Abstract
The human adenovirus type 5 (Ad5) E2 transcription unit is divided into a promoter-proximal region, E2A, and a distal region, E2B, each with its own polyadenylation site. Together these regions encode the three virus-derived proteins necessary for genome replication. Ad5 variants were produced that carried linker insertion mutations immediately 5′ and/or 3′ to the coding sequence for the E2A gene DNA binding protein (DBP). Two variants carrying solely a 5′ lesion showed decreased usage of the adjacent 3′ splice site, via which the DBP mRNA is produced, and an increased usage of the alternative downstream splice sites in the E2B region, wherein viral DNA polymerase and terminal protein precursor are encoded; these viruses showed somewhat reduced growth. A variant carrying a 3′ lesion showed a marginal increase in DBP expression and slightly accelerated growth. When lesions 5′ and 3′ to the DBP coding sequence were combined in cis, the resulting virus was severely defective for growth and expressed E2B products to the virtual exclusion of E2A DBP. These data indicate that interactions must occur between the E2A 3′ splice site and polyadenylation site before this region can be treated as an exon by the RNA processing machinery, and that a sequence alteration at the polyadenylation site that alone has only minor effects on the pattern of RNA processing can drastically affect terminal exon usage when placed in cis with a mutation that reduces splicing efficiency at the upstream 3′ splice site. The data further indicate that, in vivo, Ad5 DNA replication is limited by prevailing DBP levels rather than by levels of polymerase or terminal protein precursor.
Similar content being viewed by others
References
HorwitzM.S. in FieldsB.N. and KnipeD.M. (eds.) Virology, Vol. 2, 2nd ed. Raven Press, New York, 1990, pp 1679–1721.
ChallbergM.D. and KellyT.J., Annu Rev Biochem 58, 671–717, 1989.
StillmanB., Annu Rev Cell Biol 5, 197–245, 1989.
TemperleyS.M. and HayR.T., EMBO J 11, 761–768, 1992.
NagataK., GuggenheimerR.A., EnomotoJ., LichyH., and HurwitzJ., Proc Natl Acad Sci USA 79, 6438–6442, 1982.
RosenfeldP., O'NeillE.A., WidesR.J., and KellyT.J., Mol Cell Biol 7, 875–886, 1987.
CleatP.H. and HayR.T., EMBO J 8, 1841–1848, 1989.
StuiverM.H. and van derVlietP.C., J Virol 64, 379–386, 1990.
BosherJ., RobinsonE.C., and HayR.T., New Biol 2, 1083–1090, 1990.
ChenM., MermodN., and HorwitzM.S., J Biol Chem 265, 18634–18642, 1990.
MulY.M. and van derVlietP.C., EMBO J 11, 751–760, 1992.
GrahamF.L., SmileyJ., RussellW.C., and NairnR., J Gen Virol 36, 59–72, 1977.
PilderS., MooreM., LoganJ., and ShenkT., Mol Cell Biol 6, 470–476, 1986.
JonesN.C. and ShenkT., Cell 17, 683–689, 1979.
MeltonD.A., KriegP.A., RebagliatiM.R., ManiatisT., ZinnK., and GreenM.R., Nucleic Acids Res 12, 7035–7056, 1984.
LeppardK.N. and ShenkT., EMBO J 8, 2329–2336, 1989.
LeppardK.N., J Gen Virol 74, 575–582, 1993.
ChroboczekJ., BieberF., and JacrotB., Virology 186, 280–285, 1992.
CaravokyriC., PringleC.R., and LeppardK.N., J Gen Virol 74, 2819–2824, 1993.
ImperialeM., FeldmanL.T., and NevinsJ.R., Cell 35, 127–1 36, 1983.
CaravokyriC. and PringleC.R., Virus Genes 6, 53–62, 1992.
GreenM.R., Annu Rev Cell Biol 7, 559–599, 1991.
KruijerW., vanSchaikF.M.A., and SussenbachJ.S., Nucleic Acids Res 9, 4439–4457, 1981.
ReichN.C., SarnowP., DupreyE., and LevineA.J., Virology 128, 480–484, 1983.
HemstromC., NordkvistK., PetterssonU., and VirtanenA., J Virol 62, 3258–3264, 1988.
MorinN., DelsertC., and KlessigD.F., J Virol 63, 5228–5237, 1989.
CleghornV.G., VoelkerdingK., MorinN., DelsertC., and KlessigD.F., J Virol 63, 2289–2299, 1989.
ChangL.-S. and ShenkT., J Virol 64, 2103–2109, 1990.
SeibergM., KesslerM., AloniY., and LevineA.J., Virus Genes 1, 97–116, 1987.
SeibergM., AloniY., and LevineA.J., J Virol 63, 1134–1141, 1989.
MountS.M., Nucleic Acids Res 10, 459–472, 1982.
WieringaB., HoferE., and WeissmanC., Cell 37, 915–925, 1984.
vanSantenV.L. and SpritzR.A., Proc Natl Acad Sci USA 82, 2885–2889, 1985.
RuskinB. and GreenM.R., Nature, 317, 732–734, 1985.
DeZazzoJ.D. and ImperialeM.J., Mol Cell Biol 9, 4951–4961, 1989.
CarswellS. and AlwineJ.C., Mol Cell Biol 9, 4248–4258, 1989.
RussnakR. and GanemD., Genes Dev 4, 764–776, 1990.
ValsamakisA., ZeichnerS., CarswellS., and AlwineJ.C., Proc Natl Acad Sci USA 88, 2108–2112, 1991.
McDevittM.A., ImperialeM.J., AliH., and NevinsJ.R., Cell 37, 993–999, 1984.
HartR.P., McDevittM.A., AliH., and NevinsJ.R., Mol Cell Biol 5, 2975–2983, 1985.
HuangM.T.F. and GormanC.M., Nucleic Acids Res 18, 937–947, 1990.
ChiouH.C., DabrowskiC., and AlwineJ.C., J Virol 65, 6677–6685, 1991.
RobbersonB.L., CoteG.J., and BergetS.M., Mol Cell Biol 10, 84–94, 1990.
NiwaM., RoseS.D., and BergetS.M., Genes Dev 4, 1552–1559, 1990.
NiwaM. and BergetS.M., Genes Dev 5, 2086–2095, 1991.
McLauchlanJ., GaffneyG., WhittonJ.L., and ClementsJ.B., Nucleic Acids Res 13, 1347–1368, 1985.
GilA. and ProudfootN.J., Cell 49, 399–406, 1987.
GilmartinG.M., McDevittM.A., and NevinsJ.R., Genes Dev 2, 578–587, 1988.
PrescottJ.C. and Falk-PedersenE., J Biol Chem 267, 8175–8181, 1992.
MooreC.L., Skolni-DavidH., and SharpP.A., EMBO J 5, 1929–1938, 1986.
MasonP.J., ElkingtonJ.A., LloydM.M., JonesM.B. and WilliamsJ.G., Cell 46, 263–270, 1986.
BrownP.H., TileyL.S., and CullenB.R., Genes Dev 5, 1277–1284, 1991.
CepkoC.L. and SharpP.A., Cell 31, 407–415, 1982.
CepkoC.L. and SharpP.A., Virology 129, 137–154, 1983.
BroughD.E., DroguettG., HorwitzM.S., and KlessigD.F., Virology 196, 269–281, 1993.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Caravokyri, C., Leppard, K.N. Human adenovirus type 5 variants with sequence alterations flanking the E2A gene: Effects on E2 expression and DNA replication. Virus Genes 12, 65–75 (1996). https://doi.org/10.1007/BF00370002
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00370002