Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1987 Oct;84(20):7213–7217. doi: 10.1073/pnas.84.20.7213

Insertion and deletion mutagenesis of the human cytomegalovirus genome.

R R Spaete 1, E S Mocarski 1
PMCID: PMC299260  PMID: 2823255

Abstract

Studies on human cytomegalovirus (CMV) have been limited by a paucity of molecular genetic techniques available for manipulating the viral genome. We have developed methods for site-specific insertion and deletion mutagenesis of CMV utilizing a modified Escherichia coli lacZ gene as a genetic marker. The lacZ gene was placed under the control of the major beta gene regulatory signals and inserted into the viral genome by homologous recombination, disrupting one of two copies of this beta gene within the L-component repeats of CMV DNA. We observed high-level expression of beta-galactosidase by the recombinant in a temporally authentic manner, with levels of this enzyme approaching 1% of total protein in infected cells. Thus, CMV is an efficient vector for high-level expression of foreign gene products in human cells. Using back selection of lacZ-deficient virus in the presence of the chromogenic substrate 5-bromo-4-chloro-3-indolyl beta-D-galactoside, we generated random endpoint deletion mutants. Analysis of these mutants revealed that CMV DNA sequences flanking the insert had been removed, thereby establishing this approach as a means of determining whether sequences flanking a lacZ insertion are dispensable for viral growth. In an initial test of the methods, we have shown that 7800 base pairs of one copy of L-component repeat sequences can be deleted without affecting viral growth in human fibroblasts.

Full text

PDF
7213

Images in this article

7214Image
on p.7214
7215Image
on p.7215
7215Image
on p.7215
7215Image
on p.7215
7215Image
on p.7215
7216Image
on p.7216
7216Image
on p.7216
7216Image
on p.7216
7216Image
on p.7216
7216Image
on p.7216
7216Image
on p.7216
7216Image
on p.7216
7216Image
on p.7216
7216Image
on p.7216
7216Image
on p.7216
7217Image
on p.7217
7217Image
on p.7217
7217Image
on p.7217

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Chakrabarti S., Brechling K., Moss B. Vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques. Mol Cell Biol. 1985 Dec;5(12):3403–3409. doi: 10.1128/mcb.5.12.3403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Demarchi J. M. Human cytomegalovirus DNA: restriction enzyme cleavage maps and map locations for immediate-early, early, and late RNAs. Virology. 1981 Oct 15;114(1):23–38. doi: 10.1016/0042-6822(81)90249-x. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Geballe A. P., Spaete R. R., Mocarski E. S. A cis-acting element within the 5' leader of a cytomegalovirus beta transcript determines kinetic class. Cell. 1986 Sep 12;46(6):865–872. doi: 10.1016/0092-8674(86)90068-1. [DOI] [PubMed] [Google Scholar]
  5. Goins W. F., Stinski M. F. Expression of a human cytomegalovirus late gene is posttranscriptionally regulated by a 3'-end-processing event occurring exclusively late after infection. Mol Cell Biol. 1986 Dec;6(12):4202–4213. doi: 10.1128/mcb.6.12.4202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hubenthal-Voss J., Roizman B. Herpes simplex virus 1 reiterated S component sequences (c1) situated between the a sequence and alpha 4 gene are not essential for virus replication. J Virol. 1985 May;54(2):509–514. doi: 10.1128/jvi.54.2.509-514.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hutchinson N. I., Sondermeyer R. T., Tocci M. J. Organization and expression of the major genes from the long inverted repeat of the human cytomegalovirus genome. Virology. 1986 Nov;155(1):160–171. doi: 10.1016/0042-6822(86)90176-5. [DOI] [PubMed] [Google Scholar]
  8. Ihara S., Takekoshi M., Watanabe Y. Cleavage maps of human cytomegalovirus genome (strain Towne) determined by the use of cosmid cloning system. Arch Virol. 1986;88(3-4):241–250. doi: 10.1007/BF01310878. [DOI] [PubMed] [Google Scholar]
  9. Mackett M., Smith G. L., Moss B. Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7415–7419. doi: 10.1073/pnas.79.23.7415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. McDonough S. H., Staprans S. I., Spector D. H. Analysis of the major transcripts encoded by the long repeat of human cytomegalovirus strain AD169. J Virol. 1985 Mar;53(3):711–718. doi: 10.1128/jvi.53.3.711-718.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Mocarski E. S., Post L. E., Roizman B. Molecular engineering of the herpes simplex virus genome: insertion of a second L-S junction into the genome causes additional genome inversions. Cell. 1980 Nov;22(1 Pt 1):243–255. doi: 10.1016/0092-8674(80)90172-5. [DOI] [PubMed] [Google Scholar]
  12. Pennock G. D., Shoemaker C., Miller L. K. Strong and regulated expression of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector. Mol Cell Biol. 1984 Mar;4(3):399–406. doi: 10.1128/mcb.4.3.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Plotkin S. A., Huang E. S. Cytomegalovirus vaccine virus (Towne strain) does not induce latency. J Infect Dis. 1985 Aug;152(2):395–397. doi: 10.1093/infdis/152.2.395. [DOI] [PubMed] [Google Scholar]
  14. Plotkin S. A., Smiley M. L., Friedman H. M., Starr S. E., Fleisher G. R., Wlodaver C., Dafoe D. C., Friedman A. D., Grossman R. A., Barker C. F. Towne-vaccine-induced prevention of cytomegalovirus disease after renal transplants. Lancet. 1984 Mar 10;1(8376):528–530. doi: 10.1016/s0140-6736(84)90930-9. [DOI] [PubMed] [Google Scholar]
  15. Post L. E., Roizman B. A generalized technique for deletion of specific genes in large genomes: alpha gene 22 of herpes simplex virus 1 is not essential for growth. Cell. 1981 Jul;25(1):227–232. doi: 10.1016/0092-8674(81)90247-6. [DOI] [PubMed] [Google Scholar]
  16. Quinnan G. V., Jr, Delery M., Rook A. H., Frederick W. R., Epstein J. S., Manischewitz J. F., Jackson L., Ramsey K. M., Mittal K., Plotkin S. A. Comparative virulence and immunogenicity of the Towne strain and a nonattenuated strain of cytomegalovirus. Ann Intern Med. 1984 Oct;101(4):478–483. doi: 10.7326/0003-4819-101-4-478. [DOI] [PubMed] [Google Scholar]
  17. Spaete R. R., Mocarski E. S. Regulation of cytomegalovirus gene expression: alpha and beta promoters are trans activated by viral functions in permissive human fibroblasts. J Virol. 1985 Oct;56(1):135–143. doi: 10.1128/jvi.56.1.135-143.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Spaete R. R., Mocarski E. S. The alpha sequence of the cytomegalovirus genome functions as a cleavage/packaging signal for herpes simplex virus defective genomes. J Virol. 1985 Jun;54(3):817–824. doi: 10.1128/jvi.54.3.817-824.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Stinski M. F., Roehr T. J. Activation of the major immediate early gene of human cytomegalovirus by cis-acting elements in the promoter-regulatory sequence and by virus-specific trans-acting components. J Virol. 1985 Aug;55(2):431–441. doi: 10.1128/jvi.55.2.431-441.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Thomsen D. R., Stinski M. F. Cloning of the human cytomegalovirus genome as endonuclease XbaI fragments. Gene. 1981 Dec;16(1-3):207–216. doi: 10.1016/0378-1119(81)90077-9. [DOI] [PubMed] [Google Scholar]
  21. Varmuza S. L., Smiley J. R. Unstable heterozygosity in a diploid region of herpes simplex virus DNA. J Virol. 1984 Feb;49(2):356–362. doi: 10.1128/jvi.49.2.356-362.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES