Incorporation of uracil into viral DNA correlates with reduced replication of EIAV in macrophages.
The retrovirus equine infectious anemia virus (EIAV) encodes a dUTPase situated between reverse transcriptase and integrase. We have described the inability of EIAV with a 270-bp dUTPase deletion, delta DU EIAV, to replicate to wild-type (WT) levels in equine macrophages (D. S. Threadgill, W. K. Steagall, M. T. Flaherty, F. J. Fuller, S. T. Perry, K. E. Rushlow, S. F. J. LeGrice, and S. L. Payne, J. Virol. 67, 2592-2600, 1993). Here we describe the construction of a second dUTPase-deficient virus (DUD71E) containing a single amino acid substitution in dUTPase. delta DU and DUD71E replicate to 2% of WT levels in macrophages by 7 days postinfection, when WT EIAV is highly cytopathic. To identify the replication block(s), we analyzed DNA synthesis, integration, and transcription. DNA synthesis was normal in macrophages, with evidence of full-length viral DNA by 24 hr postinfection. The level of integrated delta DU and DUD71E DNA appeared to be decreased 2- to 3-fold compared to WT. Steady-state levels of full-length viral transcripts were decreased over 100-fold, indicating that replication of dUTPase-deficient EIAV is blocked between viral DNA synthesis and transcription. As dUTP hydrolysis normally plays a role in preventing incorporation of uracil into newly synthesized DNA, we investigated the possibility that dUTPase-deficient EIAV DNA contains uracil. In vitro assays showed that while WT virions do not utilize dUTP, dUTPase-deficient virus and recombinant RT synthesize uracil-containing DNA. The presence of uracil in viral DNA recovered from delta DU- and DUD71E-infected macrophages was also demonstrated. In macrophages, a virally encoded dUTPase may be necessary to prevent the incorporation of uracil into viral DNA.
Steagall
W K
WK
Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4960, USA.
Robek
M D
MD
Perry
S T
ST
Fuller
F J
FJ
Payne
S L
SL
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D003588:Cytopathogenic Effect, Viral; D045647:DNA Glycosylases; D004261:DNA Replication / Q000502:physiology; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry*; D006736:Horses; D004860:Infectious Anemia Virus, Equine / Q000201:enzymology* / Q000502:physiology; D008264:Macrophages / Q000821:virology*; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases; D017354:Point Mutation; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D012194:RNA-Directed DNA Polymerase; D014158:Transcription, Genetic / Q000235:genetics; D014498:Uracil / Q000032:analysis / Q000378:metabolism*; D051981:Uracil-DNA Glycosidase; D016662:Virus Integration; D014779:Virus Replication / Q000502:physiology
D004279:DNA, Viral; D014498:Uracil; D012194:RNA-Directed DNA Polymerase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase