Sequences in the 5" Non-coding Region of Human Rhinovims 14 RNA that Affect in vitro Translation

J. gen. Virol. (1989), 70, 2799-2804. Printed in Great Britain 2799 Key words: rhinovirus, human type 14/5' non-coding region~in vitro translation Sequences in the 5" Non-coding Region of Human Rhinovims 14 RNA that Affect in vitro Translation By SAAD ALSAADI,* STUART HASSARD AND GLYN STANWAY Department of Biology, University of Essex, Wivenhoe Park, Colchester C04 3SQ, U.K. (Accepted 13 June 1989) SUMMARY A subgenomic cdna clone from human rhinovirus 14 (HRV-14), comprising the 5' non-coding region and the first 1182 nucleotides of the coding sequence, has been inserted into a vector under the control of the T7 promoter, and RNA was transcribed. Deletions in the 5' non-coding sequence modulated viral polyprotein synthesis significantly in a reticulocyte lysate system. Removal of the first 491 nucleotides had little effect, but deletion of a further 55 nucleotides (491 to 546) significantly increased the efficiency of the translation process. Further deletion to nucleotide 621 almost abolished translation, suggesting an essential role for the 546 to 621 nucleotide sequence. The efficiency of the translation process can also be influenced by the addition of ribosomal salt wash prepared from uninfected HeLa cells. Picornaviruses have a single-stranded RNA genome of 7200 to 8500 nucleotides which is of positive polarity and encodes one polyprotein. The genomic RNA is analogous to a typical eukaryotic message as it is monocistronic and has a 3' poly(a) tract. However the RNA possesses no 5" cap structure and has an unusually long 5' non-coding region, often including several AUG codons (up to 14) in addition to the one involved in the initiation of polyprotein translation. The function of this region, often the most conserved area of the genome among related viruses, is at present unknown and is being studied very extensively. Of particular interest is the role of the region in translation of the virus polyprotein. Work has been done on three of the four picornavirus genera, the aphthoviruses (Grubman & Bachrach, 1979; Sangar et al., 1980; Clarke & Sangar, 1988), the cardioviruses (Jang et al., 1988) and the enteroviruses (Brown & Ehrenfeld, 1979; Bienkowska-Szewczyk & Ehrenfeld, 1988; Pelletier et al., 1988 a; Pelletier & Sonenberg, 1989) and this has demonstrated the presence of critical regions for translation, particularly one involved in internal ribosome binding (Jang et al., 1989; Pelletier & Sonenberg, 1988). Here we describe an in vitro system for studying human rhinovirus 14 (HRV-14) a member of the other picornavirus genus, the rhinoviruses, and demonstrate the presence of elements in the 5' non-coding region which are of importance in HRV-14 polyprotein synthesis. HRV-14 has been cloned and sequenced previously (Stanway et al., 1984). Initially the subgenomic clones already available were assembled into a full-length cdna copy of the viral genome and introduced into the PstI site of the vector Bluescribe. The cdna is under the control of the T7 promoter and the orientation is such that genomic sense RNA is produced during in vitro transcription. To avoid complications arising from processing of the polyprotein during in vitro translation, it was decided to truncate the cdna so that a shorter RNA encoding only the first part of the polyprotein would be produced. This product should lack the P-2A and P-3C proteases known to be responsible for polyprotein processing. This was achieved by using the restriction enzyme HpaI which has no restriction site in the vector but cuts at positions 1810, 3561, 6337 and 7168 in HRV- 14 cdna. Digestion with HpaI followed by ligation removed three fragments and left a unique HpaI site at the 1810/7168 boundary. Subsequent cutting of this construct (termed pt7) with HpaI produced the linear template required for transcription. The 0000-9042 1989 SGM

2800 Short communication cdna 1 Transcript J I pt7 1 II~ (AUG) 626 1808 I p(1-263) ] p(1 360) ] p(1-468) I p(1 491) ~ I p(1-546) ~ I p(1-621)!1 I Fig. 1. Schematic illustration of the HRV-14 cdna and its derived transcripts. The striped and crossed boxes represent the polylinker of the Bluescribe vector. Closed and open boxes represent the non-coding and the coding regions respectively. The numbers on the left denote the lengths of the deleted nucleotide sequence. The site of the initiation codon AUG is shown on the pt7 transcript. The pbal (491) transcript (*) was produced as mentioned in the text. transcript encodes VP4, VP2 and the first 62 amino acids of VP3 and has a predicted Mr of approximately 43 000. In addition to pt7, a nested set of constructs were made in which cdna in the 5' non-coding region was deleted by cutting at appropriate restriction enzyme sites in the cdna. Following complete or partial digestion the ends were made blunt by filling in or digesting back with the DNA polymerase I Klenow fragment and a second cut was made using Sinai, which cuts in the polylinker region of Bluescribe just prior to the PstI site. Ligation produced the constructs psnabi (263), pbsteii (360), pbsml (468), pbal (491), pavaii (546) and pbcli (621), each deleted from position 1 to the indicated site (Fig. 1). All constructs were analysed either by restriction endonuclease analysis or by nucleotide sequencing to confirm the deletion of the appropriate fragment. Construct pbal (491) was produced by treating the cdna with nuclease Bal31 according to the method described by Misra (1985). RNA was transcribed from pt7 and from the deleted transcripts following linearization, in a system which included 40 mm-tris-hcl ph 7-5, 6 mra-mgcl2, 2 mm-spermidine, 10 mm-naci, 2-5 mm of each of the four NTPs, 2 ~g of linearized cdna, 100 units of RNase inhibitor (Pharmacia) and 10 units oft7 RNA potymerase (Stratagene). The reaction took place for 1 h at 37 C. DNA was removed by the addition of 2 units of DNase and further incubation for 20 min. The RNA was purified by extraction with phenol-chloroform, then ethanol-precipitated and dissolved in diethylpyrocarbonate-treated water. RNA concentrations were standardized by measuring the absorbance at 260 nm. In vitro translation was carried out at 30 C for 1 h in a 50 ~tl reaction mixture containing 35 Ixl of nuclease-treated rabbit reticulocyte lysate, 1 Ixl of an amino acid mixture minus methionine (Promega Biotec), 0.75 ~tg (15 lag/ml final concentration) of RNA and 50 ~tci of [35S]methionine (Amersham). Equal amounts of RNA were added in each case. The level of RNA used was shown by the titration experiment to be approximately 30~0 less than the saturating level (data

Short communication 2801 1 2 3 4 5 6 7 8 92-5K-- 69K-- 46K--? 3 0 K - - ) Fig. 2. SDS-PAGE analysis of proteins synthesized in vitro from HRV-14 RNAs. Lanes: 1, no mrna; 2, psnabi (263); 3, pbsteii (360); 4, pbsml (468); 5, pbal31 (491); 6, pavali (546); 7, pbcli (621); 8, pt7. M r values are shown on the left. not shown). The products were analysed by SDS-PAGE and autoradiography. The results are shown in Fig. 2. The transcription and translation were repeated several times giving results identical to those presented. It can be seen that all the RNAs tested were translated to some extent but that there are marked differences in the efficiencies of translation, particularly the original pt7 transcripts with a complete 5' non-coding region which translated very poorly. It is possible that this is because it is the only construct that retains the poly(g) tail (15 residues), added during the original cdna cloning, which may interfere with translation. The four RNAs, deleted in the region 1 to 491, gave very similar results, whereas the pavali (546) transcript, lacking the first 546 nucleotides was by far the most efficient in this system. The transcript from pbcli (621) in which virtually all the 5' non-coding region had been deleted but which retained the A--AUGG Kozak consensus sequence (thought to be important for translation of eukaryotic messages; Kozak, 1981, 1984), showed a marked reduction in efficiency compared to pavali (546). One unexpected observation is the presence of two additional minor translation products, seen in PAGE below the major one, in all samples. The reason for their appearance is not clear but it could be due to spurious internal initiation. The results imply the presence of at least two viral elements that modulate translation in this in vitro system. If the pt7 transcript is ignored on the basis of a possible effect from the poly(g) tail, removal of the first 491 nucleotides appears to have little effect on translation. However as seen for pavali (546), removal of a further 55 nucleotides (to position 546), causes a marked increase in efficiency suggesting the existence of some inhibitory element in this part of the 5' non-coding region. Such an element has been found by in vitro studies on poliovirus RNA although there it was located between nucleotides 70 to 381 (Pelletier et al., 1988 b). In the case of poliovirus, its effect could be overcome by the addition of an extract derived from HeLa cells, and therefore it is likely that this element has little significance in vivo in the infected HeLa cells. This is also indicated by the lack of correlation between the positions of the elements in HRV-14 and poliovirus, despite their relatively close overall relationship. Of more interest is the low efficiency of translation seen for pbcli (621), which lacks all but the last five nucleotides of the 5' non-coding region. The difference in efficiency between pavali (546) and pbcli (621) is presumably due to sequences in the 546 to 621 region, not present in pbcli (621). Hence the

2802 Short communication u a a c a u a g a u a u a c g g c a c a u a u - a A-U A-U A-U A C A-U U - A U - A U - A U - A U - A A- U A- U A- U A- U A-U c U - A U - A U - A U - A U - A a A * u. g A- U A- U A- U A- U c -g U - A U -A U-A c A U - A U -A A- U A- U A- U * g -c A- U A-U A- U A- U A- U A- U A- U A- U C - G C - G C - G C - G C - G C - G A-u A g A g A-u A g A-u G - C G - C G - C *c - g G - C G. U U - A U - A U - A U - A U - A U - A G.U G.U G-c G.U G.U G.U G - C G - C G - C G - C G - C G - C A- A- A- A A- A-U U. U. U. U U A.N 129" I-~ U.G U.G U.G U.G UoG u.g c-g HRV-89 HRV-IB HRV-2 HRV-14 HRV-85 Poliovirus 1 Fig. 3. Computer-predicted secondary structure of RNAs from different serotypes of HRV and poliovirus type 1 immediately prior to the AUG codon that initiates the long open reading frame. Conserved nucleotides are in upper case and non-conserved ones in lower case. Sequences are taken from Duechler et al. (1987), Hughes et al. (1988), Skern et al. (1985), Stanway et al (1984) and Toyoda et al. (1984). nucleotide sequence in this region must have an important role in viral protein synthesis, at least in this system. Using the secondary structure prediction program PCFOLD we have discovered a stable, putative structural feature, consisting of a stem and loop, in the 546 to 621 part of the HRV-14 5' non-coding region (Fig. 3). A similar structure has recently been described by others (Rivera et al., 1988). The feature is present in all HRVs sequenced to date which strongly suggests a functional role. Other evidence that the structure is important is that the nucleotides within the stem are almost identical between the different rhinoviruses whereas the loop regions are highly diverse in length and sequence. Furthermore, the few differences seen in the stems still allow maintenance of the overall structure since they are either C to U changes or are compensated by a complementary change in the paired nucleotide. Notable examples are a U-G pair in the HRV-89 stem instead of the A-U seen in the other rhinoviruses and C-G and G-C in HRV-14 replacing the usual G-C and A-U (all marked with an asterisk in Fig. 3). The stem-loop structure includes the AUG that initiates the long open reading frame (boxed) and this is further evidence that it is involved in some way in the translation of the rhinovirus polyprotein. A similar stemloop structure is found in all the enteroviruses analysed (poliovirus type 1 is shown in Fig. 3). Interestingly it has been shown recently that in poliovirus type 1, sequences in the 567 to 627 region, which includes this stem-loop structure, seem to be essential for poliovirus translation in vitro (Bienkowska-Szewczyk & Ehrenfeld, 1988). In view of our results, which have located a stimulatory element to a region within nucleotides 546 to 621, we suggest that this stem-loop is the key sequence involved in both HRV and enterovirus translation. However it should be noted that the 546 to 621 region of HRV-14 also contains U-rich sequences that are also well conserved between related viruses and may therefore be important in translation. Enteroviruses are closely related to rhinoviruses, but one difference is the length of the 5' non-coding region which is due, primarily, to the presence of an extra 90 to 130 nucleotides located precisely between the stemloop and the AUG that initiates the open reading frame. If this structural feature is important it indicates differences in translation between the two groups of viruses; this is presently being investigated. a

Short communication 2803 200K-- 3 4 5 6 7 8 92'5K- 69K- 46K-- : ::::!: :"' ; - : ] 30K--, : Fig. 4. Effect of ribosomal salt wash on the efficiency of translation of the pavali (546) transcript. Lanes: 1, no mrna; 2 and 3, no mrna but with 10 and 20 p_l of ribosomal salt wash respectively; 4, pavali (546) mrna; 5, 6, 7 and 8, pavaii (546) mrna with 2.5 gl, 5 gl, 10 gl and 20 gl, respectively, of ribosomal salt wash. Several studies have presented evidence that the efficiency of poliovirus polyprotein synthesis in vitro can also be modified by other elements of non-viral origin. Addition of a crude postmitochondrial cellular fraction (Sa0) prepared from uninfected HeLa cells preferentially enhanced the initiation of P1 proteins which are encoded by the Y-terminal region of the poliovirus genome (Phillips & Emmert, 1986). Other studies have reported the enhancing effect of a ribosomal high salt wash, prepared from mock-infected or poliovirus-infected HeLa cells, on the translation efficiency of poliovirus RNA; it was concluded that HeLa cells contain a trans-acting factor that facilitates the efficient translation of poliovirus mrna (Brown & Ehrenfeld, 1979; Pelletier et al., 1988b). We therefore tested the effect of ribosomal salt wash on the efficiency of translation of HRV-14 RNA. Ribosomal salt wash was prepared from uninfected HeLa cells according to the method described by Brown & Ehrenfeld (1979). In vitro protein synthesis was carried out as before except that a different concentration of ribosomal salt wash was added to each reaction mixture. Fig. 4 shows the effect of these factors on the translation of the pavaii (546) transcript. It can be seen that these factors produced no effect when added to the reticulocyte lysate containing no exogenous mrna, i.e. no translationally active RNA was copurified with the prepared factors. It is also clear that the stimulatory effect induced by these factors correlates with their concentration in the reaction mixture. Due to reaction volume limits no more than 20 ~tl of ribosomal salt wash was used. It is noteworthy that this stimulatory effect was noticed among all tested transcripts although with different efficiencies (data not shown). The stimulatory effect was particularly evident with the pavaii (546) transcript which translates very efficiently. Since the addition of partially purified initiation factors eif-4a, -4B, -4F and -2 either alone or in combination, did not induce a stimulatory effect, Phillips & Emmert (1986) suggested the presence of other unknown factor(s) that produce the enhanced translation. However, it is possible that specific conditions may be required by these factors in order to achieve a detectable level of stimulatory effect. In summary, we have identified nucleotide sequences within the 5' non-coding region that play a significant role in HRV-14 polyprotein synthesis in vitro and have shown that the efficiency of the translation process can be modulated by certain factor(s) present within the ribosomal salt wash of HeLa cells. Further work is needed to assess the significance of these observations in vivo and to determine the relationship between the stimulatory region (564 to 621) and the site involved in internal entry of ribosomes into the picornavirus RNA.

2804 Short communication This work was funded by a grant from the Wellcome Trust. S. Hassard is supported by an MRC studentship. REFERENCES BIENKOWSKA-SZEWCZYK, K. & EHRENFELD, E. (1988). An internal Y-non-coding region required for translation of poliovirus RNA in vitro. Journal of Virology 62, 3068 3072. BROWN, B. A. & EHRENFELD, E. (1979). Translation of poliovirus RNA in vitro: changes in cleavage pattern and initiation sites by ribosomal salt wash. Virology 97, 396-405. CLARKE, B. E. & SANGAR, D. V. (1988). Processing and assembly of foot-and-mouth disease virus proteins using subgenomic RNA. Journal of General Virology 69, 2313-2325. DUECHLER, M., SKERN, T., SOMMERGRUBER, W., NEUBAUER, C., GRUENDLER, P., FOGY, I., BLAAS, D. & KUECHLER, E. (1987). Evolutionary relationships within the human rhinovirus genus: comparison of serotypes 89, 2 and 14. Proceedings of the National Academy of Sciences, U.S.A. 84, 2605-2609. GRUBMAN, M. J. & BACHRACH, H. L. (1979). Isolation of FMDV mrna from membrane-bound polyribosomes and characterization of its 5' and 3" termini. Virology' 98, 466-470. HUGHES, P. J., NORTH, C., JELLIS, C. H., MINOR, P. D. & STANWAY, G. (1988). The nucleotide sequence of human rhinovirus 1B: molecular relationships within the rhinovirus genus. Journal of General Virology 69, 49-58. JANG, S. K., KRAUSSLICH, H., NICKLIN, M. J. H., DUKE, G. M., PALMENBERG, A. C. & WIMMER, E. (1988). A segment of the 5' nontranslated region of encephalomyocarditis virus RNA direct internal entry of ribosomes during in vitro translation. Journal of Virology 62, 2636-2643. JANG, S. K., DAVIES, M. V., KAUFMAN, R. J. & WIMMER, E. (1989). Initiation of protein synthesis by internal entry of ribosomes into the 5' nontranslated region of encephalomyocarditis virus RNA in vivo. Journal of Virology 63, 1651-1660. KOZAK, M. (I98t). Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes. Nucleic Acids Research 9, 5233-5252. KOZ~'~:, M. (1984). Compilation and analysis of sequences upstream from the translational start site in eukaryotic mrnas. Nucleic Acids Research 12, 857-872. MISRA, K. (1985). A new strategy to create ordered deletions for rapid nucleotide sequencing. Gene 34, 263-268. PELLETIER, J. & SONENBERG, N. (1988). Internal initiation of translation of eukaryotic mrna directed by a sequence derived from poliovirus RNA. Nature, London 334, 320-325. PELLETIER, J. & SONENBERG, N. (1989). Internal binding of eucaryotic ribosomes on poliovirus RNA: translation in HeLa cell extracts. Journal of Virology 63, 441-444. PELLETIER, J., KAPLAN, G., RACANIELLO, V. R. & SONENBERG, N. (1988 a). Cap-independent translation of poliovirus mrna is conferred by sequence elements within the 5' noncoding region. Molecular and Cellular Biology 8, 1103-1112. PELLETIER, J., KAPLAN, G., RACANIELLO, V. R. & SONENBERG, N. (1988b). Translational efficiency of poliovirus mrna : mapping inhibitory cis-acting elements within the 5' noncoding region. Journal of Virology 62, 2219-2227. PHILLIPS, B. A. & EMMERT, A. (1986). Modulation of the expression of poliovirus proteins in reticulocyte lysates. Virology 148, 255-267. RIVERA, V. M., WELSH, J. D. & MAIZER, J. V. (1988). Comparative sequence analysis of the 5' noncoding region of the enteroviruses and rhinoviruses. Virology 165, 42-50. SANGAR, D. V., BLACK, D. N., ROWLANDS, D., HARRIS, T. J. g. & BROWN, F. (1980). Location of the initiation site for protein synthesis on foot-and-mouth disease virus RNA by in vitro translation of defined fragments of the RNA. Journal of Virology 33, 59-68. STANWAY, G., HUGHES, P. J., MOUNTFORD, R. C., MINOR, P. D. & ALMOND, J. W. (1984). The complete nucleotide sequence of a common cold virus: human rhinovirus 14. Nucleic Acids Research 12, 7859-7875. SKERN, T., SOMMERGRUBER, W., BLAAS, D., GRUENDLER, P., FRAUNDORFER, F., PIELER, C., FOGY, I. & KUECHLER, E. (1985). Human rhinovirus 2: complete nucleotide sequence and proteolytic processing signals in the capsid protein region. Nucleic Acids Research 13, 2111-2126. TOYODA, H., KHARA, M., KATAOKA, Y., SUGANAMA, T., OMATA, T., IMURA, N. & NOMOTO, A. (1984). Complete nucleotide sequences of all three poliovirus serotype genomes: implication for genetic relationship, gene function and antigenic determinants. Journal of Molecular Biology 174, 561-585. (Received 28 April 1989)