Lichtenstein D L, Rushlow K E, Cook R F, Raabe M L, Swardson C J, Kociba G J, Issel C J, Montelaro R C
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA.
J Virol. 1995 May;69(5):2881-8. doi: 10.1128/JVI.69.5.2881-2888.1995.
As an important enzyme in DNA synthesis, dUTPase is present in a wide variety of organisms and viruses and has been identified as a component of the equine infectious anemia virus (EIAV) pol gene. The role of EIAV dUTPase, designated DU, in virus replication in vitro and in vivo was investigated with a recently described infectious molecular clone of EIAV. A deletion mutant that was deficient in dUTPase activity was constructed, and its replication kinetics was examined in fetal equine kidney (FEK) cells and primary equine bone marrow macrophage (EBMM) cells. In FEK cells, which are permissive for EIAV replication, the mutant virus replicated as well as the parental virus. In primary cultures of EBMM cells, which are primary targets of EIAV infection in vivo, the DU mutant showed delayed replication kinetics and replicated to a lower extent than did the parental virus. As the multiplicity of infection decreased, the difference between the parental and mutant viruses increased, such that at the lowest multiplicity of infection tested, there was over a 100-fold difference in virus production. The mutant virus was also much less cytopathic. The role of DU in replication in vivo was examined using a Shetland pony model of EIAV infection. Shetland ponies that were infected with the parental and mutant viruses showed transient virus RNA levels in plasma approximately 5 to 10 days postinfection. The peak virus levels in plasma (as measured by a quantitative reverse transcriptase PCR assay) were 10- to 100-fold lower in the mutant virus-infected animals than in the animals infected with the parental virus. However, ponies infected with the mutant virus mounted similar antibody responses despite the marked differences in virus replication. These studies demonstrate that EIAV DU is important for the efficient replication of the virus in macrophages in vitro and in vivo and suggests that variations in the DU sequence could markedly affect the biological and pathogenic properties of EIAV.
作为DNA合成中的一种重要酶，脱氧尿苷三磷酸酶（dUTPase）存在于多种生物体和病毒中，并且已被确定为马传染性贫血病毒（EIAV）pol基因的一个组成部分。利用最近描述的EIAV感染性分子克隆，研究了EIAV dUTPase（命名为DU）在体外和体内病毒复制中的作用。构建了一个缺乏dUTPase活性的缺失突变体，并在胎马肾（FEK）细胞和原代马骨髓巨噬细胞（EBMM）中检测其复制动力学。在允许EIAV复制的FEK细胞中，突变病毒与亲本病毒一样能进行复制。在EBMM细胞的原代培养物中，EBMM细胞是EIAV体内感染的主要靶细胞，DU突变体显示出延迟的复制动力学，并且复制程度低于亲本病毒。随着感染复数的降低，亲本病毒和突变病毒之间的差异增大，以至于在测试的最低感染复数下，病毒产生量存在超过100倍的差异。突变病毒的细胞病变效应也小得多。利用EIAV感染的设得兰矮种马模型研究了DU在体内复制中的作用。感染亲本病毒和突变病毒的设得兰矮种马在感染后约5至10天血浆中显示出短暂的病毒RNA水平。血浆中的病毒峰值水平（通过定量逆转录酶PCR测定）在感染突变病毒的动物中比感染亲本病毒的动物低10至100倍。然而，尽管病毒复制存在显著差异，但感染突变病毒的矮种马产生了相似的抗体反应。这些研究表明，EIAV DU对于该病毒在体外和体内巨噬细胞中的有效复制很重要，并表明DU序列的变异可能显著影响EIAV的生物学和致病特性。
