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(NAS Colloquium) Genetic Engineering of Viruses and Viral Vectors (1996) / Chapter Skim
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A deletion mutant in the human cytomegalovirus gene encoding IEl 49laa is replication defective due to a failure in autoregulation
Pages 11321-11326

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From page 11321...
... However, at mods between 0.01 and 1, mutant virus replicated slowly on normal fibroblasts, a pattern that suggested initiation of productive infection required multiple hits. Replication of RC3031tAcc correlated with the ability to express IE2s79aa, consistent with a role for IE149~aa in positive autoregulation of the iel/ie2 promoter-enhancer and with data suggesting that virion transactivators compensate for the lack of IEl49'aa under high moi conditions.
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From page 11322...
... SalI-l~nearized plasmid was added in a molar amount equivalent to an individual of PacI-linearized cosmids and cotransfection of this mixture was carried out on ihfiel.3 cells to complement IE149:aa in trans. When the genome structure of the resulting recombinants was analyzed, two of five independent pools arising from the pON303AAcc-containing set carried a uniform population of the expected mutant, and two pools made with pON303 contained the expected Towne/Toledo wt chimera (31~.
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From page 11323...
... Although mutant virus exhibited a linear relationship between dilution and ability to form plaques on ihfiel.3 cells, plaque formation on noncomplementing HFs appeared to be nonlinear with respect to dilution, with a 10-fold dilution of stock producing an ~100-fold decrease in plaque formation. Consistent with the slow growth and nonlinear relationship between input dose and plaque formation, mutant virus exhibited a plaquing efficiency on HF that was much lower (5 x 10-4 to 3 x 10-3 1o6 105 L~ 1 04 103.
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From page 11324...
... Despite the fact that progeny accumu- 43 lated more slowly in noncomplementing cells and took ap proximately three times as long to reach peak levels, the total amount of viral DNA that could be recovered from non complementing cells was comparable to that recovered from ~ ~ complementin, cells (Fig.
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From page 11325...
... Recombinant CMV lacking IEl49~:,a may be an appropriate avirulent vector to introduce genes into hematopoietic progenitors without a risk of reactivation or dissemination. We thank Kirsten Lofgren and Tai-An Cha for providing sequence information on the cosmic ends, Danushka Formankova for in~munoblot analysis, and Maria Kirichenko for performing plaque assays.
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From page 11326...
... 11326 Colloquium Paper: Mocarski et al.
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Key Terms

  • noncomplementing cells
  • complementing cells
  • plaque formation
  • replication defective
  • blot hybridization

  • dna blot
  • productive replication
  • virus exhibited
  • cell types
  • human cytomegalovirus

  • hematopoietic progenitors
  • low mois
  • murine monoclonal
  • digests hybridized
  • gene products

  • towne strain
  • genome structure
  • viral dna
  • virion transactivators
  • productive infection

  • inefficient growth
  • virus failed
  • moi conditions
  • dna fragments
  • following infection

  • slow growth
  • previously described
  • successive passages
  • growth properties
  • virus produced


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