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Detailed analysis of the requirements of hepatitis A virus internal ribosome entry segment for the eukaryotic initiation factor complex eIF4F - PubMed

Detailed analysis of the requirements of hepatitis A virus internal ribosome entry segment for the eukaryotic initiation factor complex eIF4F

A M Borman et al. J Virol. 2001 Sep.

Abstract

The hepatitis A virus (HAV) internal ribosome entry segment (IRES) is unique among the picornavirus IRESs in that it is inactive in the presence of either the entero- and rhinovirus 2A or aphthovirus Lb proteinases. Since these proteinases both cleave eukaryotic initiation factor 4G (eIF4G) and HAV IRES activity could be rescued in vitro by addition of eIF4F to proteinase-treated extracts, it was concluded that the HAV IRES requires eIF4F containing intact eIF4G. Here, we show that the inability of the HAV IRES to function with cleaved eIF4G cannot be attributed to inefficient binding of the cleaved form of eIF4G by the HAV IRES. Indeed, the binding of both intact eIF4F and the C-terminal cleavage product of eIF4G to the HAV IRES was virtually indistinguishable from their binding to the encephalomyocarditis virus IRES, as assessed by UV cross-linking and filter retention assays. Rather, we show that HAV IRES activity requires, either directly or indirectly, components of the eIF4F complex which interact with the N-terminal fragment of eIF4G. Effectively, HAV IRES activity, but not that of the human rhinovirus IRES, was sensitive to the rotavirus nonstructural protein NSP3 [which displaces poly(A)-binding protein from the eIF4F complex], to recombinant eIF4E-binding protein (which prevents the association of the cap binding protein eIF4E with eIF4G), and to cap analogue.

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Figures

FIG. 1
FIG. 1

(A) Schematic representation of plasmids used to generate monocistronic IRES-carrying mRNAs. The HIV-1 p24 coding region is shown as an open box. The 5′ UTRs derived from picornavirus sequences are depicted as thick lines; the numbers below the HRV2 and HAV IRESs are based on the viral genome sequences and denote the first and last nucleotides of picornavirus sequence present. The junction between the 5′ UTR and the p24 coding region is detailed, and the initiation codon for p24 synthesis is underlined. (B) In vitro translation of monocistronic uncapped HAV p24 mRNA in the presence of the 2A or Lb proteinase. Standard rabbit reticulocyte lysate translation reactions (see Materials and Methods) were preincubated for 10 min at 30°C with H100 buffer (no RNA and buffer lanes), recombinant 2A and Lb proteinases (20 μg/ml; Active 2A and Active Lb lanes), or recombinant 2A and Lb proteinases which had been previously inactivated by incubation for 10 min at 4°C with elastatinal and E-64, respectively (20 μg/ml; Inactive 2A and Inactive Lb lanes). Reactions were then programmed with uncapped mRNA transcribed in vitro from pHAVp24 (10 μg/ml) (see Materials and Methods). A control reaction was programmed with water (no RNA lane). Trans- lations were processed as described in Materials and Methods. The autoradiograph of the dried 20% polyacrylamide gel is shown. The position of the HIV-1 p24 translation product is marked. Translation efficiency derived from densitometric quantification is plotted below each lane. (C) Integrity of eIF4G and PABP in translation reactions treated with the 2A and Lb proteinases. Standard translation reactions were incubated at 30°C with the indicated concentrations of purified recombinant 2A and Lb proteinases for the times indicated above each lane prior to analysis by Western blotting as described in Materials and Methods, using antibodies raised against CpN (top) and PABP (bottom). The positions of intact eIF4G, CpN, and PABP are indicated. (D) PABP is cleaved upon extended incubation with high concentrations of 2A but not Lb proteinase. Standard translation reactions were incubated at 37°C with the indicated concentrations of purified recombinant 2A and Lb proteinases for the times indicated above each lane prior to analysis by Western blotting as described in Materials and Methods, using antibodies raised against PABP. The position of intact PABP is indicated. The amount of PABP detected in each reaction was quantified by densitometry and is indicated below each lane as a percentage of the amount detected in the absence of proteinase treatment (0 min lane).

FIG. 2
FIG. 2

(A) Purified rabbit eIF4F was treated with 2A proteinase, and the CpN and CpC were separated as described in Materials and Methods; 180, 180, 170, or 80 ng of eIF4F, 2A proteinase-treated eIF4F, or purified CpN or CpC, respectively, was submitted to Western blotting using antibodies raised against CpN (top) and CpC (bottom). The positions of intact eIF4G, CpN, and CpC are indicated. (B) UV Laser cross-linking of eIF4F to the HAV and EMCV 5′ UTRs. UV cross-linking reactions containing 180 ng of intact eIF4F or 2A proteinase-treated eIF4F, 170 ng of purified CpN, or 80 ng of purified CpC were assembled exactly as described in Materials and Methods and supplemented with radiolabeled probes corresponding to the full EMCV (left) or HAV IRES (right). After cross-linking and RNase digestion, reactions were analyzed by SDS-PAGE. The autoradiograph of the dried 15% gel is shown; positions of the approximately 220-, 97-, and 43-kDa proteins labeled after cross-linking are indicated. The right-hand panel was exposed approximately three times longer than the left-hand panel. (C) The EMCV and HAV IRESs bind eIF4G and CpC with similar affinities. Filter binding assays were performed exactly as described in Materials and Methods, using radiolabeled probes corresponding to the HAV (circles) or EMCV (squares) IRES and the given concentrations of intact eIF4F (top) purified CpC (filled symbols, bottom), or purified CpN (open symbols, bottom). The amount of retained RNA (expressed as a percentage of the maximal retained RNA) is plotted against protein concentration.

FIG. 3
FIG. 3

Effects of cap analogue, 4E-BP1, and rotavirus NSP3 on HAV and HRV IRES-driven translation. Standard translation reactions programmed with the indicated uncapped RNAs (10 μg/ml) were supplemented with the concentrations indicated above each lane of recombinant rotavirus NSP3 protein (A; concentrations in micrograms per milliliter), recombinant 4E-BP1 protein (B; reactions (a) through i were made 0, 25, 50, 100, 200, 400, 800, 1,600, and 3,200 nM, respectively, in recombinant 4E-BP1), and cap analogue (C; reactions a through g were made 0, 0.22, 1.1, 5.5, 11, 22 and 66 μM, respectively, in cap analogue). An asterisk denotes the position of a strongly labeled smaller p24-derived product in reactions programmed with HRV p24 RNA, whose synthesis results from leaky scanning and initiation of ribosomes at an internal AUG in the p24 coding region. The protein larger than the p24 polypeptide synthesized from HRV p24 mRNAs results from translation initiation at an upstream AUG at position 449 in the HRV2 5′ UTR as previously described (4). Relative translation efficiency (as a percentage of that measured in the absence of each inhibitor) derived from densitometric quantification is plotted below each panel (filled circles, HAV IRES activity; open squares, HRV IRES activity). The mean values from two independent experiments (±standard deviation) are plotted in each case. (D) Displacement of eIF4E from eIF4G as measured by Lb proteinase-mediated cleavage of eIF4G. Translation reactions were incubated for 10 min on ice with H100 buffer or 4E-BP1 in H100 buffer (final concentrations, from left to right, of 30, 60, 120, and 250 nM) before being treated with H100 buffer or Lb proteinase in H100 buffer (Lb + lanes; final proteinase concentration of 15 μg/ml) for 15 min at 30°C. The integrity of eIF4G was then assessed by Western blotting as described in Materials and Methods. The positions of intact eIF4G and the primary and secondary N-terminal cleavage products of eIF4G (1° CpN and 2° CpN, respectively) are indicated. The quantity of intact eIF4G or 2° CpN in each reaction was evaluated by densitometry and is plotted below each lane. Since the anti-eIF4G antibody used recognizes the primary cleavage product of eIF4G much more efficiently than it recognizes either the intact moleule or the secondary cleavage product, the signal for 1° CpN was saturating in all reactions which had been treated with Lb proteinase. (E) Effect of NSP3 on coimmunoprecipitation of PABP and eIF4G. The degree of displacement of PABP from eIF4G was assessed by immunoprecipitation of complexes using antibodies raised against eIF4G followed by Western blot analysis of immunoprecipitates using antibodies directed against PABP as described previously (8, 34). The data presented are modified from reference .

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