Czauderna F, Fischer N, Boller K, Kurth R, Tönjes R R
Paul-Ehrlich-Institut, D-63225 Langen, Germany.
J Virol. 2000 May;74(9):4028-38. doi: 10.1128/jvi.74.9.4028-4038.2000.
The use of pig xenografts is being considered to alleviate the shortage of allogeneic organs for transplantation. In addition to the problems overcoming immunological and physiological barriers, the existence of numerous porcine microorganisms poses the risk of initiating a xenozoonosis. Recently, different classes of type C porcine endogenous retoviruses (PERV) which are infectious for human cells in vitro have been partially described. We therefore examined whether completely intact proviruses exist that produce infectious and replication-competent virions. Several proviral PERV sequences were cloned and characterized. One molecular PERV class B clone, PERV-B(43), generated infectious particles after transfection into human 293 cells. A second clone, PERV-B(33), which was highly homologous to PERV-B(43), showed a G-to-A mutation in the first start codon (Met to Ile) of the env gene, preventing this provirus from replicating. However, a genetic recombinant, PERV-B(33)/ATG, carrying a restored env start codon, became infectious and could be serially passaged on 293 cells similar to virus clone PERV-B(43). PERV protein expression was detected 24 to 48 h posttransfection (p. t.) using cross-reacting antiserum, and reverse transcriptase activity was found at 12 to 14 days p.t. The transcriptional start and stop sites as well as the splice donor and splice acceptor sites of PERV mRNA were mapped, yielding a subgenomic env transcript of 3. 1 kb. PERV-B(33) and PERV-B(43) differ in the number of copies of a 39-bp segment in the U3 region of the long terminal repeat. Strategies to identify and to specifically suppress or eliminate those proviruses from the pig genome might help in the production of PERV-free animals.
人们正在考虑使用猪异种移植物来缓解同种异体移植器官的短缺。除了克服免疫和生理障碍的问题外，众多猪源微生物的存在还带来了引发异种动物传染病的风险。最近，已经部分描述了不同类型的C型猪内源性逆转录病毒（PERV），它们在体外对人类细胞具有感染性。因此，我们研究了是否存在能产生具有感染性和复制能力的病毒粒子的完全完整的前病毒。克隆并鉴定了几个前病毒PERV序列。一个分子PERV B类克隆，PERV-B(43)，转染到人293细胞后产生了感染性颗粒。第二个克隆，PERV-B(33)，与PERV-B(43)高度同源，在env基因的第一个起始密码子（从甲硫氨酸到异亮氨酸）处有一个G到A的突变，阻止了该前病毒的复制。然而，携带恢复的env起始密码子的基因重组体PERV-B(33)/ATG具有了感染性，并且可以像病毒克隆PERV-B(43)一样在293细胞上连续传代。转染后24至48小时（p.t.）使用交叉反应抗血清检测到了PERV蛋白表达，并且在转染后12至14天发现了逆转录酶活性。绘制了PERV mRNA的转录起始和终止位点以及剪接供体和剪接受体位点，产生了一个3.1 kb的亚基因组env转录本。PERV-B(33)和PERV-B(43)在长末端重复序列U3区域中一个39 bp片段的拷贝数上有所不同。识别并特异性抑制或从猪基因组中消除这些前病毒的策略可能有助于生产无PERV的动物。
