AVIAN-SARCOMA AND LEUKOSIS VIRUS POL-ENDONUCLEASE RECOGNITION OF THE TANDEM LONG TERMINAL REPEAT JUNCTION - MINIMUM SITE REQUIRED FOR CLEAVAGE IS ALSO REQUIRED FOR VIRAL GROWTH
AVIAN-SARCOMA AND LEUKOSIS VIRUS POL-ENDONUCLEASE RECOGNITION OF THE TANDEM LONG TERMINAL REPEAT JUNCTION - MINIMUM SITE REQUIRED FOR CLEAVAGE IS ALSO REQUIRED FOR VIRAL GROWTH
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DOI：
10.1128/jvi.61.6.1999-2008.1987
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发表时间：
1987-06-01
影响因子：
5.4
通讯作者：
LEIS, J
中科院分区：
文献类型：
--
作者：
COBRINIK, D;KATZ, R;LEIS, J
逆转录病毒DNA的整合是涉及由病毒pol基因编码的核酸内切酶（pol-endo）的位点特异性反应。在体外，来自禽肉瘤和白血病病毒（ASLV）的pol-endo在单链和复制型（RF）-I底物中切割串联长末端重复序列的U 5-U3连接（LTR-LTR连接）附近的两条DNA链。我们先前已经报道了α-半乳糖苷酶切割单链底物所需的序列。β的正链和负链的pol-endo形式不同（G. Duyk，M. Longiaru，D.科布里尼克河科瓦尔，P. deHaseth，A.M. Shalka，和J. Leis，J. Virol. 56：589-599，1985）。这与RF-1底物的情况不同，其中α需要最多22个碱基对的U 5和8个碱基对的U3。β的每条链中的pol-endo切割。在LTR-LTR连接处插入回文八核苷酸（CATCGATG）消除了RF-1中的切割，但在单链DNA底物中没有。在连接处的四个核苷酸（TTAA）的缺失防止了RF-1 DNA的正链中的切割，但不影响单链DNA的切割。此外，α..β的ASLV pol-endo形式不识别来自网状内皮组织增生病病毒或莫洛尼鼠白血病病毒的异源LTR-LTR连接序列，尽管它们与ASLV连接的序列和结构相似。这些结果支持切割所需的ASLV pol-endo和LTR-LTR连接结构域之间的序列特异性相互作用的作用。通过使用感染性劳斯肉瘤病毒克隆pATV 8-K，我们将一组缺失引入U 5区，其将在病毒复制时并入LTR-LTR连接处。在未整合的前病毒中，缺失从LTR-LTR连接处的43个碱基对开始，并向连接处延伸不同的长度。用这些克隆进行的转染研究的结果表明，体内病毒生产所需的U 5序列对应于体外切割RF-1 DNA所需的序列。
Integration of retroviral DNA is a site-specific reaction involving an endonuclease encoded by the viral pol gene (pol-endo). In vitro the pol-endo from avian sarcoma and leukosis viruses (ASLVs) cleaves both DNA strands near the U5-U3 junction of tandem long terminal repeats (LTR-LTR junction) in single-stranded and replicative form (RF)-I substrates. We have reported previously that the sequences that are required for cleavage of single-stranded substrates by the .alpha..beta. form of the pol-endo differ for the plus and minus strands (G. Duyk, M. Longiaru, D. Cobrinik, R. Kowal, P. deHaseth, A.M. Shalka, and J. Leis, J. Virol. 56:589-599, 1985). This is not the case with RF-I substrates, in which a maximum of 22 base pairs of U5 and 8 base pairs of U3 were required for .alpha..beta. pol-endo cleavage in each strand. Insertion of a palindromic octanucleotide (CATCGATG) at the LTR-LTR junction abolished cleavage in RF-1 but not in single-stranded DNA substrates. Deletion of the four nucleotides (TTAA) at the junction prevented cleavage in the plus strand of RF-I DNA, but did not affect cleavage of single-stranded DNA. Furthermore, the .alpha..beta. form of ASLV pol-endo did not recognize heterologous LTR-LTR junction sequences from the reticuloendotheliosis virus or Moloney murine leukemia virus in either substrate form, despite their sequence and structural similarities to the ASLV junction. These results support a role for a sequence-specific interaction between the ASLV pol-endo and the LTR-LTR junction domains that are required for cleavage. By using the infectious Rous sarcoma virus clone pATV8-K, we introduced a set of deletions into the U5 region that would be incorporated into the LTR-LTR junction on viral replication. In the unintegrated provirus, the deletions started 43 base pairs from the LTR-LTR junction and extended various lengths toward the junction. Results of transfection studies with these clones indicated that the U5 sequences that are required for virus production in vivo correspond to those that are required for cleavage of RF-I DNA in vitro.