Involvement of a Nonstructural Protein in Poliovirus Capsid Assembly

Figures

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  FIG 1. Identification of thermally selected mutations. (A) Cartoon structure of the PV genome, showing the 5′ and 3′ UTRs flanking the open reading frame comprising the structural (P1) and nonstructural (P2 and P3) regions. (B) Evolution of PV-1 under thermal selection. Viral RNA was extracted from each passage of virions selected at 51°C (i.e., VS51), 53°C (i.e., VS53), and 57°C (i.e., VS57) (25). The entire genome of the evolving population at each passage was reverse transcribed and amplified by PCR. The virus pool was sequenced and aligned against the wt PV-1 sequence by ClustalOmega. A cartoon representation of selected mutations is shown. Solid black vertical lines represent wt sequences of the P1 and 2A regions. Nonsynonymous mutations are presented as colored shapes in VP4, VP2, VP3, VP1, and 2Apro, as shown in the key inset. (C) Sequence comparisons of 2Apro among enteroviruses. Alignment of a 60-residue region of 2Apro of thermally selected viruses VS51, VS53, and VS57 against enterovirus A (coxsackievirus A1 [CVA-1]), enterovirus B (echovirus 1 [EV1]), enterovirus C (PV-1, PV-2, PV-3, and CVA-2), enterovirus D: human enterovirus 94 (EV-94); enterovirus E: bovine enterovirus…

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  FIG 2. Effect of 2Apro mutations on cis-cleavage activity at the P1/2A junction. (A) Cartoon of the construct used in the transcription/translation (TNT) assay. (B) Autoradiographs of SDS-PAGE of TNT samples. Following incubation at 30°C for 90 min, further incorporation of [35S]Cys-Met was prevented by the addition of excess unlabeled Cys-Met and samples collected at 30-minute intervals. Samples were separated by SDS-PAGE and protein bands detected by autoradiography. Arrows correspond to the P1/2Apro precursor and processed P1. Time points represent chase. (C and D) Normalized densitometry of the P1/2Apro precursor (C) and cleaved P1 (D) over time. Graphs represent the intensity of the P1 band of phosphoscreen scans of autoradiograph. Scanned images were analyzed by ImageJ version 1.47t (n = 2; error bars show standard errors of the means [SEM]; **, P < 0.001 compared to wt).

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  FIG 3. Polyprotein processing of wt and 2Apro mutant PV-1. (A) Both 2A mutations were introduced into an infectious clone of wt PV-1 (i.e., pt7Rbz). RNA transcripts of pt7Rbz or 2A-I99V/-G102R were used in HeLa cell-free reactions. Following incubation at 34°C for 2 h, excess Cys-Met was added and samples taken at various time points. Samples (lanes 1 to 12) were separated by 8% SDS-PAGE and radiolabeled proteins detected by autoradiography. To identify specific bands, pcDNA constructs of P1, P2, noncleavable P1-2A, and noncleavable P1-P2 were expressed in TNT reactions in the presence of [35S]Cys-Met and incubated for 3 h (lanes 13 to 17). (B and C) Band intensities were quantified from a phosphoscreen image and the levels of P1-P2 (B) and P1-2A (C) presented as normalized percent intensity over background phosphorescence. Scanned images were analyzed by ImageJ version 1.47t (n = 3; error bars show SEM; *, P < 0.05; **, P < 0.001 [compared to wt at each time point]).

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  FIG 4. Effect of 2Apro mutations on PV-1 replicon replication. (A) HeLa cells were transfected with T7 RNA transcripts and replication monitored by GFP fluorescence over time using an IncuCyte Zoom. A replication-deficient mutant, 3D-GNN, was included as a control for input translation. (B) The data from panel A at 22 h posttransfection (total GFP-positive cells) were also plotted as a bar graph for clarity (n = 3; error bars show SEM; *, P < 0.05; **, P < 0.001; ***, P < 0.0001).

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  FIG 5. Effects of 2Apro mutations on virus recovery. RNA transcripts were generated from infectious clones of the wt or VP1-I194V in the presence or absence of the 2Apro mutations 2A-I99V/-G102R, transfected into mouse L cells or HeLa cells, and incubated at 37°C for 24 h. (A) Virus titers recovered from transfected mouse L cells (n = 3; error bars show SEM; ***, P < 0.0001) (B) Virus titers recovered from transfected HeLa cells. (n = 3; error bars show SEM; ***, P < 0.0001) (C) Virus samples recovered from HeLa cells were diluted in serum-free medium to equal starting titers, incubated at a range of temperatures between 37°C and 55°C for 30 min, cooled to 4°C, and titrated by plaque assays using HeLa cells (n = 2; error bars show standard deviations [SD]; *, P < 0.05 compared to wt).

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  FIG 6. Effects of reduced translation on an assembly-deficient VP1-I194V mutant. (A) Mouse L cells were treated with increasing concentrations of cycloheximide and assayed for toxicity by using MTS. The IC50 was evaluated by using dose-dependent curves (n = 2; error bars show SD). (B) Mouse L cells were transfected with T7 RNA transcripts of the wt and treated with cycloheximide at increasing concentrations. Cell lysates were harvested using RIPA buffer. Supernatants and cell lysates were separated by SDS-PAGE and immunoblotted against anti-PV-1 VP1 MAb 8560. Representative results from two biological repeats are shown. (C) Mouse L cells were transfected with T7 RNA transcripts of the wt and VP1-I194V mutant and treated at two concentrations of cycloheximide. Supernatants were clarified by low-speed centrifugation. Virus titers of supernatants from cycloheximide-treated wt- and VP1-I194V mutant-transfected cells were determined by plaque assays using HeLa cells (n = 2; error bars show SEM; *, P < 0.05; ***, P < 0.0001 [compared to untreated wt]).