Wk. Steagall et al., INCORPORATION OF URACIL INTO VIRAL-DNA CORRELATES WITH REDUCED REPLICATION OF EIAV IN MACROPHAGES, Virology, 210(2), 1995, pp. 302-313
The retrovirus equine infectious anemia virus (EIAV) encodes a dUTPase
situated between reverse transcriptase and integrase. We have describ
ed 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. T
hreadgill, W. K. Steagall, M. T. Flaherty, F. J. Fuller, S. T. Ferry,
K. E. Rushlow, S. F. J. LeGrice, and S. L Payne, J. Virol. 67, 2592-26
00, 1993). Here we describe the construction of a second dUTPase-defic
ient virus (DUD71E) containing a single amino acid substitution in dUT
Pase. Delta DU and DUD71E replicate to 2% of WT levels in macrophages
by 7 days postinfection, when WT EIAV is highly cytopathic. To identif
y the replication block(s), we analyzed DNA synthesis, integration, an
d transcription. DNA synthesis was normal in macrophages, with evidenc
e of full-length viral DNA by 24 hr postinfection. The level of integr
ated Delta DU and DUD71E DNA appeared to be decreased 2- to 3-fold com
pared to WT. Steady-state levels of full-length viral transcripts were
decreased over 100-fold, indicating that replication of dUTPase-defic
ient 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 tha
t dUTPase-deficient EIAV DNA contains uracil. in vitro assays showed t
hat 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. (C
) 1995 Academic Press, Inc.