A deletion mutant in the human cytomegalovirus gene encoding IE1(491aa) is replication defective due to a failure in autoregulation.
Human cytomegalovirus (CMV) replication begins with the expression of two regulatory proteins, IE1(491aa) and IE2(579aa), produced from differentially spliced transcripts under control of the ie1/ie2 promoter-enhancer. A deletion mutation removing all 406 IE1(491aa)-specific amino acids was engineered into the viral genome and this mutant (RC303 delta Acc) was propagated on an IE1(491aa)-expressing human fibroblast cell line (ihfie1.3). RC303 delta Acc failed to replicate on normal human fibroblasts at low multiplicities of infection (mois). At mois > 3 plaque-forming units per cell, virus replication and production of progeny were comparable to wild type. However, at mois 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 RC303 delta Acc correlated with the ability to express IE2(579aa), consistent with a role for IE1(491aa) in positive autoregulation of the ie1/ie2 promoter-enhancer and with data suggesting that virion transactivators compensate for the lack of IE1(491aa) under high moi conditions. ie1-deficient CMV should be completely avirulent, suggesting its utility as a gene therapy vector for hematopoietic progenitors that are normal sites of CMV latency.
Mocarski
E S
ES
Department of Microbiology and Immunology, Stanford University School of Medicine, CA 94305-5402, USA. mocarski@leland.stanford.edu
Kemble
G W
GW
Lyle
J M
JM
Greaves
R F
RF
D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics* / Q000502:physiology; D003673:Defective Viruses / Q000235:genetics* / Q000502:physiology; D005091:Exons; D005347:Fibroblasts; D005809:Genes, Regulator; D005814:Genes, Viral; D005816:Genetic Complementation Test; D006706:Homeostasis; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis / Q000235:genetics*; D007231:Infant, Newborn; D008297:Male; D011995:Recombination, Genetic; D017384:Sequence Deletion; D012867:Skin / Q000166:cytology; D014162:Transfection / Q000379:methods*; D010948:Viral Plaque Assay; D014764:Viral Proteins; D014779:Virus Replication
C088645:IE1 protein, cytomegalovirus; D017874:Immediate-Early Proteins; D014764:Viral Proteins