Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2

Abstract

The Epstein-Barr virus protein (EB2) allows the nuclear export of a particular subset of early and late viral RNAs derived from intronless genes. EB2 is conserved among most herpesvirus members and its presence is essential for the production of infectious particles. Here we show that, besides its role as a nuclear export factor, EB2 strongly stimulates translation of unspliced mRNAs without affecting overall cellular translation. Interestingly, this effect can be reversed by the addition of an intron within the gene. The spliced mRNA is then efficiently exported and translated even in the absence of EB2. Moreover, we show that EB2 associates with translating ribosomes and increases the proportion of its target RNA in the polyribosomal fraction. Finally, testing of EB2 homolog proteins derived from EBV-related herpesviruses, shows that, even if they play similar roles within the replication cycle of their respective virus, their mechanisms of action are different.

Figure 1.

EB2 stimulates expression of the late EBV viral protein BDLF1. (A) Schematic representation of the BDLFI-encoding construct pTRE2-Flag-BDLF1. (B) Western analysis of Flag-BDLF1 expression in HeLa cells transfected with pTRE2-Flag-BDLF1, together with pTet-On and an expression plasmid for Flag-EB2 (pCI-FEB2) as indicated in the figure. Doxycycline was added as indicated. The M2 anti-Flag MAb was used to visualize both Flag-BDLF1 and Flag-EB2 proteins. (C) Quantification of the BDLF1 cytoplasmic RNA by semi-quantitative RT-PCR in HeLa cells transfected as described above.

Figure 2.

EB2 stimulates expression of a Renilla luciferase intronless gene at the translational level, independently of any viral-derived sequence. (A) Schematic representation of the luciferase intronless coding vector used in this study (pcDNAGlobinRen) showing positions of the CMV promoter and BGH polyadenylation signal. (B) Immunoblot of HeLa cells cotransfected with pcDNAGlobinRen together with the empty pCI vector or increasing amounts of the FlagEB2-encoding plasmid, pCI-FlagEB2 (250 and 500 ng). The M2 anti-Flag MAb was used to visualize Flag-EB2. Asterisk denotes an unspecific band detected by the M2 anti-Flag antibody. (C) Quantification of the amount of U6 snRNA present respectively in the nuclear and cytoplasmic fractions of cellular extracts used in D. U6 snRNA was amplified by RT-PCR using the specific primer set indicated in Table 1, and analyzed on a 2% agarose gel. (D) Measure of luciferase activity and quantification of cytoplasmic luciferase-encoding mRNAs by quantitative RT-PCR using GAPDH as an internal control. Total luciferase activity was measured 24 h post-transfection (top panel) and the amount of cytoplasmic luciferase coding mRNAs was quantified (middle panel). Translational efficiency (bottom panel) was calculated by normalizing the total luciferase activity by reference to the amount of cytoplasmic luciferase mRNA. AU: arbitrary units.

Figure 3.

EB2 does not affect protein stability or global cellular mRNA translation and its effect on translation is independent of the amount of cytoplasmic luciferase coding mRNA. (A) Time-lapse measure of total luciferase activity from HeLa cells mock transfected (pCI) or transfected with an EB2 expression vector (pCI-FlagEB2) (250 ng) after addition of cycloheximide to block translation. Luciferase activity was measured 0, 15, 30, 60, 120, 180 and 240 min after addition of cycloheximide to the cell medium. (B) Metabolic labeling of HeLa cells mock transfected (pCI) or transfected with the EB2-encoding plasmid (250 ng), using ³⁵S-labeled methionine. After a 30-min pulse labeling, cells were lyzed and total cellular proteins resolved on 12% SDS-PAGE. Total translation was quantified by phosphorimaging using a Fujifilm FLA5100. (C) Top panel: Luciferase activity was plotted against the amount of cytoplasmic luciferase coding mRNAs in HeLa cells transfected with increasing amounts of luciferase-encoding plasmid in the absence (pCI) or presence (pCI-FlagEB2) of EB2 (250 and 500 ng). Bottom panel: Translation rates per unit of luciferase-encoding mRNAs (calculated by normalizing luciferase activity by reference to the amount of cytoplasmic luciferase-encoding mRNAs) in HeLa cells transfected with increasing amounts of luciferase-encoding plasmid in the absence or presence of EB2 expression plasmid. AU: arbitrary units.

Figure 4.

Translation stimulation does not occur with spliced mRNAs. (A) Schematic representation of the intronless (pcDNAGlobinRen) and the intron-containing (pcDNAIntronGlobinRen) vectors encoding the Renilla luciferase showing positions of the human β-globin intron within the 5′UTR of the luciferase construct (gray arrows correspond to positions of the PCR primers used to test efficient splicing of the intron). (B) RT-PCR (using primers shown in A) from cells cotransfected with pcDNAGlobinRen and pCI or the EB2-encoding plasmid pCI-FlagEB2 (lanes 2 and 3) or from cells cotransfected with pcDNAIntronGlobinRen and pCI or pCI-FlagEB2 (lanes 5 and 6), or directly from the purified DNA vector (lanes 4 and 7). (C) Luciferase activity normalized by reference to the amount of cytoplasmic luciferase-encoding mRNAs from HeLa cells cotransfected with pcDNAGlobinRen and pCI or pCI-FlagEB2, or pcDNAIntronGlobinRen and pCI or pCI-FlagEB2. The amount of luciferase-encoding mRNAs was monitored by quantitative RT-PCR.

Figure 5.

EB2 cosediments with polyribosomes and enhances utilization of its target mRNA by the translation machinery. (A, B and C) Cell cytosolic extracts of HEK293T transfected with pcDNAGlobinRen alone or pcDNAGlobinRen together with pCI-Flag.EB2 or pcDNAIntron-GlobinRen alone, were fractionated across 10–50% sucrose gradients. (A) Fractions from the gradient corresponding to HEK293T transfected with pcDNAGlobinRen together with pCI-Flag.EB2 were analyzed by western blotting with antibodies against the Flag epitope to detect Flag.EB2, poly(A)-binding protein (PABP) or α-tubulin. (Top) UV absorbance (254 nm) profile of cytoplasmic ribonucleoprotein complexes. (B) 18S and 28S RNA profile determined by quantitative RT-PCR. (C) Quantification of the Renilla luciferase reporter mRNA fractionated across 10–50% sucrose gradients by quantitative RT-PCR. (Top panel) HEK293T transfected with pcDNAGlobinRen. (Middle panel) HEK293T transfected with pcDNAGlobinRen together with pCI-Flag.EB2. (Bottom panel) HEK293T transfected with pcDNAIntron-GlobinRen. (D, E and F) An EDTA-treated cytoplasmic extract of HEK 293T cells transfected with pcDNAGlobinRen together with pCIF.EB2 was fractionated across a 10–50% sucrose gradients. (D) EB2 is relocalized to the top of the gradient. (Top) UV absorbance (254 nm) profile of cytoplasmic ribonucleoprotein complexes. (E) 18S and 28S RNA profile determined by quantitative RT-PCR. (F) Quantification of the Renilla luciferase reporter mRNA fractionated across a 10–50% sucrose gradient by quantitative RT-PCR.

Figure 6.

Differential effects on translation from EB2-related proteins derived from different herpesviruses. (A) Cytoplasmic luciferase mRNA levels monitored by quantitative PCR in HeLa cells mock transfected (pCI) or transfected with 500 ng of EBV EB2, HSV-1 ICP27, HKSV ORF57 and HCMV UL69-encoding plasmids. (B) Translation rate. Luciferase activity was normalized by the amount of cytoplasmic mRNAs from cells mock transfected (pCI) or transfected with EBV EB2, HSV-1 ICP27, HKSV ORF57 and HCMV UL69-encoding vectors, together with the reporter plasmid pcDNAGlobinRen. AU: arbitrary units.