Abstract
The pp28 (UL99) gene of human cytomegalovirus is expressed as a true late gene, in that DNA synthesis is absolutely required for mRNA expression. Our previous studies demonstrated that pp28 promoter sequences from position -40 to +106 are sufficient for late gene expression in the context of the viral genome (C. P. Kohler, J. A. Kerry, M. Carter, V. P. Muzithras, T. R. Jones, and R. M. Stenberg, J. Virol. 68:6589-6597, 1994). To extend these studies, we have examined the sequences in the downstream leader region of the pp28 gene for their role in late gene expression. Deletion of sequences from position -6 to +46 (deltaSS) results in a threefold increase in gene expression in transient assays. In contrast, deletion of sequences from position +46 to +88 (deltaA) has little effect on gene expression. These results indicate that the sequences from position -6 to +46 may repress gene expression. To further analyze this region, site-directed mutagenesis was performed. Mutation of residues from either position +1 to +6 (SS1) or position +12 to +17 (SS2) duplicated the effect of the deltaSS deletion mutant, indicating that sequences from position +1 to +17 were important for the inhibitory effect. To assess the biological significance of these events, a recombinant virus construct containing the deltaSS mutant promoter regulating expression of the chloramphenicol acetyltransferase (CAT) reporter gene was generated. Analysis of this virus (RV delta SSCAT) revealed that deletion of sequences from position -6 to +46 does not alter the kinetic class of this promoter. However, the ratio of CAT protein to CAT mRNA levels in RV delta SSCAT-infected cells was 8- to 12-fold higher than that observed in the parental RV24/26CAT-infected cells. These results imply that the leader sequences within the pp28 gene can regulate the translation of this late gene.
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- Adam B. L., Jervey T. Y., Kohler C. P., Wright G. L., Jr, Nelson J. A., Stenberg R. M. The human cytomegalovirus UL98 gene transcription unit overlaps with the pp28 true late gene (UL99) and encodes a 58-kilodalton early protein. J Virol. 1995 Sep;69(9):5304–5310. doi: 10.1128/jvi.69.9.5304-5310.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Avni D., Shama S., Loreni F., Meyuhas O. Vertebrate mRNAs with a 5'-terminal pyrimidine tract are candidates for translational repression in quiescent cells: characterization of the translational cis-regulatory element. Mol Cell Biol. 1994 Jun;14(6):3822–3833. doi: 10.1128/mcb.14.6.3822. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Colberg-Poley A. M., Santomenna L. D., Harlow P. P., Benfield P. A., Tenney D. J. Human cytomegalovirus US3 and UL36-38 immediate-early proteins regulate gene expression. J Virol. 1992 Jan;66(1):95–105. doi: 10.1128/jvi.66.1.95-105.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Depto A. S., Stenberg R. M. Functional analysis of the true late human cytomegalovirus pp28 upstream promoter: cis-acting elements and viral trans-acting proteins necessary for promoter activation. J Virol. 1992 May;66(5):3241–3246. doi: 10.1128/jvi.66.5.3241-3246.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Depto A. S., Stenberg R. M. Regulated expression of the human cytomegalovirus pp65 gene: octamer sequence in the promoter is required for activation by viral gene products. J Virol. 1989 Mar;63(3):1232–1238. doi: 10.1128/jvi.63.3.1232-1238.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Geballe A. P., Leach F. S., Mocarski E. S. Regulation of cytomegalovirus late gene expression: gamma genes are controlled by posttranscriptional events. J Virol. 1986 Mar;57(3):864–874. doi: 10.1128/jvi.57.3.864-874.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Geballe A. P., Mocarski E. S. Translational control of cytomegalovirus gene expression is mediated by upstream AUG codons. J Virol. 1988 Sep;62(9):3334–3340. doi: 10.1128/jvi.62.9.3334-3340.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gibson W., Marcy A. I., Comolli J. C., Lee J. Identification of precursor to cytomegalovirus capsid assembly protein and evidence that processing results in loss of its carboxy-terminal end. J Virol. 1990 Mar;64(3):1241–1249. doi: 10.1128/jvi.64.3.1241-1249.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guo Y. W., Huang E. S. Characterization of a structurally tricistronic gene of human cytomegalovirus composed of U(s)18, U(s)19, and U(s)20. J Virol. 1993 Apr;67(4):2043–2054. doi: 10.1128/jvi.67.4.2043-2054.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guzowski J. F., Singh J., Wagner E. K. Transcriptional activation of the herpes simplex virus type 1 UL38 promoter conferred by the cis-acting downstream activation sequence is mediated by a cellular transcription factor. J Virol. 1994 Dec;68(12):7774–7789. doi: 10.1128/jvi.68.12.7774-7789.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guzowski J. F., Wagner E. K. Mutational analysis of the herpes simplex virus type 1 strict late UL38 promoter/leader reveals two regions critical in transcriptional regulation. J Virol. 1993 Sep;67(9):5098–5108. doi: 10.1128/jvi.67.9.5098-5108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hershey J. W. Translational control in mammalian cells. Annu Rev Biochem. 1991;60:717–755. doi: 10.1146/annurev.bi.60.070191.003441. [DOI] [PubMed] [Google Scholar]
- Homa F. L., Otal T. M., Glorioso J. C., Levine M. Transcriptional control signals of a herpes simplex virus type 1 late (gamma 2) gene lie within bases -34 to +124 relative to the 5' terminus of the mRNA. Mol Cell Biol. 1986 Nov;6(11):3652–3666. doi: 10.1128/mcb.6.11.3652. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Huang L., Malone C. L., Stinski M. F. A human cytomegalovirus early promoter with upstream negative and positive cis-acting elements: IE2 negates the effect of the negative element, and NF-Y binds to the positive element. J Virol. 1994 Apr;68(4):2108–2117. doi: 10.1128/jvi.68.4.2108-2117.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Iskenderian A. C., Huang L., Reilly A., Stenberg R. M., Anders D. G. Four of eleven loci required for transient complementation of human cytomegalovirus DNA replication cooperate to activate expression of replication genes. J Virol. 1996 Jan;70(1):383–392. doi: 10.1128/jvi.70.1.383-392.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jackson R. J., Hunt S. L., Gibbs C. L., Kaminski A. Internal initiation of translation of picornavirus RNAs. Mol Biol Rep. 1994 May;19(3):147–159. doi: 10.1007/BF00986957. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jenkins D. E., Martens C. L., Mocarski E. S. Human cytomegalovirus late protein encoded by ie2: a trans-activator as well as a repressor of gene expression. J Gen Virol. 1994 Sep;75(Pt 9):2337–2348. doi: 10.1099/0022-1317-75-9-2337. [DOI] [PubMed] [Google Scholar]
- Jones T. R., Muzithras V. P. Fine mapping of transcripts expressed from the US6 gene family of human cytomegalovirus strain AD169. J Virol. 1991 Apr;65(4):2024–2036. doi: 10.1128/jvi.65.4.2024-2036.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jones T. R., Muzithras V. P., Gluzman Y. Replacement mutagenesis of the human cytomegalovirus genome: US10 and US11 gene products are nonessential. J Virol. 1991 Nov;65(11):5860–5872. doi: 10.1128/jvi.65.11.5860-5872.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kashiwagi K., Ito K., Igarashi K. Spermidine regulation of ornithine decarboxylase synthesis by a GC-rich sequence of the 5'-untranslated region. Biochem Biophys Res Commun. 1991 Aug 15;178(3):815–822. doi: 10.1016/0006-291x(91)90964-9. [DOI] [PubMed] [Google Scholar]
- Kerry J. A., Priddy M. A., Jervey T. Y., Kohler C. P., Staley T. L., Vanson C. D., Jones T. R., Iskenderian A. C., Anders D. G., Stenberg R. M. Multiple regulatory events influence human cytomegalovirus DNA polymerase (UL54) expression during viral infection. J Virol. 1996 Jan;70(1):373–382. doi: 10.1128/jvi.70.1.373-382.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kerry J. A., Priddy M. A., Stenberg R. M. Identification of sequence elements in the human cytomegalovirus DNA polymerase gene promoter required for activation by viral gene products. J Virol. 1994 Jul;68(7):4167–4176. doi: 10.1128/jvi.68.7.4167-4176.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kim S. J., Park K., Koeller D., Kim K. Y., Wakefield L. M., Sporn M. B., Roberts A. B. Post-transcriptional regulation of the human transforming growth factor-beta 1 gene. J Biol Chem. 1992 Jul 5;267(19):13702–13707. [PubMed] [Google Scholar]
- Klucher K. M., Rabert D. K., Spector D. H. Sequences in the human cytomegalovirus 2.7-kilobase RNA promoter which mediate its regulation as an early gene. J Virol. 1989 Dec;63(12):5334–5343. doi: 10.1128/jvi.63.12.5334-5343.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klucher K. M., Spector D. H. The human cytomegalovirus 2.7-kilobase RNA promoter contains a functional binding site for the adenovirus major late transcription factor. J Virol. 1990 Sep;64(9):4189–4198. doi: 10.1128/jvi.64.9.4189-4198.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kohler C. P., Kerry J. A., Carter M., Muzithras V. P., Jones T. R., Stenberg R. M. Use of recombinant virus to assess human cytomegalovirus early and late promoters in the context of the viral genome. J Virol. 1994 Oct;68(10):6589–6597. doi: 10.1128/jvi.68.10.6589-6597.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Koromilas A. E., Lazaris-Karatzas A., Sonenberg N. mRNAs containing extensive secondary structure in their 5' non-coding region translate efficiently in cells overexpressing initiation factor eIF-4E. EMBO J. 1992 Nov;11(11):4153–4158. doi: 10.1002/j.1460-2075.1992.tb05508.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kouzarides T., Bankier A. T., Satchwell S. C., Weston K., Tomlinson P., Barrell B. G. Large-scale rearrangement of homologous regions in the genomes of HCMV and EBV. Virology. 1987 Apr;157(2):397–413. doi: 10.1016/0042-6822(87)90282-0. [DOI] [PubMed] [Google Scholar]
- Kozak M. Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs. Mol Cell Biol. 1989 Nov;9(11):5134–5142. doi: 10.1128/mcb.9.11.5134. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kozak M. How do eucaryotic ribosomes select initiation regions in messenger RNA? Cell. 1978 Dec;15(4):1109–1123. doi: 10.1016/0092-8674(78)90039-9. [DOI] [PubMed] [Google Scholar]
- Kozak M. Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc Natl Acad Sci U S A. 1986 May;83(9):2850–2854. doi: 10.1073/pnas.83.9.2850. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liu B., Stinski M. F. Human cytomegalovirus contains a tegument protein that enhances transcription from promoters with upstream ATF and AP-1 cis-acting elements. J Virol. 1992 Jul;66(7):4434–4444. doi: 10.1128/jvi.66.7.4434-4444.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lövkvist-Wallström E., Stjernborg-Ulvsbäck L., Scheffler I. E., Persson L. Regulation of mammalian ornithine decarboxylase. Studies on the induction of the enzyme by hypotonic stress. Eur J Biochem. 1995 Jul 1;231(1):40–44. [PubMed] [Google Scholar]
- Macejak D. G., Sarnow P. Internal initiation of translation mediated by the 5' leader of a cellular mRNA. Nature. 1991 Sep 5;353(6339):90–94. doi: 10.1038/353090a0. [DOI] [PubMed] [Google Scholar]
- Pelletier J., Sonenberg N. Insertion mutagenesis to increase secondary structure within the 5' noncoding region of a eukaryotic mRNA reduces translational efficiency. Cell. 1985 Mar;40(3):515–526. doi: 10.1016/0092-8674(85)90200-4. [DOI] [PubMed] [Google Scholar]
- Pelletier J., Sonenberg N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature. 1988 Jul 28;334(6180):320–325. doi: 10.1038/334320a0. [DOI] [PubMed] [Google Scholar]
- Rivera-Gonzalez R., Imbalzano A. N., Gu B., Deluca N. A. The role of ICP4 repressor activity in temporal expression of the IE-3 and latency-associated transcript promoters during HSV-1 infection. Virology. 1994 Aug 1;202(2):550–564. doi: 10.1006/viro.1994.1377. [DOI] [PubMed] [Google Scholar]
- Schleiss M. R., Degnin C. R., Geballe A. P. Translational control of human cytomegalovirus gp48 expression. J Virol. 1991 Dec;65(12):6782–6789. doi: 10.1128/jvi.65.12.6782-6789.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Scully A. L., Sommer M. H., Schwartz R., Spector D. H. The human cytomegalovirus IE2 86-kilodalton protein interacts with an early gene promoter via site-specific DNA binding and protein-protein associations. J Virol. 1995 Oct;69(10):6533–6540. doi: 10.1128/jvi.69.10.6533-6540.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sommer M. H., Scully A. L., Spector D. H. Transactivation by the human cytomegalovirus IE2 86-kilodalton protein requires a domain that binds to both the TATA box-binding protein and the retinoblastoma protein. J Virol. 1994 Oct;68(10):6223–6231. doi: 10.1128/jvi.68.10.6223-6231.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spector D. H., Klucher K. M., Rabert D. K., Wright D. A. Human cytomegalovirus early gene expression. Curr Top Microbiol Immunol. 1990;154:21–45. doi: 10.1007/978-3-642-74980-3_2. [DOI] [PubMed] [Google Scholar]
- Staprans S. I., Rabert D. K., Spector D. H. Identification of sequence requirements and trans-acting functions necessary for regulated expression of a human cytomegalovirus early gene. J Virol. 1988 Sep;62(9):3463–3473. doi: 10.1128/jvi.62.9.3463-3473.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stasiak P. C., Mocarski E. S. Transactivation of the cytomegalovirus ICP36 gene promoter requires the alpha gene product TRS1 in addition to IE1 and IE2. J Virol. 1992 Feb;66(2):1050–1058. doi: 10.1128/jvi.66.2.1050-1058.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stenberg R. M., Kerry J. A. Cytomegalovirus genes: their structure and function. Scand J Infect Dis Suppl. 1995;99:3–6. [PubMed] [Google Scholar]
- Stenberg R. M., Thomsen D. R., Stinski M. F. Structural analysis of the major immediate early gene of human cytomegalovirus. J Virol. 1984 Jan;49(1):190–199. doi: 10.1128/jvi.49.1.190-199.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stinski M. F., Thomsen D. R., Stenberg R. M., Goldstein L. C. Organization and expression of the immediate early genes of human cytomegalovirus. J Virol. 1983 Apr;46(1):1–14. doi: 10.1128/jvi.46.1.1-14.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vagner S., Gensac M. C., Maret A., Bayard F., Amalric F., Prats H., Prats A. C. Alternative translation of human fibroblast growth factor 2 mRNA occurs by internal entry of ribosomes. Mol Cell Biol. 1995 Jan;15(1):35–44. doi: 10.1128/mcb.15.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wade E. J., Klucher K. M., Spector D. H. An AP-1 binding site is the predominant cis-acting regulatory element in the 1.2-kilobase early RNA promoter of human cytomegalovirus. J Virol. 1992 Apr;66(4):2407–2417. doi: 10.1128/jvi.66.4.2407-2417.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Welch A. R., McNally L. M., Gibson W. Cytomegalovirus assembly protein nested gene family: four 3'-coterminal transcripts encode four in-frame, overlapping proteins. J Virol. 1991 Aug;65(8):4091–4100. doi: 10.1128/jvi.65.8.4091-4100.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wing B. A., Huang E. S. Analysis and mapping of a family of 3'-coterminal transcripts containing coding sequences for human cytomegalovirus open reading frames UL93 through UL99. J Virol. 1995 Mar;69(3):1521–1531. doi: 10.1128/jvi.69.3.1521-1531.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Winkler M., Rice S. A., Stamminger T. UL69 of human cytomegalovirus, an open reading frame with homology to ICP27 of herpes simplex virus, encodes a transactivator of gene expression. J Virol. 1994 Jun;68(6):3943–3954. doi: 10.1128/jvi.68.6.3943-3954.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wright D. A., Spector D. H. Posttranscriptional regulation of a class of human cytomegalovirus phosphoproteins encoded by an early transcription unit. J Virol. 1989 Jul;63(7):3117–3127. doi: 10.1128/jvi.63.7.3117-3127.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]