Transient expression of cellular polypyrimidine-tract binding protein stimulates cap-independent translation directed by both picornaviral and flaviviral internal ribosome entry sites In vivo

Abstract

The regulation of cap-independent translation directed by the internal ribosome entry sites (IRESs) present in some viral and cellular RNAs is poorly understood. Polypyrimidine-tract binding protein (PTB) binds specifically to several viral IRESs. IRES-directed translation may be reduced in cell-free systems that are depleted of PTB and restored by reconstitution of lysates with recombinant PTB. However, there are no data concerning the effects of PTB on IRES-directed translation in vivo. We transfected cells with plasmids expressing dicistronic transcripts in which the upstream cistron encoded PTB or PTB deletion mutants (including a null mutant lacking amino acid residues 87 to 531). The downstream cistron encoded a reporter protein (chloramphenicol acetyltransferase [CAT]) under translational control of the poliovirus IRES which was placed within the intercistronic space. In transfected BS-C-1 cells, transcripts expressing wild-type PTB produced 12-fold more reporter protein than similar transcripts encoding the PTB null mutant. There was a 2.4-fold difference in CAT produced from these transcripts in HeLa cells, which contain a greater natural abundance of PTB. PTB similarly stimulated CAT production from transcripts containing the IRES of hepatitis A virus or hepatitis C virus in BS-C-1 cells and Huh-7 cells (37- to 44-fold increase and 5 to 5.3-fold increase, respectively). Since PTB had no quantitative or qualitative effect on transcription from these plasmids, we conclude that PTB stimulates translation of representative picornaviral and flaviviral RNAs in vivo. This is likely to reflect the stabilization of higher ordered RNA structures within the IRES and was not observed with PTB mutants lacking RNA recognition motifs located in the C-terminal third of the molecule.

FIG. 1

(A) Organization of dicistronic transcripts expressing human PTB by cap-dependent translation and expressing CAT under the translational control of an IRES within the intercistronic space. Plasmid designations appear at the left; in each, “x” is “S” for constructs containing the Sabin type 1 poliovirus 5′NTR in the intercistronic space or “A” for constructs containing the HAV 5′NTR. Transcription is under control of a composite CMV-T7 promoter. Intact PTB is expressed by pPwt/SC or pPwt/AC and contains a total of 531 amino acid residues. In pΔ87-118/xC, 32 amino acid residues have been removed from the PTB sequence by an in-frame deletion. Frameshift mutations in pΔ87-531/xC (null mutant) and pΔ361-531/xC result in termination of PTB translation at Thr⁸⁶ and Val³⁶⁰, respectively. At the bottom is a graphical representation of the PTB molecule showing locations of the four RRMs with respect to these deletion mutations. (B) Organization of dicistronic transcripts encoding the wt or null mutant PTB upstream of the HCV IRES and a downstream CAT reporter protein. The upstream sequences are identical to those shown for the related transcripts in panel A, but the intercistronic space contains the complete HCV 5′NTR fused naturally to the 5′-most 8 nt of the HCV open reading frame (Δ Core) and in-frame CAT coding sequence.

FIG. 2

Products of cell-free translation reactions programmed with synthetic dicistronic RNA transcripts containing the poliovirus 5′NTR within the intercistronic space (Fig. 1A). Reaction mixes (25 μl) were programmed with 1 μg of the indicated T7 transcripts (lanes 2 to 5) or no RNA (lane 1). Intact PTB appears as a doublet band with an apparent molecular mass of ∼57 kDa (lane 2), while mutant PTBs appear as more rapidly migrating doublet bands (lanes 3 and 5). No PTB product is evident from translation of the null mutant, pΔ87-531/SC (lane 4), consistent with the small predicted size of this product (∼10.7 kDa). CAT is produced from all four transcripts and migrates with an apparent molecular mass of ∼24 kDa (lanes 2 to 5).

FIG. 3

Impact of PTB transient expression on levels of CAT reporter protein activity in cultured mammalian cells which were transfected with plasmids containing the poliovirus 5′NTR within the intercistronic space (Fig. 1A). CAT activities were measured 46 to 50 h following transfection and were normalized to those obtained following transfection of the null mutant pΔ87-531/SC (100%). Error bars indicate the standard deviations of results obtained in two separate experiments, each involving two replicate transfections (total of four transfections). (A) Relative CAT activities following DNA transfection of BS-C-1 cells which contain a low cytoplasmic abundance of PTB. Transfection with the null mutant, pΔ87-531/SC, generated a mean CAT activity value of 777 cpm, while CAT activity in mock-transfected cells (m) was 69 cpm. (B) Relative CAT activities in transfected H1-HeLa cells which contain a greater natural abundance of PTB than BS-C-1 cells. Transfection with the null mutant, pΔ87-531/SC, generated a mean CAT activity value of 22,715 cpm, while CAT activity in mock-transfected cells was 67 cpm.

FIG. 4

Impact of PTB transient expression on levels of CAT reporter protein activity in cells which were transfected with plasmids (Fig. 1A) containing the HAV 5′NTR within the intercistronic space (results shown are means of four separate transfections from a total of two experiments; see the legend to Fig. 3). (A) Relative CAT activities following DNA transfection of BS-C-1 cells. Transfection with the null mutant, pΔ87-531/AC, generated a mean CAT activity value of 2531 cpm, while CAT activity in mock-transfected cells (M) was 60 cpm. (B) Relative CAT activities in transfected Huh-7 cells, which are derived from a human hepatocellular carcinoma. Transfection with the null mutant, pΔ87-531/AC, generated a mean CAT activity value of 536 cpm, while CAT activity in mock-transfected cells was 65 cpm. CAT assay incubation times were extended compared to those used in the experiments shown in Fig. 4 because of the expected low basal rate of HAV translation (78).

FIG. 5

Northern blot analysis of the poly(A) fraction of RNA extracted from BS-C-1 cells following DNA transfection with constructs containing the HAV IRES, pPwt/AC (lane 1), or its related null mutant pΔ87-531/AC (lane 2). Lane 3 was loaded with RNA from mock-transfected cells. The probe for hybridization was complementary to the CAT sequence.

FIG. 6

Enhancement of the translational activity of the poliovirus IRES within dicistronic transcripts is dependent on the cap-dependent translation of PTB from the upstream cistron. (A) Organization of plasmids encoding protease 2A (2A^pro) of poliovirus under control of the CMV IE promoter. pCMV-2A expresses wt 2A^pro, while pCMV-2A(H20N) expresses a mutant 2A^pro which lacks proteolytic activity. (B) BS-C-1 cells were cotransfected with dicistronic plasmid DNAs plus either pCMV-2A (grey bars) or pCMV-2A(H20N) (black bars). Results shown represent mean values obtained in a total of four transfections of plasmid DNAs ± standard deviation, normalized for each series to that obtained with the null mutant, pΔ87-531/SC (100%). Absolute CAT activities following transfection of the null mutant, pΔ87-531/SC, were 28,908 cpm with pCMV-2A cotransfection and 2,726 cpm with pCMV-2A(H20N) cotransfection, compared to 60 cpm for mock transfection.

FIG. 7

Indirect immunofluorescence detection of PTB in normal HeLa (A) Huh-7 (B), and BS-C-1 cells (C) and BS-C-1 cells 24 h following electroporation of the pPwt/AC plasmid (D). The primary antibody for these studies was rabbit anti-GST-PTB, while the secondary antibody was TRITC-labeled swine antibody to rabbit immunoglobulin.

FIG. 8

Immunoblot detection of PTB in transfected BS-C-1 and Huh-7 cells in comparison to increases in translation directed by the wt IRES of HAV. (A) Relative expression of renilla luciferase (RLuc) following DNA transfection of cells with a dicistronic plasmid expressing wt PTB from the upstream cistron, compared with a matched control plasmid encoding the PTB null mutant, Δ87-531, in the upstream cistron. RLuc was translated from the downstream cistron by a cap-independent process under control of the wt 5′NTR of HAV. Transfection was by a liposome-mediated procedure. (B) Immunoblot analysis of BS-C-1 and Huh-7 cells following DNA transfection under conditions identical to those used in panel A with monocistronic plasmids expressing either wt PTB or the null mutant, Δ87-531. C, cytoplasmic fraction; N, nuclear fraction.

FIG. 9

PTB stimulates the HCV IRES following transfection of mammalian cells with plasmids containing dicistronic transcriptional units (see Fig. 1B and the legend to Fig. 3). (A) CAT activities in lysates collected following DNA transfection of BS-C-1 cells with the null mutant, pΔ87-531/CC, or pPwt/CC. Transfection was by a cationic liposome-mediated method (see Materials and Methods) and with the null mutant, pΔ87-531/CL, generated a mean CAT activity of 22,907 cpm. Because of variation in transfection efficiency was greater with liposome-mediated transfection than with electroporation as in the experiments shown in Fig. 3 and 4, the results are shown as the ratio of CAT activities produced by the wt versus null mutant expression vectors. The data shown represent the mean values obtained in seven separate transfection experiments ± standard deviation. (B) CAT activities following DNA transfection of Huh-7 cells with the same plasmids. Transfection with the null mutant, pΔ87-531/CL, generated a mean CAT activity of 30,170 cpm. Results shown represent the mean values obtained in two separate transfection ± range.