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Journal of General Virology (1994), 75, 2663-2670. Printedin Great Britain 2663 Mechanism of translation of the bicistronic mRNA encoding human papillomavirus type 16 E6-E7 genes Theresa M. C. Tan,* Bernd Gloss,~ Hans-Ulrich Bernard and Robert C. Y. Ting Institute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Republic of Singapore The transforming genes E6 and E7 of human papillomavirus (HPV) type 16 and other HPV types are expressed from a bicistronic mRNA with a characteristic spacing of 3 to 6 bp between the termination codon of E6 and the initiation codon of E7. Plasmid pSP64E6E7 which contains the reading frames of both E6 and E7 was constructed in order to study the expression of both proteins in a coupled transcription/rabbit reticulocyte translation system. Both E6 and E7 proteins were expressed simultaneously. This translation could be interfered with by antisense oligonucleotides corresponding to various regions of the transcript. Antisense oligonucleotides targeted at sequences flanking either side of the translation initiation codon of the E6 open reading frame were effective in inhibiting the synthesis of both proteins, whereas oligonucleotides complementary to the coding regions downstream of the first start codon showed either a considerably reduced effect or none at all. In particular, there was limited inhibition of E7 translation by antisense oligonucleotides flanking the translation start region of the E7 gene. In the presence of RNase H, it was possible to selectively inhibit the synthesis of either E6 or E7 by several gene-internal antisense oligonucleotides. We conclude that HPV16 E6-E7 bicistronic mRNA is fully functional and that both proteins are translated with equal efficiency via the scanning mechanisms with reinitiation at the second open reading frame. In addition, both AE6 and AE7 may have therapeutical potential as they are capable of inhibiting the proliferation of CaSki cells which contain the HPV16 genome. Introduction encoded in the E6 and E7 genes whose products interfere with the functions of the p53 and the Rb tumour suppressor genes (Hudson et al., 1990; Mtinger et al., 1989; Hawley-Nelson et al., 1989; Werness et al., 1990; Dyson et al., 1989). The fact that the E6 and E7 proteins are consistently expressed in HPV-positive cervical cancers and in cell lines derived from this cancer (Schwarz et al., 1985; Schneider-G/idicke & Schwarz, 1986; Smotkin & Wettstein, 1986; Androphy et al., 1987; Bedell et al., 1989; Cullen et al., 1991) supports the hypothesis that expression of E6 and E7 plays a role in cervical carcinogenesis. The HPV16 E6 and E7 proteins are transcribed from the same promoter, P97, in the form of a bicistronic message (Schneider-G/idicke & Schwarz, 1986; Smotkin & Wettstein, 1986). This study examines whether the E6-E7 message allows E7 to be translated efficiently. The use of antisense oligonucleotides to elucidate the mechanism for translation of this bicistronic message will be described. The implications of the results for understanding the mechanism of translation of this bicistronic message and for the design of specific and effective antisense oligonucleotides will be discussed. Antisense oligonucleotides are short segments of single stranded RNA or DNA with nucleotide sequences complementary to a specific gene or its mRNA. When targeted at mRNA or its precursor (pre-mRNA), antisense oligonucleotides hybridize and form duplexes which prevent the translation of the message into the encoded protein (Paterson et al., 1977; Izant & Weintraub, 1985; Melton, 1985; Blake et al., 1985). A refined understanding of this inhibitory mechanism could lead to using it as a tool for interfering with pathogenic processes. To study this mechanism we used the transforming genes of the human papillomavirus. Papillomaviruses have a circular DNA genome of approximately 8 kb. To date, more than 60 human papillomavirus types (HPV) have been identified. Among these a subgroup, including HPV16 and HPV18, is frequently detected in cervical cancer (DeVilliers, 1989). The transforming properties of HPV16 and HPV18 are -~ Presentaddress:Schoolof Medicine,Universityof California,San Diego, 9500 GilmanDrive, La Jolla, California92093, U.S.A. 0001 2654 © 1994SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 09:22:45 T. M. C. Tan and others 2664 Methods Synthesis of oligonucleotides. Oligonucleotides were synthesized by B. Li at the Institute of Molecular and Cell Biology or custom-made by New England Biolabs. The oligonucleotides were purified by HPLC. All oligonucleotides were dried and resuspended at a concentration of ~ ~f '=PeTS I r,. ~ I~SP6 TS ~I~P97 ~ o Region A /~TTTAGGTGACACAI"rAGAATACAAGCTTAACTGCA~TGTn'CAGGACCCACAG sPrPromotor HindIII Met PheGtn Asp Pro Gin ASP6 AE6 Region B CAGCTGTAATCATGCATGGAGATACACCT Gin Leu Ter Met His Gly Asp Thr Pro q b AE7 Region C AAACCA~AATCTACCATGGCTGATCCTGCAG Lys Pro Ter PstI Oligonucleotides ASP6 AE6 A B AE7 c D Control Sequences (5-3") Position targeted AGTTAAGC'FrGTATI'C SP6 TS GTGGGTCCTGAAACAT 104-119 TATTGCTGTTCTAATG 363-378 GATGATCTGCAACAAG 516-531 GTGTATCTCCATGCAT 562-577 TGGTTTCTGAGAACAG 840-855 CTGCTTGTCCAGCTGG 682-697 (455-469) TTGGCCGCTGCCATCC .... Fig. 1. Structure of the plasmid pSP64E6E7 and positions of antisense oligonucleotides. The numbering of HPV16E6/E7 follows that published by Seedorf et al., 1985. Sequences in oligonucleotide D that are complementary to E6 are underlined. The ORFs of HPV16 E6 and E7 are cloned using the HindlII and PstI sites of the pSP64 plasmid. The SP6 transcription start (SP6 TS) is 12 bases upstream of the HPV 16 sequences starting at P97 and ending at the PstI restriction site. Plasmid pSP64E6 is similar to pSP64E6E7 but ends with the bases TC after the E6 termination codon followed by the PstI site. Plasmid pSP64E7 is also similar to pSP64E6E7 but starts with the sequence TGTAATC after the HindlII site followed by the ATG of E7. 1 mM in water. The sequence of the oligonucleotides and their expected positions upon hybridization are shown in Fig. 1. Phosphorothioate analogs were custom-made by Oligos Etc and resuspended at a concentration of 2 mM in water. Plasmids. Segments of the HPV-16 genome that either encoded the E6 protein, E7 protein or both proteins together as a bicistronic sequence were obtained from a HPV-16 clone using the PCR. The forward PCR primer contained a HindIII restriction site whereas the reverse primer had a PstI site (Table 1). After cleavage with these enzymes, each of the three PCR products were ligated into the plasmid pSP64. The recombinant vectors will be referred to as pSP64E6, pSP64E7 and pSP64E6E7 (Fig. 1). In vitro protein translation. The TNT SP6 coupled reticulocyte lysate system (Promega) was used for the coupled transcription-translation reactions. The pre-mix contained 52 ~tl lysate, 4 lal reaction buffer, 4 lal SP6 polymerase, 2 ~tl amino acid mixture without cysteine (all the above reagents were supplied with the system), 4 lal recombinant RNasin ribonuclease inhibitor (40 U/~I; Promega), 6 lal of H20 and 8 lal of 35S-labelled L-cysteine containing 74 pmol (NEN, DuPont). The specific activities of the four batches of zsS-labelled L-cysteine ranged from 1150 to 1220Ci/mmol. Pre-mix (8 lal) was added to 1 lag of recombinant vector DNA and the reaction volume was adjusted to 10 lal before incubation at 37 °C for 120 min. Hybrid arrested in vitro translation. The 10 lal reaction mixture containing 1 lag of pSP64E6E7 was similar to that described above except for the presence of the antisense oligonucleotides at a final concentration of 50, 100 or 200 taM. Translation of pSP64E7 was also performed in the presence of 50 and 100 tam of antisense oligonucleotides. Inhibition of translation by RNase H and antisense oligonucleotides. The 10 p.l reaction mixture containing 1 lag of pSP64E6E7 was similar to that described above except for the presence of 25 taMof the antisense oligonucleotide and 1.5 U of RNase H (Promega). For reactions conducted in the presence of 25 tam of AE6 or the control oligonucleotide, 0-25 lag of a luciferase expression plasmid (provided as a positive control in the TNT SP6 coupled reticulocyte lysate system) was added as a control. Denaturing gel analysis of translation products. After 120 min of incubation at 37 °C, 2 ~tl samples were removed from each reaction mixture for analysis on 12.5 % SDS-PAGE. The electrophoresis was stopped when the bromophenol blue dye had run off the bottom of the gel. The gel was fixed in acetic acid :isopropylalcohol:water (10: 25 : 65) for 20 min, soaked in Amplify (Amersham) for 10 min followed by immersion in 5 % glycerol for 20 rain before being dried and then fluorographed overnight at 70 °C. The bands were quantified with t h e Table 1. Primers used for obtaining the E6, E7 and E6-E7 open reading frames* Open reading frames/ (recombinant vector) E6(pSP64E6) E7(pSP64E7) E6/E7 (pSP64E6E7) PCR primers Forward primer a 5" GGAAGCTTAACTGCAATGTTTCAGGACCC 3' Reverse primer b 5' GGCTGCAGGATTACAGCTGGGTT 3' Forward primer c 5' GGAAGCTTTGTAATCATGCATGGAGATAC 3' Reverse primer d 5' GGCTGCAGGATCAGCCATGGTAGATTATG 3' Forward primer a and reverse primer d * The underlined bases in the forward primers show the Hindlll restriction site whereas those on reverse primers show the PstI site. Bases in bold indicate either the start codon (ATG) or the termination codon (TTA). the Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 09:22:45 Translation o f H P V 1 6 E 6 - E 7 m R N A image analysing system Visage 110 (Biolmage) using the Sunview program. Cell proliferation as determined by tetrazolium reduction. An established cervical epithelial tumour cell line, CaSki, containing HPV16 was used in this study. The cells were grown in MEM supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 Ixg/ml streptomycin and 2 mM-glutamine. Another cervicaltumour cell line, C-33A, lacking the HPV genome was used as a control cell line. The C-33A cells were grown in medium similar to that used for CaSki cells with the addition of 0.8 mg/1 of MEM non-essential amino acids and 1 mu-pyruvate. Cells (5 x 10a) were seeded in each well of the 96-weU plate (Nunc) and allowed to recover for 48 h prior to the treatment with phosphorothioate oligonucleotides.Fresh medium containing 5 ~tg/mlof the transfection reagent, DOTAP (Boehringer-Mannheim) and 20 laM of phosphorothioate analogs was then added to the cellsand the cellswere treated for 48 h with a change of medium after 24 h. Twenty lal of freshly prepared MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium]PMS (phenazine methosulphate) was then added to each well. (MTS and PMS were supplied as solutions of 2 mg/ml and 0.92 mg/ml respectively in the CellTitre 96 AQueous Non-radioactive cell proliferation assay kit from Promega). After incubation at 37 °C for 3 h, the A490was read using an ELISA plate reader. Assays were performed in triplicate and the results were expressed as percentage inhibition of the untreated culture. This was determined by finding the difference in A490 values between the untreated and oligonucleotide-treated cultures, and subsequently by calculating the ratio between this and the A490value of the untreated culture as a percentage. Results Synthesis of E6 and E7 O u r initial a i m was to identify the H P V 1 6 proteins E6 a n d E7 after expression in a c o u p l e d in vitro t r a n s c r i p t i o n - r a b b i t reticulocyte t r a n s l a t i o n system, a n d to m o n i t o r their expression f r o m a bicistronic m R N A . T h e plasmids pSP64E6 a n d pSP64E7 were used to confirm the identities o f the E6 a n d E7 t r a n s l a t i o n p r o d u c t s a n d pSP64E6E7 was c o n s t r u c t e d as a source for the bicistronic m R N A (Fig. 1). T h e p r o d u c t s were analysed by S D S - P A G E a n d f l u o r o g r a p h y (Fig. 2). The E6 p r o t e i n m i g r a t e d as predicted as a 17K protein. By contrast, the H P V 1 6 E7 p r o t e i n m i g r a t e d slowly to a n a b n o r m a l extent; with a Mr of 19K instead o f the expected 12K value. This a b e r r a n t m i g r a t i o n b e h a v i o u r o f H P V 16 E7 has been previously described (Mfinger et al., 1991 ; Scheffner et al., 1992). The bicistronic m R N A t r a n scribed f r o m pSP64E6E7 resulted in the synthesis o f b o t h proteins. The intensity of the E7 signal was d e t e r m i n e d to be 51.9 % +3"7 % ( N = 4) o f the E6 signal. Effects of antisense oligonucleotides on E6 and E7 translation T o study the effects o f interference b y antisense oligonucleotides o n the t r a n s l a t i o n o f E6 a n d E7 f r o m the bicistronic E 6 - E 7 m R N A , p r o t e i n synthesis was carried 2665 o u t in a c o m b i n e d t r a n s c r i p t i o n - t r a n s l a t i o n system with pSP64E6E7 as described above b u t with antisense oligonucleotides a d d e d at the start o f the reaction. Oligonucleotides A E 6 a n d ASP6, which were c o m p l e m e n t a r y to sequences flanking the E6 start c o d o n or o v e r l a p p i n g the start of the SP6 t r a n s c r i p t respectively, were effective in i n h i b i t i n g b o t h E6 a n d E7 p r o t e i n synthesis. I n h i b i t i o n levels were greater t h a n 90 % with 1 2 3 4 - 32 - 27 - 21-5 18 E71~ E6P - 14 Fig. 2. In vitro translation of HPV16 E6 and E7 in a combined transcription-rabbit reticulocytetranslation system. Lanes 1and 2 show the synthesis of E7 from pSP64E7 and E6 from pSP64E6, respectively. Lane 3 shows the negative control without any plasmid. Lane 4 shows simultaneous synthesis of E6 and E7 from pSP64E6E7. The positions of Mr markers are indicated on the right and the positions of E6 and E7 are indicated by arrowheads on the left. T a b l e 2. Inhibition o f H P V 1 6 E6 and E7 translation by antisense oligonucleotides Antisense oligonucleotides/ concentrations (pM) A 50 100 200 B 50 100 200 C 50 100 200 D 50 100 200 ASP6 50 100 200 AE6 50 100 200 AE7 50 100 200 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 09:22:45 Percent inhibition of translation of E6 38"7 37'8 60"5 1"7 5"9 5"0 0"0 0"0 0"0 7"6 0"0 0-0 27-0 90"8 92-2 80.5 96"9 100"0 5"6 19-5 65"0 Percent inhibition of translation of E7 0"0 0"0 28"2 0"0 0"0 10"6 0"0 0"0 0"0 0"0 0'0 0'0 52"1 94'0 92'2 86"2 97"2 100"0 25-0 39'5 66"3 T. M. C. Tan and others 2666 1 2 3 4 5 6 1 7 2+ 3+ 4 5+ 6 7 - 49 L~ - 32 97 69 49 27 E71~ 21.5 - 18 32 14 27 Fig. 3. Inhibition of E7 translation by antisense oligonucleotides AE6 and AE7. Lane 1 is a negative control without any plasmid. Lanes 2 and 7 show the translation of E7 from pSP64E7. Lane 3 has 100 laMof AE7 and lane 4 has 50 laMof AE7. Lane 5 has 100 laMof AE6 and lane 6 has 50 gM of AE6. The positions of Mr markers are indicated on the fight and the position of E7 is indicated by the arrowhead on the left. 1 2 3 4+ 5 6+ 7 8+ 9 E7 E61~ 10+ 14 32 - 27 21.5 -18 - -14 Fig. 4. Selective inhibition of E6/E7 translation by antisense oligonucleotides A, B, C and D in the presence of RNase H. All lanes with (+) indicate the addition of RNase H. Lane 1 shows the normal translation of E6 and E7. Lane 2 is a negative control without any plasmid. Lanes 3 and 4 have oligonucleotide A, lanes 5 and 6 oligonucleotide B, lanes 7 and 8 oligonucleotide C and lanes 9 and 10 oligonucleotideD. All oligonucleotidesare at a concentration of 25 laN. In the presence of RNase H, oligonucleotides A and B selectively inhibit E6 synthesis whereas D inhibits E7 and to a lesser extent E6 as well. The positions of Mr markers are indicated on the fight and the positions of E6 and E7 are indicated by arrowheads on the left. 100 or 200 gM of either oligonucleotide for E6, as well as for the d o w n s t r e a m E7 gene (Table 2). I n the case o f the oligonucleotide AE7, which is c o m p l e m e n t a r y to sequences flanking the E7 i n i t i a t i o n c o d o n , only approx. 40 % a n d 65 % i n h i b i t i o n levels were observed at 100 a n d 200 gM-AE7. A E 7 also h a d some effect o n the expression of the E6 gene especially w h e n a d d e d in a m o u n t s of 200 JaM. This non-specific i n h i b i t i o n is due to the partial identity b e t w e e n oligonucleotides A E 6 a n d A E 7 (nine o u t of 16 bp). Both 100 JaM-AE6 a n d -AE7 did n o t affect the t r a n s c r i p t i o n o f pSP64E6E7 (data n o t shown). Oligonucleotides A, B, C a n d D, which were c o m p l e m e n t a r y to sequences w i t h i n the E6 or the E7 gene or Fig. 5. Inhibition of E6/E7 translation by antisense oligonudeotides AE6 and AE7 in the presence of RNase H. A control protein, luciferase (62K) was simultaneously translated. The translation reactions were similar to those described in Methods with the exception of the addition of 0.25 lag of the luciferase plasmid (provided as a positive control with the Promega kit). Lanes with (+) indicate the addition of RNase H. Lane 1 is without any plasmid. Lane 2 shows the translation of L, E6 and E7 in the presence of RNase H. The synthesis of all three proteins is slightly affected by the presence of RNase H. Lanes 3 and 4 have an unrelated control oligonncleotide (5' TTGGCCGCTGCCATCC 3') and lanes 5 and 6 have oligonucleotide AE6. Lane 7 shows the normal translation of all three proteins. The concentration of all oligonucleotideswas 25 laM.The positions of Mr markers are indicated on the right and the positions of luciferase (L), E6 and E7 are indicated by arrowheads on the left. to the sequences at the 3' t e r m i n u s of the genes, h a d little or n o effect o n E6 or E7 t r a n s l a t i o n (Table 2). I n addition, the effect o f A E 6 a n d A E 7 o n the t r a n s l a t i o n o f E7 from pSP64E7 was also examined. A E 6 did n o t i n h i b i t the synthesis o f E7 w h e n added at the 50 ~tM level b u t there was some i n h i b i t i o n at the 100 jam level which was due to the p a r t i a l identity b e t w e e n A E 6 a n d AE7. I n contrast, A E 7 affected E7 synthesis m o r e significantly w h e n a d d e d i n 50 gM a m o u n t s a n d there was almost complete i n h i b i t i o n w h e n a d d e d at 100gM a m o u n t s (Fig. 3). RNase H and antisense inhibition of E6 and E7 translation E x o g e n o u s R N a s e H cleaves R N A - D N A hybrids a n d it has been d e m o n s t r a t e d that its a d d i t i o n to in vitro t r a n s l a t i n g systems c a n increase the extent of h y b r i d arrest t r a n s l a t i o n o f m R N A s ( M i n s h u l l & H u n t , 1986). I n this study, we e x a m i n e d the effects of R N a s e H o n the Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 09:22:45 Translation of HPV16 E6-E7 m R N A 70 T 60 synthesis of luciferase and E7 (Fig. 5). The unrelated control oligonucleotide also had no effect on the expression of the three proteins. Effect of the antisense oligonucleotides on cell proliferation 50 20 Treatment of the CaSki cell cultures for 48 h with either AE6 or AE7 phosphorothioate analogs, resulted in the inhibition of proliferation of CaSki cells by 38"6 % and 52.6 %, respectively. In contrast, treatment of C-33A cell cultures only led to slight inhibition levels of 6.6 % and 18-2 % by AE6 and AE7, respectively. Treatment of both cell lines with the unrelated control oligonucleotide also resulted in minimal inhibition. 10 Discussion 40 e~ o 2667 30 ,.Q .= 0 Control AE6 AE7 Fig. 6. Inhibition of cell proliferation by AE6 and AE7. Treatment of CaSki cells by phosphorothioate analogs of AE6 and AE7 led to inhibition of cell growth ( , ) whereas little inhibition was observedfor similarlytreated C-33Acells(D). There was also little inhibition of cell growth for both cell lines by the unrelated control oligonucleotide (control). The results are expressed as mean (+S.D.) percentage inhibition of growth. expression of E6 and E7 proteins from the bicistronic mRNA. Fig. 4 illustrates the effects of RNase H in combination with 25 gM o f oligonucleotides A, B, C or D. In the absence of RNase H, these oligonucleotides had little or no effect (Table 2). However, together with RNase H, oligonucleotides A and B could selectively inhibit the synthesis of E6 (Fig. 4). Oligonucleotide C in the absence or presence of RNase H had little effect, whereas oligonucleotide D caused the complete inhibition of E7 synthesis in the presence of RNase H and also a significant inhibition (77"6 %) o f E6 synthesis. The inhibition in E6 expression is due to the 75 % identity between the oligonucleotide and the E6 coding region. As a means o f examining the specificity of the RNase H and antisense oligonucteotide combined inhibitory effects, a series of experiments using a luciferase expression plasmid as the control were carried out. Fig. 5 (lane 2) shows the synthesis of E6, E7 and the 62K luciferase protein in the presence of RNase H. The synthesis of all three proteins was slightly decreased in the presence of RNase H and this is because of the effects of minimal unspecific degradation o f the luciferase and bicistronic E6-E7 RNAs. When 25 gM of oligonucleotide AE6 and RNase H were used it was possible to selectively inhibit E6 synthesis without significant effect on the Our research examined the efficiency of translation of both proteins from a bicistronic viral m R N A , and the possibility o f interfering differentially with the expression of both genes by antisense oligonucleotides. In cervical cancers and cervical carcinoma-derived cell lines containing HPV16, mRNAs encoding E6 and E7 proteins are transcribed from the same promoter, P97 (SchneiderG~idicke & Schwarz, 1986; Smotkin & Wettstein, 1986), in the form of a bicistronic mRNA. The unspliced E6-E7 m R N A encodes the full-length E6 as well as E7 proteins. In addition, two spliced transcripts E6*I-E7 and E6*II-E7 whose biological functions are unknown are usually detected (Shirasawa et al., 1991). It has been proposed that it is a function of these spliced transcripts to stimulate E7 translation (Schneider-Gfidicke & Schwarz, 1986; Smotkin et al., 1989). The unspliced transcripts of all HPV types that infect genital epithelia, have a short spacing region (5 bp in the case of HPV16) between the termination codon of E6 and the initiation codon of E7. As it is not possible to examine the translation of the bicistronic HPV16 E6-E7 in cultures using cell lines containing the HPV16 genome since splicing events occur, we examined the translation in vitro using an eukaryotic-derived rabbit reticulocyte system which should mimic the intracellular conditions that are present for translation of HPV mRNAs. Most mRNAs of eukaryotes and their viruses are monocistronic but in the case of those m R N A s which are bicistronic, the ribosomes can reinitiate translation at the next A U G codon downstream (Kozak, 1984; Liu et al., 1984; Peabody et al., 1986). The efficiency of reinitiation in such cases is usually low (Kozak, 1984; Liu et al., 1984) but this progressively improves as the intercistronic sequence lengthens (Kozak, 1987). This reinitiation only occurs when the upstream open reading frame (ORF) is a 'minicistron' and encodes for a small Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 09:22:45 2668 T. M . C. Tan and others peptide. For plant and animal virus mRNAs that are structurally bicistronic, encoding two full-length proteins, only the 5'-proximal end ORF is usually translated, i.e. they are functionally monocistronic (Kozak, 1986). This study demonstrates that despite the close proximity of the ORFs of E6 and E7 and the fact that the message codes for two full-length proteins, the translation of the protein E7 is as efficient as that of E6 in vitro. This quantification was done by comparing the E7 and E6 signals in radiolabelling experiments using [35S]cysteine. The E7 signal was expected to be half that of E6 as there are seven cysteine residues in E7 as compared with the 14 cysteine residues in E6. In our experiments the E7 signals had an intensity which was 51% of that of E6, leading us to conclude that both proteins are translated with almost equal efficiency. Similar effective expression of both proteins was also observed when E6 and E7 were translated in a rabbit reticulocyte lysate system using RNAs transcribed from pSP64E6E7 (data not shown). The efficient translation of the E6 and E7 proteins of other HPVs has also been reported (Roggenbuck et al., 1991). For antisense inhibition studies, a series of antisense oligonucleotides was constructed. The AE6 and AE7 oligonucleotides were constructed to include sequences flanking the start codons of E6 and E7 respectively as these regions are probably most sensitive to antisense inhibition (Gupta, 1987; Maher & Dolnick, 1987). As such, there is partial identity between AE6 and AE7 (nine out of 16 bp). However, at concentrations of 50 ~tM or less, AE6 could selectively bind to sequences flanking the E6 start codon and did not inhibit the translation of E7 from pSP64E7 which encodes for E7 alone. In contrast, 50 gM of AE6 inhibited the synthesis of both E6 and E7 from pSP64E6E7 which encodes the bicistronic E6-E7 message whereas AE7 did not affect this synthesis. At high concentrations, especially at 200 gM levels, there were some unspecific effects from both oligonucleotides. Our data on antisense inhibition also indicate that there is efficient translation of E6 and E7 from the bicistronic message. Oligonucleotides ASP6 and AE6 targeted at sequences around the E6 initiation codon can inhibit translation of both proteins efficiently at 100 and 50 gM levels respectively. This observation fully supports the scanning process model. In its simplest form, this model (Kozak, 1989) postulates that a 40S ribosomal subunit and an imperfectly defined set of initiation factors (Pain, 1986) enters at the 5' end of the mRNA and migrates along the mRNA until it reaches the first start codon, whereupon a 60S ribosomal subunit joins it and the first peptide bond is formed. In line with this model, the inhibition of expression of E6 by ASP6 is probably due to the interference with the binding of the 40S ribosomal subunit and the initiation factors or their passage to the start codon, whereas AE6 probably interferes with the binding of the 60S subunit. Oligonucleotides B and AE7 that are targeted at sequences flanking the E7 initiation codon, however, are less effective in reducing E7 translation that arises from the bicistronic message, although partial inhibition was observed at 200 [.tM levels of these oligonucleotides. These observations together with the inhibition by ASP6 and AE6, strengthen the hypothesis that E6 and E7 are translated from a bicistronic sequence and that the ribosomes reinitiate translation efficiently after termination of E6 translation. The hindrance caused by the antisense oligonucleotides B and AE7 is not sufficient to dislodge the ribosomes and prevent E7 synthesis. Alternatively, a mechanism whereby the ribosomes bind mRNA internally at the initiation A U G of E7 could be operating. Our data do not support this possibility since oligonucleotide AE7 would then have blocked translation of E7 more effectively. In addition, the ratio of the E7:E6 [35S]cysteine signal would not be 51% if E7 was translated by the internal ribosome-binding mechanism since one would not expect equal molecules of both proteins to be translated. The coding region-specific antisense oligonucleotides also did not prevent protein synthesis. This again is probably due to the fact that the translating ribosome can dislodge the oligonucleotide from the RNA template (Sankar et al., 1989). RNase H was used to examine whether the genes 1 internal binding antisense oligonucleotides which did not lead to a significant inhibition of E6 and E7 expression were interacting specifically with the targeted regions under the experimental conditions used. We observed that RNase H has a dramatic effect on translation inhibition even in the case of oligonucleotides other than AE6 and ASP6. Also, the concentration of oligonucleotide AE6, which is effective without RNase H at a concentration of 50 gM, can be reduced to 25 ~tMin the presence of RNase H. The interaction of the oligonucleotide AE6 with the mRNA was observed to be highly specific since the translation of an unrelated protein, luciferase, was not affected at all. Taken together, these results are in agreement with published data demonstrating that oligonucleotides directed against gene-internal sequences form heteroduplexes with mRNA efficiently. These heteroduplexes can be detected due to their destruction by RNase H, but they are unstable during the translation process (Minshull & Hunt, 1986; Walder & Walder, 1988; Gagnor et al., 1987). Therefore, for effective interference of translation from such bicistronic messages, one should employ antisense oligonucleotides that are complementary to regions at the 5' end or at the initiation codon of the first open reading frame. Hence, antisense inhibition of translation from such a bicistronic message closely Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 09:22:45 Translation of H P V 1 6 E 6 - E 7 m R N A resembles that of monocistronic messages in that regions flanking the first initiation codon at the 5' end are most sensitive to antisense oligonucleotide inhibition of translation (Sankar et al., 1989; Maher & Dolnick, 1987; Gupta, 1987). In vivo, a large fraction of the E6-E7 transcript is spliced to produce two shorter transcripts, E6*I-E7 and E6*II-E7. Since these two shorter transcripts have the same 5' oligonucleotide sequence in common with the full-length E6-E7 transcript, one would assume that the effects of the AE6 oligonucleotide would be on all three transcripts. To monitor this possibility, we studied the growth inhibitory effects of AE6 and AE7 on CaSki and C-33A cells. It is well documented that the expression of the HPV E6 and E7 genes is directly linked to the proliferative capacity of cervical cancer cells (von Kenbel Doeberitz et al., 1988, 1990; Watanabe et al., 1993). Interference with the expression of these proteins in CaSki cells by the phosphorothioate analog of AE6 is sufficient to inhibit cell proliferation. The AE7 oligonucleotide was also effective in inhibiting cell proliferation. This inhibition of cell proliferation is specific since both antisense oligonucleotides did not affect C-33A cells that do not harbour HPV genome. These results suggest that these oligonucleotides may be useful therapeutically. In conclusion, our research provides evidence for the mechanism of translation of a bicistronic mRNA of a human virus. The use of antisense oligonucleotides as a means of dissecting the mechanism of translation provides an useful method for analysing protein synthesis. 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