⁵S]methionine-labeled products of the translation reactions used in the Arf and GGA3 immunoblots.

FIG. 2.

Mutant 3CD F441S is defective in recruitment of Arf and specific GEFs to membranes. RNAs coding for wt 3CD or for the F441S 3CD mutant were translated in HeLa S10 extracts, and the membranous fraction was collected by centrifugation and assessed by immunoblotting with anti-Arf, -BIG1, -BIG2, and -Rab9 antibodies.

FIG. 3.

Association of poliovirus proteins with membranes in vitro. RNAs coding for wt 3CD, the 3CD F441S mutant, 3C, 3D, or 3A were translated in HeLa S10 extracts in the presence of [³⁵S]methionine, and the membranous fraction was collected by centrifugation. Total material recovered after centrifugation and 1/10th of the material from unfractionated translation reactions were resolved by SDS-PAGE, and the distribution of proteins between fractions was determined using phosphorimaging.

FIG. 4.

Association of 3CD with cellular structures in HeLa cells. (A) HeLa cells were transfected with plasmids expressing either free EGFP or ER-targeted Sec61-YFP fusion protein. After 24 h, the cells were treated with digitonin, fixed, and examined by fluorescence microscopy. (B) HeLa cells were transfected with plasmids expressing either wt 3CD or the F441S 3CD mutant and treated with digitonin as for panel A. The cells were fixed, stained with anti-3D antibody, and examined by fluorescence microscopy.

FIG. 5.

Viability of poliovirus RNAs with mutations in 3CD. HeLa cell monolayers were transfected with serial dilutions of full-length RNA transcripts, overlaid with agarose, incubated for ∼48 h, and stained for plaques. Plaques from appropriate dilutions/representative experiments are shown.

FIG. 6.

Translocation of Arf to membranes induced by mutant 3CD proteins. RNAs coding for wt 3CD or for 3CDs with the indicated mutations in position 441 were translated in HeLa S10 extracts. The membranous fraction was collected by centrifugation and assessed by immunoblotting with anti-Arf antibodies (upper panel). The same membrane used for the Arf immunoblot was stripped and probed with antibodies to Rab9, as a loading control (middle panel). The lower panel shows the [³⁵S]methionine-labeled products of the translation reactions.

FIG. 7.

Stimulation of poliovirus virion production in vitro by 3CD. Poliovirus RNA (vRNA) alone or in combination with RNA coding for wt 3CD or the F441S or F441I 3CD mutant were translated and replicated in HeLa S10 extracts for 12 to 15 h. Formation of infectious virus was determined by plaque assay on HeLa cells monolayers. Error bars indicate standard deviations.

FIG. 8.

RNA polymerase elongation activity of mutant 3D proteins. (A) RNAs coding for wt 3D or for 3Ds with the indicated mutations in position 258, corresponding to amino acid 441 of 3CD, were translated in HeLa S10 extracts, and 2 μl of translation mix was used as a source of 3D protein in an RNA chain elongation assay with poly(A) template and oligo(U) primer. (B) Autoradiogram of the gel showing [³⁵S]methionine labeled products of translation reactions used in the elongation assay. Error bars indicate standard deviations.

FIG. 9.

Replication of poliovirus replicons harboring mutations that inhibit 3D elongation activity. Renilla luciferase poliovirus replicon RNA transcripts were transfected into HeLa cells growing in 96-well plates and incubated in the presence of cell-permeative Renilla luciferase substrate. Light readings were taken hourly with a Molecular Devices MV reader. RLU, relative light units. Error bars indicate standard deviations.