Steagall W K, Robek M D, Perry S T, Fuller F J, Payne S L
Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4960, USA.
Virology. 1995 Jul 10;210(2):302-13. doi: 10.1006/viro.1995.1347.
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.
逆转录病毒马传染性贫血病毒（EIAV）编码一种位于逆转录酶和整合酶之间的dUTPase。我们曾描述过缺失270个碱基对dUTPase的EIAV（δDU EIAV）在马巨噬细胞中无法复制到野生型（WT）水平（D.S. Threadgill、W.K. Steagall、M.T. Flaherty、F.J. Fuller、S.T. Perry、K.E. Rushlow、S.F.J. LeGrice和S.L. Payne，《病毒学杂志》67，2592 - 2600，1993年）。在此我们描述了第二种dUTPase缺陷病毒（DUD71E）的构建，该病毒的dUTPase中有一个单氨基酸替换。感染后7天，当WT EIAV具有高度细胞病变性时，δDU和DUD71E在巨噬细胞中的复制水平为WT的2%。为了确定复制障碍，我们分析了DNA合成、整合和转录。巨噬细胞中的DNA合成正常，感染后24小时有全长病毒DNA的证据。与WT相比，整合的δDU和DUD71E DNA水平似乎降低了2至3倍。全长病毒转录本的稳态水平降低了100多倍，表明dUTPase缺陷型EIAV的复制在病毒DNA合成和转录之间受阻。由于dUTP水解通常在防止尿嘧啶掺入新合成的DNA中起作用，我们研究了dUTPase缺陷型EIAV DNA含有尿嘧啶的可能性。体外试验表明，虽然WT病毒粒子不利用dUTP，但dUTPase缺陷型病毒和重组逆转录酶会合成含尿嘧啶的DNA。从感染δDU和DUD71E的巨噬细胞中回收的病毒DNA中尿嘧啶的存在也得到了证实。在巨噬细胞中，病毒编码的dUTPase可能是防止尿嘧啶掺入病毒DNA所必需 的。
