CHARACTERIZATION OF CHIMERIC FULL-LENGTH MOLECULAR CLONES OF ALEUTIAN MINK DISEASE PARVOVIRUS (ADV) - IDENTIFICATION OF A DETERMINANT GOVERNING REPLICATION OF ADV IN CELL-CULTURE
CHARACTERIZATION OF CHIMERIC FULL-LENGTH MOLECULAR CLONES OF ALEUTIAN MINK DISEASE PARVOVIRUS (ADV) - IDENTIFICATION OF A DETERMINANT GOVERNING REPLICATION OF ADV IN CELL-CULTURE
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DOI：
10.1128/jvi.67.10.5976-5988.1993
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发表时间：
1993-10-01
影响因子：
5.4
通讯作者：
WOLFINBARGER, JB
中科院分区：
文献类型：
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作者：
BLOOM, ME;BERRY, BD;WOLFINBARGER, JB
阿留申水貂病细小病毒(ADV)ADV-G株对水貂无致病性，但在细胞培养中允许复制，而ADV-Utah 1株对水貂高度致病，但在细胞培养中复制能力较差。为了将这些表型差异与主要基因组特征联系起来，我们构建了一系列代表MAP单位(MU)15至88的ADV-G全长复制分子克隆与ADV-Utah 1亚基因组克隆之间的嵌合质粒。将其导入细胞培养和细胞裂解产物连续传代后，我们确定在感染性AdV-G质粒中替换ADV-Utah 1基因组的几个片段(MU15-54和65-73)并不影响这些构建体在体外产生感染性病毒的能力。像ADV-G一样，从这些复制能力强的克隆中提取的病毒在接种10天后既不会引起可检测到的病毒血症，也不会在成年水貂中引起阿留申病的任何证据。另一方面，其他嵌合质粒不能产生感染性病毒，因此在体外是复制缺陷的。ADV-Utah 1的MU 54-65 EcoRI-EcoRV片段是导致AdV-G复制缺陷的最小片段。将AdV-G EcoRI-EcoRV片段替换为复制缺陷克隆恢复了复制能力，这表明基因组的这一0.53kb部分完全位于衣壳蛋白VP1和VP2的共同编码序列中，含有控制细胞培养复制的决定因素。5天后用复制缺陷克隆进行细胞培养时，不能挽救二聚体复制DNA和单链后代DNA。这一缺陷不能通过与具有复制能力的结构的共转染来补充。
The ADV-G strain of Aleutian mink disease parvovirus (ADV) is nonpathogenic for mink but replicates permissively in cell culture, whereas the ADV-Utah 1 strain is highly pathogenic for mink but replicates poorly in cell culture. In order to relate these phenotypic differences to primary genomic features, we constructed a series of chimeric plasmids between a full-length replication-competent molecular clone of ADV-G and subgenomic clones of ADV-Utah 1 representing map units (MU) 15 to 88. After transfection of the plasmids into cell culture and serial passage of cell lysates, we determined that substitution of several segments of the ADV-Utah 1 genome (MU 15 to 54 and 65 to 73) within an infectious ADV-G plasmid did not impair the ability of these constructs to yield infectious virus in vitro. Like ADV-G, the viruses derived from these replication-competent clones caused neither detectable viremia 10 days after inoculation nor any evidence of Aleutian disease in adult mink. On the other hand, other chimeric plasmids were incapable of yielding infectious virus and were therefore replication defective in vitro. The MU 54 to 65 EcoRI-EcoRV fragment of ADV-Utah 1 was the minimal segment capable of rendering ADV-G replication defective. Substitution of the ADV-G EcoRI-EcoRV fragment into a replication-defective clone restored replication competence, indicating that this 0.53-kb portion of the genome, wholly located within shared coding sequences for the capsid proteins VP1 and VP2, contained a determinant that governs replication in cell culture. When cultures of cells were studied 5 days after transfection with replication-defective clones, rescue of dimeric replicative form DNA and single-stranded progeny DNA could not be demonstrated. This defect could not be complemented by cotransfection with a replication-competent construction.