INTRODUCTION

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Epstein-Barr virus (EBV), a human herpesvirus, infects lymphoid and epithelial cells. Infection by this virus is closely related to several neoplastic diseases, including Burkitt's lymphoma, T-cell lymphoma, Hodgkin's disease, and nasopharyngeal carcinoma (45). EBV has two distinct life cycles. After infecting B lymphocytes, the virus immortalizes the cells and is maintained under latent conditions. During this stage, the virus expresses six nuclear antigens (EBNAs), three membrane proteins, and two species of small RNA (EBERs) (31). The viral lytic cycle is initiated when the cells are exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA), sodium butyrate, calcium ionophores, or anti-immunoglobulin (6, 14, 35, 51, 55). EBV lytic activation is associated with the expression of two EBV-encoded immediate-early proteins, Rta and Zta (7, 18, 51). Zta, a transcription factor of the b-Zip family (15), not only activates the transcription of EBV immediate-early and early genes (11, 17, 25, 29, 49) but also binds to the lytic origin of EBV, which is a prerequisite for EBV lytic replication (16, 48). Rta protein is another transcription factor, often acting synergistically with Zta to activate EBV lytic genes (11, 25, 30).

Rta and Zta are encoded by BRLF1 and BZLF1, respectively. These two genes are situated adjacent to each other on the EBV genome (5). During the immediate-early stage of the EBV lytic cycle, a 1.0-kb monocistronic BZLF1 mRNA is transcribed from a promoter (P_Z) located immediately upstream from the gene (Fig. 1A) (37). BZLF1 is also transcribed from the promoter of BRLF1 (P_R) (37). The mRNA transcribed from this promoter is 4 kb long. This mRNA is spliced into a 3.3-kb mRNA or an 0.8-kb mRNA (Fig. 1A) (37). The 0.8-kb transcript (Fig. 1A) encodes an Rta-Zta fusion protein called RAZ. This protein forms a heterodimer with Zta to inhibit the function of Zta (19). The 3.3-kb transcript is bicistronic, containing both BRLF1 and BZLF1 (Fig. 1A) (37). In an early study, we used a plasmid containing a BRLF1-ZLUC bicistronic transcription unit transcribed from the cytomegalovirus immediate-early promoter (P_C) and demonstrated that only the bicistronic mRNA is transcribed from the plasmid in P3HR1 cells and EBV-negative Akata cells; the P_Z promoter remains inactive, and the monocistronic ZLUC mRNA is not transcribed in the cells (9). That study also demonstrated that both Rta and Zta are efficiently translated from the bicistronic mRNA. Furthermore, the bicistronic transcript is not further spliced or cleaved, indicating that the Zta protein is not translated from a degradation product of the 3.3-kb mRNA (9). Genetic studies also demonstrated that deletion, insertion, frameshift, and nonsense mutations in BRLF1 abolish the translation of Zta from the bicistronic mRNA. Additionally, these mutations can be complemented by a functional BRLF1, indicating that Rta protein is required for translating Zta from the bicistronic mRNA (9).

View larger version (21K): [in this window] [in a new window]   FIG. 1.   (A) mRNAs transcribed from PR and PZ during the EBV lytic cycle. The pre-mRNA transcribed from PR (4.0 kb) is spliced into a 3.3-kb and an 0.8-kb mRNA. An inhibitor of Zta called RAZ is translated from the 0.8-kb transcript. Meanwhile, the 1.0-kb BZLF1 transcript is transcribed from PZ. The black and white boxes represent the exons of BZLF1 and BRLF1, respectively. (B) The intercistronic region of the BRLF1-BZLF1 bicistronic mRNA is 210 nucleotides long. The region includes five ORFs. The initiation codons of these ORFs are boxed and numbered. This area also contains two large inverted repeats, SI and SII. Regions I and II are the two crucial regions for Zta translation from the bicistronic mRNA. Meanwhile, KspI and HindIII are the restriction enzyme sites created by site-directed mutagenesis. TATA, TATA sequence of PZ; +1, transcription start site of BZLF1 monocistronic mRNA. (C) Plasmids pCMV-RZLUC(KspI) and pCMV-R-LUC are the reporter plasmids used for the analysis of bicistronic translation.

In eukaryotic cells, mRNAs are rarely bicistronic. However, if an mRNA contains two open reading frames (ORFs), the presence of the upstream ORF (uORF) often regulates the translation of the downstream ORF, leading to a scenario in which the downstream ORF is either poorly translated or not translated (34). This phenomenon is explained by translation typically being initiated from the cap site of mRNA, subsequently leading to translation of uORF. However, after translating the uORF, ribosomes may lose all the initiation factors associated with 40S ribosome and be unable to continue to scan the intercistronic region to initiate translation of the downstream ORF (33). According to a translation reinitiation model, if a bicistronic mRNA includes a short uORF, ribosomes which translated uORF may still retain some initiation factors following translation (36). Such a retaining effect may allow 40S ribosome to scan the intercistronic region to begin translation of the downstream ORF (34). For example, efficiency of translation reinitiation in human immunodeficiency virus type 1 mRNA is determined by the length of uORF and by intercistronic distance (36). Another example of translation reinitiation is the translation of GCN4 of Saccharomyces cerevisiae, which acts as a transcription activator of amino acid biosynthesis genes (28). The GCN4 ORF in mRNA is preceded by four short uORFs (uORF1 to uORF4). As is generally known, translation of GCN4 first involves translation of uORF1. Meanwhile, after uORF1 is translated, 40S ribosome continues to scan the sequence downstream. Under nonstarvation conditions, the translation initiation complex is reassembled rapidly, allowing translation to reinitiate from the initiation codon of uORF4. This translation apparently inhibits the translation of GCN4. However, under starvation conditions, a greater scanning distance is required for 40S ribosome to recruit the translation initiation factors necessary to form a translation initiation complex. Such a requirement explains why 40S ribosome neglects the initiation codon of uORF4 and initiates translation from the initiation codon of GCN4 (1, 39). Besides the translation reinitiation mechanism, translation of a bicistronic mRNA can also involve a mechanism called leaky scanning (10, 34). If the AUG codon of a uORF is surrounded by a reading context that does not favor translation initiation, 40S ribosome may skip this AUG codon and use a downstream AUG codon for translation initiation. In addition to ribosome scanning and leaky scanning, the genomes of certain viruses are translated by directly attaching ribosome subunits to an internal ribosome entry site (IRES) in the 5' untranslated region (internal initiation) (41). Several types of IRESs have been defined, which differ in sequence and structure (42, 53). Meanwhile, eIF-4B and several other proteins binding to the polypyrimidine tract in the IRES are involved in internal initiation (43). In addition to internal initiation, translation of viral protein may occasionally involve ribosome shunting (22, 23, 54). In the case of cauliflower mosaic virus 35S RNA, shunting allows ribosomes to bypass the elements which block ribosome scanning in the untranslated region (20, 22) and requires the activity of a virus-encoded translational transactivator (21). In this study, we demonstrate that Zta protein is translated from the BRLF1-BZLF1 bicistronic mRNA by a discontinuous ribosome-scanning mechanism similar to ribosome shunting or internal initiation. In addition, a sequence that complements helix 36 of 18S rRNA in the intercistronic region is required for initiating the translation.

	MATERIALS AND METHODS

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Cell line. An EBV-positive Burkitt's lymphoma cell line, P3HR1, was used for transfection studies. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum.

Plasmids. Plasmids pGL2-Basic and pGEM-7Zf() were purchased from Promega Corp. (Madison, Wis.). Plasmid pCMV-RZLUC (9) contains a BRLF1-BZLF1 transcription unit transcribed from P_C, in which BZLF1 is fused with the firefly luciferase gene (luc). Plasmid pCMV-RZLUC(KspI), a derivative of pCMV-RZLUC (9), contained a newly created KspI site located 5 nucleotides downstream from the termination codon of BRLF1. This KspI site was created by the method of Ho et al. (24), using PCR primers KSPA (5'-ATTTTAGACACCGCGGAAAACTGCC) and 3-ATG (5'-CTATGAGGTATATTAGCAATGCC) and primers KSPB (5'-GGCAGTTTTCCGCGGTGTCTAAAAT) and RZR (5'-GATGAATGTCTGCTGCATGCCATGC). PCR was performed, using pCMV-RZLUC as a template under conditions of 94°C for 30 s, 56°C for 60 s, and 72°C for 60 s, for 35 cycles. Approximately 1/10 of the PCR fragments was mixed and reamplified without adding any primers under conditions of 94°C for 30 s, 45°C for 60 s, and 72°C for 60 s, for 25 cycles. The PCR fragment was further amplified with primers RZR and 3-ATG. The amplified fragment was cut with NsiI and was used to replace the NsiI fragment in pCMV-RZLUC to generate pCMV-RZLUC(KspI). Plasmids pRZLUC(XBA) and pCMV-RZLUC(XBA) were generated by adopting a similar strategy. Plasmid pCMV-RZLUC(KSP-SL) was generated by inserting a double-stranded oligonucleotide, SL-A (5'-CCGCGGCGCGTGGTGGCGGCTGCAGCCGCCACCACGCGCGGCGG) into the KspI site of pCMV-RZLUC(KspI). Plasmid pCMV-RZLUC(STY-SL) was generated by inserting a double-stranded oligonucleotide, SL-B (5'-GAATTCCCATCTTGGGAATTC), into the StyI site of pCMV-RZLUC(KspI). Plasmid pGEM-SS was constructed by inserting the SphI fragment of pCMV-RZLUC, which contained the 3' region of BRLF1, the intercistronic region, and the 5' region of ZLUC, into the SphI site of pGEM-7Zf(). Plasmid pGEM-SS(KspI) was identical to pGEM-SS except that the SphI fragment from pCMV-RZLUC(KspI) was inserted. Plasmid pGEM-SS(DZ) was identical to pGEM-SS(KspI) except that the BZLF1 sequence in ZLUC was deleted and a HindIII site was present at the 3' terminus of the intercistronic region. Plasmid pCMV-R-LUC was constructed by replacing the SphI fragment of pCMV-RZLUC(KspI) with the SphI fragment of pGEM-SS(DZ). Plasmid pCMV-R-LUC(BC) is a derivative of pCMV-R-LUC in which the intercistronic region (the KspI-HindIII fragment) was replaced by a 133-nucleotide sequence (nucleotide 8628 to nucleotide 8760 of the EBV genome) amplified from the BamHI-C fragment of EBV. The forward primer for amplifying this 133-nucleotide fragment contained a KspI site at the 5' end; the reverse primer contained a HindIII site at the 5' end. The amplified DNA fragment was digested by KspI and HindIII and was used to replace the KspI-HindIII fragment of pGEM-SS(KspI) to generate pGEM-SS(BC). The SphI fragment of this plasmid was then isolated by restriction digestion and was used to replace the SphI fragment of pCMV-RZLUC to generate pCMV-R-LUC(BC). Next, a double-stranded oligonucleotide, consisting of the sequence from nucleotides 86 to 125 of the intercistronic region (region I), was inserted into the SpeI site, which is located at the 40th nucleotide of the 133-nucleotide BamHI-C fragment in pGEM-SS(BC). The fragment was then isolated by SphI digestion and was used to replace the SphI fragment of pCMV-RZLUC to generate pCMV-R-LUC(BC-RI). Plasmid pCMV-R-LUC(BC-RII) was constructed with a similar method with a double-stranded oligonucleotide consisting of the region II sequence (from nucleotides 126 to 165 of the intercistronic region). Plasmids pRZLUC(D33) and pCMV-RZLUC(D33) were generated by amplifying a fragment lacking the 33-bp region downstream from the StyI site of the intercistronic region. The forward primer used for amplification contained an StyI site at the 5' end. The amplified fragment was digested with StyI and HindIII and was used to replace the StyI-HindIII fragment in pGEM-SS. The fragment was reisolated from pGEM-SS by SphI digestion to replace the SphI fragment in pRZLUC and pCMV-RZLUC. Plasmids pRZLUC(D62) and pCMV-RZLUC(D62) were generated by similar methods. Mutants 5'-45, 5'-85, 5'-165, and 5'-205 contained a deletion from the KspI site to the 45th, 85th, 165th, and 205th nucleotides in the intercistronic region, respectively. Deletion 5'-45 was generated by amplifying a fragment containing the region between nucleotide 45 of the intercistronic region and the 15th nucleotide of BZLF1, using pGEM-SS(KspI) as a template. The forward primer used for amplification contained a KspI site at the 5' end. The amplified fragment was cut by KspI and StyI and was then used to replace the KspI-StyI fragment of pGEM-SS(DZ). The SphI fragment of this plasmid was isolated by restriction digestion and was used to replace the SphI fragment in pCMV-R-LUC to generate a plasmid containing the 5'-45 deletion. Mutation 5'-85 was generated by a similar strategy. Mutation 5'-165 was generated by digesting pGEM-SS(KspI) with NotI and XhoI, treating the fragment with T4 DNA polymerase, and ligating with T4 DNA ligase. This procedure removed the NotI-XhoI fragment as well as the XbaI site on the vector portion of the plasmid. This plasmid, pSS-KspI(XbaI), was used as a template to amplify a DNA fragment containing the region from nucleotide 165 of the intercistronic region to the 5' region of luc. The forward primer used for PCR contained a KspI site at the 5' end. The PCR fragment was digested with KspI and XbaI; the fragment was used to replace the KspI-XbaI fragment in pSS-KspI(XbaI). The resulting plasmid was cut with SphI, and the restriction fragment was used to replace the SphI fragment in pCMV-RZLUC. Deletion 5'-128 was generated by first deleting the KspI-StyI fragment of pGEM-SS(DZ). Next, the SphI fragment of the resulting plasmid was isolated and used to replace the SphI fragment in pCMV-RZLUC. Deletion 5'-205 was generated with a method similar to that used for generating the 5'-128 deletion. Deletion 3'-86 was generated by amplifying a DNA fragment covering the region between the last 7 nucleotides of BRLF1 and the 85th nucleotide of the intercistronic region, using pCMV-RZLUC(KspI) as a template. The primers used for amplification were D1 (5'-ATTTTAGACACCGCGGAAAACTGCC) and D2 (5'-CAAGCTTTTTAGGTGTGTCTATGAGGT). In primer D2, a HindIII site was present at the 5' end. The PCR fragment was digested with KspI and HindIII; the fragment was used to replace the KspI-HindIII fragment of pCMV-R-LUC. Deletion 3'-166 was generated by the same method except that primer D3 (5'-GAAGCTTTGAGTTACCTGTCTAACATC) instead of D2 was used. Deletion 3'-129 was generated by cutting pGEM-SS(DZ) with StyI and HindIII. The plasmid was then self-ligated after the DNA was repaired with the Klenow fragment of DNA polymerase I. The SphI fragment was then isolated and was used to replace the SphI fragment in pCMV-R-LUC. Mutant R86-165 was generated by cutting a pGEM-SS(DZ) derivative, containing the 5'-85 deletion, with MluI and StyI. The fragment was isolated and was used to replace the MluI-StyI fragment of the 3'-166 deletion mutant of pGEM-SS(DZ). The SphI fragment was then isolated to replace the SphI fragment in pCMV-RZLUC. 18SC and 18S fragments, containing the region between nucleotide 1401 and nucleotide 1560 of 18S rRNA, were amplified, using the DNA of P3HR1 cells as a template. In the 18SC fragment, a KspI site was created at the end with nucleotide 1560 and a HindIII site was created at the other end. This DNA was used to replace the KspI-HindIII fragment of pGEM-SS(KspI). The fragment was isolated by SphI digestion and was used to replace the SphI fragment in pCMV-RZLUC to generate pCMV-R-LUC(18SC). Plasmid pCMV-R-LUC(18S) was generated by the same method except that the KspI and the HindIII sites were created at the opposite ends of the 18S DNA fragment. The constructs described herein were also generated in pRZLUC and pR-LUC, which have sequences identical to those of pCMV-RZLUC and pCMV-R-LUC, except without P_C. Plasmid pBMLF1-luc contains a BMLF1 promoter (nucleotides 84850 to 84289 of the EBV genome) inserted into the SmaI site upstream from a luc gene in pGEM-7Zf(). Moreover, linker-scanning mutants were generated by site-directed mutagenesis (24). In each linker-scanning mutant, every nucleotide A was changed to nucleotide C and vice versa, and every nucleotide G was changed to nucleotide T and vice versa. The five initiation codons in the intercistronic region were changed to GTG, TTG, ATA, TTG, and GTG, respectively, by site-directed mutagenesis (24).

Northern blot analysis. Total RNA was isolated from P3HR1 cells with guanidinium isothiocyanate and was purified by CsCl centrifugation according to a method described elsewhere (46). RNA was separated by electrophoresis on a 1% agarose-formaldehyde gel and was transferred to a nylon membrane (Amersham). A glyceraldehyde-3-phosphate dehydrogenase probe (8) and a luc probe (9) were prepared by in vitro transcription with T7 RNA polymerase. Hybridization was performed according to a method described elsewhere (9).

Transfection and luciferase assay. Plasmids used for the transfection studies were prepared by CsCl gradient centrifugation according to a method described elsewhere (46). For plasmid transfection, 10 µg of plasmid DNA was mixed with 5 × 10⁶ cells in 300 µl of culture medium. Electroporation was performed at 960 µF and 0.2 kV with a Bio-Rad (Richmond, Calif.) Gene Pulser electroporator. After electroporation, cells were transferred to 10 ml of fresh culture medium. TPA at a final concentration of 30 ng/ml and sodium butyrate at a final concentration of 3 mM were added to induce the EBV lytic cycle. Cell lysate was prepared 24 h after transfection according to a method previously described (13). Each transfection experiment was repeated at least three times, and each sample in the experiment was prepared in duplicate.

DNA sequencing. DNA sequencing was performed according to the chain termination method of Sanger et al. (47). The constructs reported herein were confirmed by sequencing.

	RESULTS

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Intercistronic region and bicistronic translation. Plasmid pCMV-RZLUC was constructed by cloning the BRLF1-BZLF1 transcription unit downstream from P_C and fusing the 5' 75 nucleotides of BZLF1 with the firefly luciferase gene (luc). In an earlier study, we confirmed that a BRLF1-ZLUC bicistronic mRNA is transcribed from this plasmid and a Zta-Luc (Zluc) fusion protein is efficiently translated from the bicistronic mRNA (9). Because pCMV-RZLUC does not contain a convenient restriction enzyme site that would allow mutations to be generated in the intercistronic region, we created a KspI site located 5 nucleotides downstream from the termination codon of BRLF1 in pCMV-RZLUC (Fig. 1B). The plasmid containing this site, pCMV-RZLUC(KspI) (Fig. 1C), exhibited a luciferase activity about equal to that displayed by pCMV-RZLUC (Fig. 2), indicating that the sequence change does not influence the translation of Zluc. The BZLF1 sequence in pCMV-RZLUC(KspI) was then removed to generate pCMV-R-LUC (Fig. 1B and C). This deletion increased luciferase activity by 1.2-fold (Fig. 2). Despite the sequence of BZLF1 being deleted in pCMV-R-LUC, the initiation codon of luc remains and has a more favorable reading context than the initiation codon of BZLF1, explaining the increased activity. The intercistronic region in pCMV-R-LUC was then deleted by KspI-HindIII digestion. This deletion reduced the expression by approximately 90% (Fig. 2, D-KH). The intercistronic region (KspI-HindIII fragment) in pCMV-R-LUC was also replaced with a 133-bp fragment isolated from the BamHI-C fragment of EBV. This replacement did not lead to the translation of luciferase (Fig. 2, BC), indicating that the intercistronic region is crucial for translating Zta.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Requirement of the intercistronic region for the bicistronic translation. Plasmids pCMV-RZLUC (RZLUC), pCMV-RZLUC(KspI) [RZLUC(KspI)], pCMV-R-LUC (R-LUC), pCMV-R-LUC(D-KH) (D-KH), and pCMV-R-LUC(BC) (BC) were transfected into P3HR1 cells. The luciferase activity exhibited by these plasmids was monitored at 24 h after transfection.

Mutation of the TATA sequence of the BZLF1 promoter. The TATA sequence of the BZLF1 promoter was also mutated (Fig. 3A) to examine how the mutation affects the expression of luciferase reporter protein. As expected, luciferase was not expressed from pRZLUC in P3HR1 cells under latent conditions (Fig. 3B) owing to the lack of transcription of BRLF1-ZLUC bicistronic mRNA and luc monocistronic mRNA. On the other hand, luciferase was expressed at a high level if the cells were treated with TPA and sodium butyrate (Fig. 3B), indicating that this treatment activates the BZLF1 promoter. This activation was not observed, however, if the TATA sequence was changed from TTTAAA to TCTAGA (an XbaI sequence) (Fig. 3B, XBA), indicating that the mutation severely inhibits the ability of the plasmid to transcribe the monocistronic mRNA under lytic conditions. The same mutation in pCMV-RZLUC decreased the expression of luciferase only 22% under latent conditions (Fig. 3C). Since Rta is known to activate P_Z (2, 44), a cotransfection study was performed to examine whether Rta activates the transcription of ZLUC monocistronic mRNA from the XBA construct. According to our results, cotransfecting pCMV-R and a reporter plasmid containing a luc gene transcribed from the BMLF1 promoter resulted in a high-level expression of luciferase activity (Fig. 3D), indicating that the Rta protein expressed from pCMV-R is capable of activating an EBV early promoter. On the other hand, Rta protein expressed from pCMV-R did not activate the transcription from the XBA mutant construct lacking a P_C (Fig. 3D), indicating that Rta protein is incapable of activating the mutant Zp promoter. In addition, under our experimental conditions, Rta does not activate the Zp in pRZLUC (Fig. 3D). The TATA sequence was also removed by deletion. Deleting 33 and 62 nucleotides from the StyI site abolished the expression of luciferase from pRZLUC under lytic conditions (Fig. 3B). On the other hand, the deletions in pCMV-RZLUC decreased the expression by only 25 and 35%, respectively (Fig. 3C), demonstrating that expression of luciferase by the TATA mutants of pCMV-RZLUC is unrelated to the transcription of ZLUC monocistronic mRNA.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Effects of TATA mutations and expression of Zluc reporter protein. (A) The TATA sequence (TTTAAA) of the BZLF1 sequence was either changed to TCTAGA (XbaI sequence) by site-directed mutagenesis or removed by deletion (D33, D62, and D-KH). (B) Plasmid pRZLUC (wild type) and its mutant derivatives (XBA, D33, D62, and D-KH) were transfected into P3HR1 cells. After transfection, cells were maintained under latent conditions (black bars) or treated with TPA and sodium butyrate (white bars) to induce the viral lytic cycle. (C) Plasmids pCMV-RZLUC (wild type) (white bars) and pRZLUC (wild type) (black bars) and their respective mutant derivatives (XBA, D33, D62, and D-KH) were transfected into P3HR1 cells, which were maintained under latent conditions. (D) Effects of Rta on the expression of luciferase from pCMV-RZLUC, the promoter of BMLF1 (pBMLF1-luc), the XBA mutant, pRZLUC, and pGL2-Basic. Luciferase activity was monitored at 24 h after transfection. ZIB, ZIC, ZIIIA, ZIIIB, ZII, and TATA sequences are cis elements regulating the transcription of BZLF1. S, StyI site; X, XbaI site; H, HindIII site.

ORFs in the intercistronic region and the translation of Zta. The intercistronic region of BRLF1-BZLF1 bicistronic mRNA contains five ORFs (Fig. 1B). To examine whether Zta protein is translated from the bicistronic mRNA by a translation reinitiation mechanism, we altered the initiation codon of these ORFs in pCMV-RZLUC to GUG, UUG, AUA, UUG, and GUG, respectively, to investigate whether these changes influence the translation. Theoretically, these sequence changes should remove the potential suppressive uORFs and increase the translation efficiency of the downstream ORF if Zluc translation indeed involves translation reinitiation. According to our results, altering the first, second, third, and fifth AUG codons did not significantly influence the translation of Zluc (Fig. 4). Meanwhile, altering the sequence of the fourth AUG codon reduced luciferase activity by roughly 40% (Fig. 4).

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Mutations of the AUG sequences in the intercistronic region and Zluc translation. The five AUG sequences in pCMV-RZLUC were altered to GUG (1st), UUG (2nd), AUA (3rd), UUG (4th), and GUG (5th), respectively. The plasmids were transfected into P3HR1 cells, and luciferase activity was monitored at 24 h after transfection.

Effect of high-energy stem-loop structures in the intercistronic region. A 44-nucleotide sequence, SL-A, was inserted into the KspI site of pCMV-RZLUC(KspI) (Fig. 1B) to elucidate how Zluc fusion protein is translated from the bicistronic mRNA. Kozak (32) demonstrated that the SL-A sequence, when transcribed into RNA, forms a hairpin structure with a G value of 64 kcal/mol, which can effectively block ribosome scanning. Inserting this sequence at the KspI site not only failed to prevent Zluc translation but also increased Zluc expression 1.8-fold (KSP-SL [Fig. 5]), suggesting that the ribosomes that translated Rta do not continue to scan the intercistronic region. Our results also show that inserting the SL-A sequence did not influence the transcription of the bicistronic mRNA (see lanes c to e, Fig. 9). We also inserted a stem-loop structure, SL-B, into the StyI site in the intercistronic region (Fig. 1B). SL-B has a G value of 14 kcal/mol, although it has an energy level lower than that of SL-A, and has also been demonstrated elsewhere to block ribosome scanning (1). Unlike the SL-A insertion, this insertion lowered Zluc translation to the background level (Fig. 5, STY-SL). The SL-A sequence was also inserted into the KspI site of pRZLUC(KspI), a plasmid that does not contain P_C and is incapable of transcribing the bicistronic mRNA. This insertion did not lead to the expression of luciferase (data not shown). Finally, we inserted the SL-A sequence into the KspI site of two mutant derivatives of pCMV-RZLUC(KspI), APA-FS and BA, which contain a mutation in BRLF1 and cannot translate Zluc (9). Inserting the SL-A sequence into the KspI site of these mutant constructs did not result in Zluc translation (Fig. 5, APA-KSL and BA-KSL).

View larger version (15K): [in this window] [in a new window]   FIG. 5.   Insertion of high-energy stem-loop structures into the intercistronic region and the translation of Zluc. SL-A and SL-B are two stem-loop structures known to block ribosome scanning. KSP, pCMV-RZLUC(KspI); KSP-SL, SL-A sequence inserted into the KspI site of pCMV-RZLUC(KspI); APA-KSL, the SL-A sequence inserted into the KspI site of a mutant derivative of pCMV-RZLUC(KspI), containing a frameshift mutation at the ApaI site in BRLF1; BA-KSL, SL-A sequence inserted into the KspI site of a mutant derivative of pCMV-RZLUC(KspI), containing a BstXI-ApaI deletion in BRLF1; STY-SL, SL-B sequence inserted into the StyI site in the intercistronic region of pCMV-RZLUC(KspI).

Deletion analysis of the intercistronic region. Deletions were generated to delineate the sequence in the intercistronic region essential for translation. Deleting the region from the KspI site to nucleotide 45 (5'-45) and nucleotide 85 (5'-85) of pCMV-R-LUC (Fig. 6A) did not influence the translation of luciferase (Fig. 6B). However, deletion from the KspI site to nucleotide 128 (the StyI site) (5'-128), nucleotide 165 (5'-165), and nucleotide 205 (5'-205) reduced luciferase activity by approximately 80 to 90% (Fig. 6B). Also examined was the effect of the 3' deletions on the translation of luciferase. Deletion from the HindIII site to nucleotide 166 (3'-166) (Fig. 6A) did not influence luciferase translation. Furthermore, deletions to nucleotide 129 (3'-129) and 86 (3'-86) (Fig. 6A) decreased luciferase activity by approximately 80 to 85% (Fig. 6B). Deleting both the regions upstream from nucleotide 85 and downstream from nucleotide 166 did not influence the translation (Fig. 6B, R86-165). These results suggested that the region between nucleotides 86 and 165 is critical for translating the downstream ORF. Northern blot analysis also revealed that these deletions did not significantly influence the transcription of the bicistronic mRNA (see lanes p to w, Fig. 9).

View larger version (25K): [in this window] [in a new window]   FIG. 6.   Deletion analysis of the intercistronic region. (A) Deletions were generated from the KspI site of pCMV-R-LUC to nucleotides 45 (5'-45), 85 (5'-85), 128 (5'-128), 165 (5'-165), and 205 (5'-205). Deletions were also generated from the HindIII site to nucleotides 166 (3'-166), 129 (3'-129), and 86 (3'-86). In R86-165, the regions upstream from nucleotide 85 and downstream from nucleotide 166 were deleted. (B) The plasmids were transfected into P3HR1 cells, and luciferase activity exhibited by the plasmids was examined at 24 h after transfection.

Linker-scanning analysis. The importance of the region between nucleotides 86 and 165 in pCMV-R-LUC(R86-165) was further studied through linker-scanning analysis. A mutation in the region between nucleotides 86 and 95 (M86-95) decreased luciferase activity by approximately 51% (Fig. 7), a mutation between nucleotides 96 and 105 (M96-105) decreased the activity by 54%, a mutation between nucleotides 106 and 115 (M106-115) decreased the activity by 70% (Fig. 7), and a mutation between nucleotides 136 and 145 (M136-145) decreased luciferase activity by 77% (Fig. 7). Changing the sequence between nucleotides 116 and 135 (M116-125 and M126-135) decreased expression by 30 to 40% (Fig. 7). Mutations in the region between nucleotides 146 and 165 (M146-155 and M156-165) only slightly decreased luciferase expression (Fig. 7). A Northern blot analysis revealed that these mutations did not significantly influence the transcription of bicistronic mRNA (see lanes f to m, Fig. 9).

View larger version (21K): [in this window] [in a new window]   FIG. 7.   Linker-scanning analysis. The intercistronic region was replaced by an RNA comprising the sequence between nucleotides 86 and 165 in the intercistronic region (R86-165). Mutations were generated in the regions in the plasmid between nucleotides 86 and 95 (M86-95), 96 and 105 (M96-105), 106 and 115 (M106-115), 116 and 125 (M116-125), 126 and 135 (M126-135), 136 and 145 (M136-145), 146 and 155 (M146-155), and 156 and 165 (M156-165). The plasmids were transfected into P3HR1 cells, and luciferase activity exhibited by the plasmids was examined at 24 h after transfection.

Involvement of a sequence complementary to the 18S rRNA in Zta translation. The sequence between nucleotides 86 and 165 is arbitrarily divided into two regionsregion I and region II. Region I, from nucleotides 86 to 125, is partially complementary to the region between nucleotides 1484 and 1528 of 18S rRNA (Fig. 8A and B). This study has already demonstrated that luciferase protein is not translated from the bicistronic mRNA transcribed from pCMV-R-LUC(BC), where the intercistronic region is replaced with a 133-bp sequence from the BamHI-C fragment of EBV (Fig. 2). Inserting the region I sequence into the intercistronic region in pCMV-R-LUC(BC) partially restored the translation of luciferase to a level approximately 50% of that exhibited by the wild-type construct (Fig. 8C, BC-RI). Meanwhile, inserting the region II sequence into the intercistronic region in pCMV-R-LUC(BC) did not result in the translation of luciferase (Fig. 8C, BC-RII). We also substituted the entire intercistronic region with a fragment containing the sequence between nucleotide 1401 and nucleotide 1560 of 18S rRNA. If the sequence was inserted in an orientation complementary to 18S rRNA, the plasmid [pCMV-R-LUC(18SC)] exhibited luciferase activity equivalent to that exhibited by the wild-type construct (Fig. 8C). Luciferase was not translated, however, if the 18S sequence was inserted in the opposite orientation (Fig. 8C, 18S). Hybridization analysis also revealed that a lack of translation by the 18S mutant is not attributed to a lack of transcription of the bicistronic mRNA (see lanes n and o, Fig. 9). Inserting SL-A into the KspI site of pCMV-R-LUC(18SC) did not affect the translation. Meanwhile, inserting the SL-A sequence into the APA-FS frameshift mutation in pCMV-R-LUC(18SC) did not lead to the translation of the reporter protein (Fig. 8C).

View larger version (22K): [in this window] [in a new window]   FIG. 8.   Sequence complementation between region I (nucleotides 86 to 125 in the intercistronic region) and the region between nucleotides 1484 and 1528 of 18S rRNA (A), the sequence and the structure of helix 36 of 18S rRNA (B), and the importance of the region I sequence in translating luciferase protein from the bicistronic mRNA (C). The intercistronic region in pCMV-R-LUC (R-LUC) was replaced with a 133-nucleotide sequence from the BamHI-C fragment of EBV (BC). The sequences between nucleotides 86 and 125 (BC-RI) and between nucleotides 126 and 165 (BC-RII) of the intercistronic region were inserted into the SpeI site of the BamHI-C fragment. The intercistronic region was also replaced with a fragment, consisting of the sequence between nucleotides 1401 and 1560 of 18S rRNA. The sequence was inserted in an orientation that complements the 18S rRNA (18SC) or in the opposite orientation (18S). The nucleotides complementary to those in region I are indicated by dots in panel B. 18SC(SL-A), SL-A sequence inserted into the KspI site in pCMV-R-LUC(18SC); 18SC(APA-FS), pCMV-R-LUC(18SC) containing a frameshift mutation at the ApaI site in BRLF1; h33 to h44, helices in 18S rRNA; numbers, nucleotide positions.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Zluc is translated from the BRLF1-ZLUC bicistronic mRNA. As is generally known, EBV in P3HR1 cells can occasionally enter a lytic cycle spontaneously. This lytic activation is presumably attributed to the transcription of monocistronic BZLF1 mRNA. Therefore, in our experiments, we used freshly cultured cells for transfection and for Northern blot analysis and RNA was isolated within 15 to 20 h after transfection. Ragoczy et al. (44) and Adamson et al. (2) recently demonstrated that Rta protein, when expressed at extremely high levels, activates the transcription of BZLF1 monocistronic mRNA. This raises the possibility that Rta protein translated from the BRLF1-ZLUC bicistronic mRNA might have activated the transcription of ZLUC monocistronic mRNA that was undetected by Northern blot analysis and resulted in the expression of Zluc. To eliminate these possibilities, we mutated the TATA sequence of the BZLF1 promoter to examine the consequence of the mutations for Zluc expression. Our study reveals that the BZLF1 promoter in the pRZLUC construct is functional in P3HR1 cells, since Zluc was expressed at a high level when the cells were treated with TPA and sodium butyrate (Fig. 3B). Varying or deleting the TATA sequence in pRZLUC apparently destroys the function of the promoter, since Zluc is not expressed after lytic induction by TPA and sodium butyrate (Fig. 3B). In the case of pCMV-RZLUC, the TATA mutations in the plasmid do not significantly influence the expression of Zluc under latent conditions (Fig. 3C), demonstrating that Zluc expression is independent of the transcription of ZLUC monocistronic mRNA. The most important evidence, in which transcription of monocistronic ZLUC mRNA is demonstrated to be unimportant, likely comes from the 18SC construct (Fig. 8). In this plasmid, the entire intercistronic region, which comprises the most important region of P_Z, is replaced by the helix 36 sequence of the 18S rRNA. Although completely destroying the function of P_Z, this replacement does not influence the expression of Zluc, demonstrating that Zluc is indeed translated from the bicistronic mRNA. Notably, our study reveals that the M106-115 and the M136-145 linker-scanning mutations in pCMV-RZLUC significantly lower the expression of Zluc (Fig. 7). These two mutations alter the ZII and the TATA sequences of P_Z (Fig. 3A). Although these two regions are important for activation of the BZLF1 promoter by Rta (2) and for transcription of ZLUC monocistronic mRNA, respectively, the activity decreases are probably unrelated to the transcriptional functions of these cis elements. In this study, we have performed a Northern blot analysis and demonstrated that the mutations that we have generated do not significantly influence the transcription of the bicistronic mRNA in P3HR1 cells (Fig. 9).

View larger version (36K): [in this window] [in a new window]   FIG. 9.   Transcription of bicistronic mRNA from the plasmids containing mutations in the intercistronic region. RNA was purified from P3HR1 cells transfected with bicistronic and monocistronic markers (lane a), pRZLUC (lane b), pCMV-RZLUC (lane c), pCMV-RZLUC(SL-A) (lane d), pCMV-RZLUC(Ksp) (lane e), pCMV-RZLUC(M86-95) (lane f), pCMV-RZLUC(M96-105) (lane g), pCMV-RZLUC(M106-115) (lane h), pCMV-RZLUC(M116-125) (lane i), pCMV-RZLUC(M126-135) (lane j), pCMV-RZLUC(M136-145) (lane k), pCMV-RZLUC(M146-155) (lane l), pCMV-RZLUC(M156-165) (lane m), pCMV-RZLUC(18S) (lane n), pCMV-RZLUC(18SC) (lane o), 5'-45 deletion (lane p), 5'-85 deletion (lane q), 5'-128 deletion (lane r), 5'-165 deletion (lane s), 5'-205 deletion (lane t), 3'-166 deletion (lane u), 3'-129 deletion (lane v), and 3'-86 deletion (lane w). A 32P-labeled luc probe was used for hybridization. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Translation of Zluc from the bicistronic mRNA involves a discontinuous ribosome-scanning process. As is generally known, uORFs typically decrease the efficiency of the translation of the downstream major ORF (20). Therefore, if Zluc is translated by translation reinitiation, uORFs in the intercistronic region should lower the efficiency of ZLUC translation. This inhibitory effect can be demonstrated by altering the AUG codons to remove the uORFs. This accounts for why we changed the five initiation codons in the intercistronic region. According to our results, the ORFs in the intercistronic region are unimportant for the bicistronic translation since altering the initiation codon of these ORFs does not affect the translation, except for the mutation in the fourth initiation codon, which decreases luciferase activity by approximately 40% (Fig. 4). This decrease is probably attributable to the disruption of the homology between region I and 18S rRNA (Fig. 8A) rather than to the ORF itself playing a role in the translation. Furthermore, we inserted a hairpin structure, SL-A, known to block ribosome scanning, into the 5' region of pCMV-RZLUC(KspI). This insertion apparently does not hinder Zluc translation (Fig. 5), suggesting that the ribosome that translated Rta does not continue to scan the 5' region of the intercistronic region and that Zta translation does not involve translation reinitiation. Notably, the SL-A insertion actually increases Zluc translation by 1.8-fold (Fig. 5). The reason for this increase remains unknown. Possibly, SL-A may have changed the conformation of the intercistronic region, thus causing the increase. A Northern blot analysis shows that creating the KspI site and inserting the SL-A sequence in pCMV-RZLUC do not influence the transcription of bicistronic mRNA (lanes c to e, Fig. 9). We also show that inserting the SL-A sequence into the APA-FS and BA mutants of pCMV-R-LUC does not cause the translation of the downstream ORF (Fig. 5). This finding excludes the possibility that the SL-A sequence itself plays a role in translation initiation. Based on the fact that Zta translation probably does not involve translation reinitiation, we hypothesize that the intercistronic region may contain a ribosome entry site (internal initiation) or an acceptor site (ribosome shunting), allowing the initiation of Zluc translation. If this hypothesis is correct, inserting a hairpin structure, which prevents ribosome scanning, downstream from the site should prevent the translation of the downstream ORF. This investigation finds that inserting the SL-B sequence, which is known to block ribosome scanning, into the StyI site in the intercistronic region does prevent the translation of Zluc (Fig. 5), suggesting that the site may exist upstream from or near the StyI site.

The sequence complementarity between region I and helix 36 of 18S rRNA is important for Zluc translation. Computer analysis indicates that the intercistronic region may form two large hairpin structures, SI and SII (Fig. 1B). These structures are unimportant for the bicistronic translation, since destroying the SI structure (Fig. 6, 5'-85) and the SII structure (Fig. 6, 3'-166) does not affect the translation. This study also demonstrates that the intercistronic region is critical in translating the downstream ORF from the bicistronic mRNA, since replacing the intercistronic region with a sequence from the BamHI-C fragment of EBV does not cause translation (Fig. 2). According to deletion analysis, the region between nucleotides 86 and 165 is especially important (Fig. 6). Computer analysis also reveals that the region between nucleotides 86 and 125 (region I) in the intercistronic region is partially complementary to helix 36 of 18S rRNA (from nucleotide 1489 to nucleotide 1524 of 18S rRNA) (Fig. 8A and B), a region exposed to the surface of the ribosome and involved in translocation and translation termination (26, 27, 38, 40, 50, 52). Linker-scanning analysis also reveals that the level of luciferase translation by the mutants correlates with the degree of homology between the two molecules (Fig. 7 and 8A). For instance, the region between nucleotide 106 and nucleotide 115 has the highest degree of homology (Fig. 8A), and a mutant with a mutation in this region (M106-115) exhibited the lowest luciferase activity (Fig. 7). Notably, the first 20 nucleotides of region I are less homologous to 18S rRNA (Fig. 8A), and mutations in this region (M86-95 and M96-105) do not decrease luciferase activity as much as does the M106-115 mutation (Fig. 7). Additionally, a point mutation at nucleotide 104 (in the fourth initiation codon) (Fig. 1B and 8A), creating one mismatch, reduces luciferase translation by 40% (Fig. 4, 4th). Furthermore, inserting the region I sequence into the intercistronic region of pCMV-R-LUC(BC) partially restores the translation, indicating that region I may act as a ribosome entry site or an acceptor site for the translation of the downstream ORF. This work also demonstrates that, although region II alone does not support the translation of luciferase from the bicistronic mRNA (Fig. 8, BC-RII), without region II, translation of luciferase from the bicistronic mRNA is inefficient (Fig. 8, BC-RI). These observations suggest that, instead of providing a site for ribosome binding, region II may assist the binding of 40S ribosome to region I. Notably, the 18S rRNA sequence in pCMV-R-LUC(18SC) does not contain a sequence which is complementary to the sequence of region II. However, pCMV-R-LUC(18SC) expresses a luciferase activity at the wild-type level (Fig. 8C), suggesting that, when the intercistronic region contains a sequence that is already 100% homologous to the helix 36 region of 18S rRNA, region II may become redundant. This sequence complementarity may indeed be important since Yueh and Schneider (54) recently demonstrated that translation by ribosome shunting on the late adenovirus mRNA and mammalian hsp70 mRNA also involves a sequence complementarity between the untranslated region of the mRNAs and sequences in the 3' region of 18S rRNA.

Zluc translation involves either ribosome shunting or internal entry. The mechanism translating Zta from the BRLF1-BZLF1 bicistronic mRNA appears complex. Our earlier study demonstrated that mutations in BRLF1 abolish the translation of Zta from the bicistronic mRNA. Furthermore, these mutations can be genetically complemented with a functional BRLF1 in cis, indicating that Rta is required for Zta translation from the bicistronic mRNA (9). Our current study suggests that translation of Zta from the bicistronic mRNA may use a shunting mechanism similar to that involved in the translation of cauliflower mosaic virus 35S RNA, in which case ribosome shunting requires a virus-encoded translational transactivator (21, 22). In the case of EBV, Rta may participate in the ribosome shunting process to assist the ribosomes that translated Rta in translocating to region I. It is also likely that the region I sequence may simply serve as an IRES. After interacting with Rta, 40S ribosomes may bind to this site (internal initiation). Meanwhile, computer analysis revealed that the sequence of the N-terminal 482 amino acids of Rta is 21% identical and 53% similar to that of eIF-4B (STM protein) of S. cerevisiae (3, 12; data not shown). As is generally known, eIF-4B may be capable of mediating complex formation between mRNA and ribosomes via interactions with rRNA (4). eIF-4B is also involved in internal initiation from IRESs in picornaviruses (43). These findings suggest that Rta might function as a translation initiation factor to facilitate Zta translation. As is generally known, yeast eIF-4B does not share extensive sequence homology with human eIF-4B. This may explain why human eIF-4B, which should be abundant in cells, cannot substitute for Rta in Zta translation (9). Results presented herein not only provide further insight into how Zta protein is translated from the BRLF1-BZLF1 bicistronic mRNA but also indicate the importance of transcription of the BRLF1-BZLF1 bicistronic mRNA in activating the EBV lytic cycle.