Virology, GlaxoWellcome Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK1
Department of Medicine, Imperial College School of Medicine at St Mary's, London W2 1NY, UK2
Author for correspondence: Ken Grace.Fax +44 1438 764810. e-mail kgg29888{at}glaxowellcome.co.uk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
Main text |
---|
![]() ![]() ![]() ![]() |
---|
Although the original GB inoculum was of human origin, neither GBV-A nor GBV-B appear to be human pathogens (Karayiannis et al., 1997 ). Following experimental infection of tamarins, only GBV-B could be detected in liver samples and was associated with biochemical evidence of an acute self-limiting hepatitis (Schlauder et al., 1995
).
The GBV-B genome is a positive-sense 9143 nucleotide (nt) RNA molecule, which encodes a polyprotein of 2864 amino acids (Muerhoff et al., 1995 ). There is significant sequence identity with HCV in specific regions, such as the putative helicase and RNA-dependent RNA polymerase of the two viruses (Simons et al., 1995b
; Muerhoff et al., 1995
). However, they only share 28% amino acid similarity across the entire polyprotein (Muerhoff et al., 1995
).
HCV has a 5' untranslated region (UTR) approximately 341 nt in length. This region is known to initiate translation of the HCV polyprotein via an internal ribosome entry site (IRES), which involves most of the untranslated region (Tsukiyama-Kohara et al., 1992 ; Wang et al., 1993
; Rijnbrand et al., 1995
; Reynolds et al., 1996
; Honda et al., 1996a
, b
). As the genome of GBV-B is similar in length and organization to that of HCV, it seems likely that GBV-B may also initiate translation in a similar manner. The 5'UTR of GBV-B is 445 nt in length compared with the 341 nt length of most HCV strains. Alignment of the two sequences revealed several segments of absolute sequence identity, with two large insertions in the GBV-B sequence at positions corresponding to nt 40 and nt 102 in the HCV sequence. Excluding these insertions, the sequences share an average identity of 60%. Indeed, a model of the secondary structure of the GBV-B 5'UTR has been suggested using comparative sequence analysis with HCV (Honda et al., 1996b
). This shows that the highly conserved secondary structure present in HCV and the pestiviruses may also be present in the 5'UTR of GBV-B.
In the present study, we investigated whether the IRES of GBV-B can function in a similar manner to the HCV IRES by using mono- and dicistronic reporter gene plasmid constructs. In addition, site-directed mutation analysis and in vitro transcription/translation experiments were employed to study IRES structurefunction relationships.
Several monocistronic or dicistronic reporter gene constructs were made as shown in Fig. 1(a). These were employed to determine whether the GBV-B 5'UTR contains an IRES, and to compare it to the IRES of HCV. All plasmids were constructed in Bluescript II KS transcription vectors (Stratagene). The HCV sequences (nt -341 to +855) were derived from a British patient infected with genotype 1b virus and were subcloned as described by Reynolds et al. (1995)
.
|
All reporter plasmids were transcribed using RiboMAX T7 large scale RNA production kit (Promega). RNA transcripts were used to programme nuclease-treated rabbit reticulocyte lysate (Promega) in vitro translation reactions (Pelham & Jackson, 1976 ) as described by Grace et al. (1991)
. In addition, the extent to which these processes are affected by altering potassium ion concentration was also studied. Potassium chloride was added to the rabbit reticulocyte lysate to create added potassium chloride concentrations ranging from 0 to 100 mM. The commercial rabbit reticulocyte lysate is supplemented with potassium ions to 79 mM in the final translation reaction (Promega).
Analysis of HCV dicistronic plasmids, pKCUC1 and pKCUS4 (Fig. 1b), demonstrated that translation of the upstream reporter gene, CAT, was reduced as the concentration of potassium chloride increased. However, the reporter genes under translational control of the HCV IRES (core/
E1 and SEAP) were not inhibited by high potassium levels. This result is consistent with the observation that potassium ion concentration has a differential effect on cap-dependent and cap-independent, or IRES driven, translation (Jackson, 1991
).
RNA transcripts were generated from the GBV-B dicistronic SEAP plasmid (pKCGBS) and were translated in vitro using rabbit reticulocyte lysate (Fig. 1c). Both CAT and SEAP were produced from the GBV-B transcripts. The fact that SEAP protein was produced indicated that translation had occurred via internal ribosome entry in the GBV-B 5'UTR, since SEAP is out of frame with the upstream reporter gene, CAT, and is not expressed from transcripts lacking GBV-B sequences in the intercistronic space. Increasing the concentration of potassium ions reduced CAT expression but appeared to have less of an effect upon translation of SEAP, which was downstream of the GBV-B 5'UTR. Levels of SEAP expression appeared to be similar to those seen when translation was under the control of the HCV IRES. These observations indicate that the 5'UTR of GBV-B contains an IRES, as predicted from the secondary structure of the RNA.
There are several reports in the literature describing site-directed mutagenesis of the HCV IRES. These studies have allowed identification of regions important for IRES function and confirmed AUG341 as the authentic initiation codon. Mutations described by Reynolds et al. (1995) and Wang et al. (1994)
affecting either the initiating AUG or the polypyrimidine tract II (Py II) domain of the HCV IRES were repeated in the corresponding regions of the GBV-B 5'UTR. The QuikChange site-directed mutagenesis kit (Stratagene) was used to generate dicistronic expression vectors carrying the GBV-B 5'UTR with the mutations shown in Table 1
(Wang et al., 1994
; Reynolds et al., 1995
). The mutations were designated mut1mut8. The effect of each mutation on the translational efficiency of the IRES was determined by in vitro translation (Fig. 2a
) and subsequent densiometric measurement of the autoradiogram using an Epson GT9000 scanner and Phoretix 1D gel analysis v4.0 software (Fig. 2b)
. The effect of each mutation was also determined by measuring SEAP activity in a cell-based transfection assay (Fig. 2c)
.
|
|
Mut2 (CUCC to AGAA) between nt 242 and 245 altered the sequence of Py II found at the base of domain III and has the potential to destroy base-pairing and secondary structure in this region. No in vitro protein translation or in vivo SEAP activity could be detected from the GBV-B IRES when this mutation was present. These results from both in vitro and in vivo experiments suggest that the RNA structure in this region is important for GBV-B IRES function, in a similar way to the equivalent region of the HCV IRES. Nucleotides 242245 are predicted to base-pair with nt 424427 (Honda et al., 1996b ). Disruption of this base-pairing might account for the observed decrease in translational efficiency of the mutated RNA. Therefore, a second mutation was studied, mut3 (GGAG to AAGA), between nt 424 and nt 427. These base substitutions should also cause disruption of the base-pairing interaction predicted in Py II found at the base of domain III. As expected, the in vitro and in vivo translational efficiency of the GBV-B IRES was greatly reduced and again indicates the importance of the RNA secondary structure in this region. However, when both compensatory mut2 and mut3 were introduced together in the same construct (mut1), the function of the IRES was partially restored, even though the primary sequence of the two regions had been altered. This suggests that the secondary structure of the RNA is more important than the primary sequence during ribosome binding.
It has been observed that the HCV IRES can accommodate certain mutations at initiation codon AUG341 with very little effect on the efficiency of translation (Reynolds et al., 1995 ). Similar mutations were applied to the GBV-B IRES and their relative effects upon translation from a dicistronic expression cassette were observed. Mutations in initiating codon AUG446 demonstrated that changes AUG
AUU (mut4) and AUG
CUG (mut5) could be tolerated, although translation occurred at a reduced level. Mutation of the authentic initiation codon to AAG, GAG or GCG (mut6, mut7 and mut8) completely abolished translation and no SEAP activity was detected in the cell-based assay. Thus, site-directed mutagenesis of the GBV-B 5'UTR confirms that AUG 446 is the authentic initiating codon of polyprotein synthesis.
Introduction of mutations similar to those shown to affect HCV IRES function (Wang et al., 1994 ; Reynolds et al., 1995
) had a similar effect on GBV-B IRES function. It was noted that mut4 (AUG
AUU) was better tolerated in vivo than in vitro. In comparison with the other mutations, particularly mut1 (Py II double mutation) and mut5 (AUG
CUG), the relative activity of mut4 was reversed between the in vitro and in vivo assays. These experiments have been repeated on several occasions and yielded identical results; the reason for this observation is not clear.
The results presented here confirm that there is an IRES in the GBV-B genome capable of conferring internal initiation of translation of a downstream heterologous reporter cistron as predicted from the model of the secondary structure of the RNA (Honda et al., 1996b ). The function of the GBV-B IRES was investigated experimentally in comparative studies with constructs containing the HCV IRES. Expression levels of the reporter gene under IRES control were similar in both the HCV and the GBV-B dicistronic plasmid constructs, suggesting a similar level of activity of the GBV-B and HCV IRES.
In vitro translation studies of transcripts containing the GBV-B 5'UTR under conditions of varying potassium chloride concentration revealed that the expression of the downstream reporter was more tolerant to increasing potassium ion concentrations. The tolerance to high potassium ion levels was similar to that seen when RNA transcripts containing the HCV 5'UTR, which is known to contain an IRES, replaced that of GBV-B. In both cases translation of the upstream cistron was greatly reduced as potassium levels increased. Potassium ion concentration is known to have a differential effect on cap-dependent and cap-independent, or IRES driven, translation (Jackson, 1991 ). Consequently, these results support the view that the 5'UTR of GBV-B contains an IRES.
Site-directed mutagenesis of the GBV-B 5'UTR confirms that AUG446 is the initiating codon of polyprotein synthesis. Introduction of mutations similar to those shown to affect HCV IRES function (Wang et al., 1994 ; Reynolds et al., 1995
) had the same effect on GBV-B IRES function. These results indicate that the secondary structure of the RNA is more important than the primary sequence during ribosome binding (Wang et al., 1994
; Reynolds et al., 1995
).
In conclusion, the above observations confirm that the 5'UTR of GBV-B contains an IRES and that this IRES functions in a similar manner to that of HCV. In view of this, GBV-B may be of value as a potential surrogate virus for evaluation of potential anti-HCV compounds.
![]() |
References |
---|
![]() ![]() ![]() ![]() |
---|
Deinhardt, F., Holmes, A. W., Capps, R. B. & Popper, H. (1967). Studies on the transmission of human viral hepatitis to marmoset monkeys. Transmission of disease, serial passage and description of liver lesions. Journal of Experimental Medicine 125, 673-687.[Medline]
Fuerst, T. R., Niles, E. G., Studier, F. W. & Moss, B. (1986). Eukaryotic transient expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proceedings of the National Academy of Sciences, USA 83, 8122-8126.[Abstract]
Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Molecular and Cellular Biology 2, 1044-1051.[Medline]
Grace, K., Amphlett, E., Day, S., Lemon, S., Sangar, D., Rowlands, D. J. & Clarke, B. E. (1991). In vitro translation of hepatitis A virus subgenomic RNA transcripts. Journal of General Virology 72, 1081-1086.[Abstract]
Holmes, A. W., Deinhardt, F., Wolfe, L., Froesner, G., Paterson, D. & Casto, B. (1973). Specific neutralization of human hepatitis type A in marmoset monkeys. Nature 243, 419-420.[Medline]
Honda, M., Ping, L.-H., Rijnbrand, C. A., Amphlett, E., Clarke, B., Rowlands, D. & Lemon, S. M. (1996a). Structural requirements for initiation of translation by internal ribosome entry within genome-length hepatitis C virus RNA. Virology 222, 31-42.[Medline]
Honda, M., Brown, E. A. & Lemon, S. M. (1996b). Stability of a stemloop involving the initiator AUG controls the efficiency of internal initiation of translation on hepatitis C virus RNA. RNA 2, 955-968.[Abstract]
Jackson, R. J. (1991). Potassium salts influence the fidelity of mRNA translation initiation in rabbit reticulocyte lysates: unique features of encephalomyocarditis virus RNA translation. Biochemica Biophysica Acta 1088, 345-358.[Medline]
Karayiannis, P., Petrovic, L. M., Fry, M., Moore, D., Enticott, M., McGarvey, M. J., Scheuer, P. J. & Thomas, H. C. (1989). Studies of GB hepatitis agent in tamarins. Hepatology 9, 186-192.[Medline]
Karayiannis, P., Hadziyannis, S. J., Kim, J., Pickering, J. M., Piatak, M., Hess, G., Yun, A., McGarvey, M., Wages, J. & Thomas, H. C. (1997). Hepatitis G virus infection: clinical characteristics and response to interferon. Journal of Viral Hepatitis 4, 37-44.[Medline]
Linnen, J., Wages, J., Zhang-Keck, Z. Y., Fry, K. E., Krawczynski, K. Z., Alter, H., Koonin, E., Gallagher, M., Alter, M., Hadziyannis, S., Karayiannis, P., Fung, K., Nakatsuji, Y., Shih, J. W.-K., Young, L., Piatak, M., Hoover, K., Fernandez, J., Chen, S., Zou, J.-C., Morris, T., Hyams, K. C., Ismay, S., Lifson, J. D., Hess, G., Foung, S. K. H., Thomas, H. C., Bradley, D., Margolis, H. & Kim, J. P. (1996). Molecular cloning and disease association of hepatitis G virus: a transfusion transmissible agent. Science 271, 505-508.[Abstract]
Muerhoff, A. S., Leary, T. P., Simons, J. N., Pilot-Matias, T. J., Dawson, G. J., Erker, J. C., Chalmers, M. L., Schlauder, G. G., Desai, S. M. & Mushahwar, I. K. (1995). Genomic organisation of GB viruses A and B: two new members of the Flaviviridae associated with GB agent hepatitis. Journal of Virology 69, 5621-5630.[Abstract]
Pelham, H. R. B. & Jackson, R. J. (1976). An efficient mRNA-dependent translation system from reticulocyte lysates. European Journal of Biochemistry 67, 247-256.[Abstract]
Reynolds, J. E., Kaminski, A., Kettinen, H. J., Grace, K., Clarke, B. E., Carroll, A. R., Rowlands, D. J. & Jackson, R. J. (1995). Unique features of internal initiation of hepatitis C virus RNA translation. EMBO Journal 14, 6010-6020.[Abstract]
Reynolds, J. E., Kaminski, A., Carroll, A. R., Clarke, B. E., Rowlands, D. J. & Jackson, R. J. (1996). Internal initiation of translation of hepatitis C virus RNA: the ribosome entry site is at the authentic initiation codon. RNA 2, 867-878.[Abstract]
Rijnbrand, R., Bredenbeek, P., van der Straten, T., Whetter, L., Inchauspe, G., Lemon, S. & Spaan, W. (1995). Almost the entire 5' non-translated region of hepatitis C virus is required for cap independent translation. FEBS Letters 365, 115-119.[Medline]
Schlauder, G. G., Dawson, G. J., Simons, J. N., Pilot-Matias, T. J., Gutierrez, R. A., Heynen, C. A., Knigge, M. F., Kurpiewski, G. S., Buijk, S. L., Leary, T. P., Muerhoff, A. S., Desai, S. M. & Mushahwar, I. K. (1995). Molecular and serologic analysis in the transmission of the GB hepatitis agent. Journal of Medical Virology 46, 81-90.[Medline]
Simons, J. N., Pilot-Matias, T. J., Leary, T. P., Dawson, G. J., Desai, S. M., Schlauder, G. G., Muerhoff, A. S., Erker, J. C., Buijk, S. I., Chalmers, M. L., Van Sant, C. L. & Mushahwar, I. K. (1995a). Identification of two flavivirus-like genomes in the GB hepatitis agent. Proceedings of the National Academy of Sciences, USA 92, 3401-3405.[Abstract]
Simons, J. N., Leary, T. P., Dawson, G. J., Pilot-Matias, T. J., Muerhoff, A. S., Schlauder, G. G., Desai, S. M. & Mushahwar, I. K. (1995b). Isolation of novel virus-like sequences associated with human hepatitis. Nature Medicine 1, 564-569.[Medline]
Tsukiyama-Kohara, K., Iizuka, N., Kohara, M. & Nomoto, A. (1992). Internal ribosome entry site within hepatitis C virus RNA. Journal of Virology 66, 1476-1483.[Abstract]
Wang, C., Sarnow, P. & Siddiqui, A. (1993). Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism. Journal of Virology 67, 3338-3344.[Abstract]
Wang, C., Sarnow, P. & Siddiqui, A. (1994). A conserved helical element is essential for internal initiation of translation of hepatitis C virus. Journal of Virology 68, 7301-7307.[Abstract]
Whetter, L. E., Day, S. P., Elroy-Stein, O., Brown, E. A. & Lemon, S. M. (1994). Low efficiency of the 5' non-translated region of hepatitis A virus RNA in promoting CAP independent translation in permissive monkey kidney cells. Journal of Virology 68, 5253-5263.[Abstract]
Received ;
accepted 17 May 1999.