Alcaraz-Estrada Sofia L, Del Angel Rosa, Padmanabhan Radhakrishnan
Division de Medicina Genomica, Centro Medico Nacional"20 de Noviembre"-ISSSTE, Mexico, D.F., Mexico.
Methods Mol Biol. 2014;1138:131-50. doi: 10.1007/978-1-4939-0348-1_9.
Dengue virus serotypes 1-4 are members of mosquito-borne flavivirus genus of Flaviviridae family that encode one long open reading frame (ORF) that is translated to a polyprotein. Both host and virally encoded proteases function in the processing of the polyprotein by co-translational and posttranslational mechanisms to yield 10 mature proteins prior to viral RNA replication. To study cis- and trans-acting factors involved in viral RNA replication, many groups [1-8] have constructed cDNAs encoding West Nile virus (WNV), DENV, or yellow fever virus reporter replicon RNAs. The replicon plasmids constructed in our laboratory for WNV [9] and the DENV4 replicon described here are arranged in the order of 5'-untranslated region (UTR), the N-terminal coding sequence of capsid (C), Renilla luciferase (Rluc) reporter gene with a translation termination codon, and an internal ribosome entry site (IRES) element from encephalomyocarditis virus (EMCV) for cap-independent translation of the downstream ORF that codes for a polyprotein precursor, CterE-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5, followed by the 3'-UTR. In the second DENV4 replicon, the Rluc gene is fused sequentially downstream to the 20 amino acid (aa) FMDV 2A protease coding sequence, neomycin resistance gene (Neo(r)), a termination codon, and the EMCV leader followed by the same polyprotein coding sequence and 3'-UTR as in the first replicon. The first replicon is useful to study by transient transfection experiments the cis-acting elements and trans-acting factors involved in viral RNA replication. The second DENV4 replicon is used to establish a stable monkey kidney (Vero) cell line by transfection of replicon RNA and selection in the presence of the G418, an analog of neomycin. This replicon is useful for screening and identifying antiviral compounds that are potential inhibitors of viral replication.
登革病毒1 - 4型是黄病毒科蚊媒黄病毒属的成员，它们编码一个长开放阅读框（ORF），该阅读框被翻译为一个多聚蛋白。宿主和病毒编码的蛋白酶通过共翻译和翻译后机制在多聚蛋白的加工过程中发挥作用，从而在病毒RNA复制之前产生10种成熟蛋白。为了研究参与病毒RNA复制的顺式和反式作用因子，许多研究小组[1 - 8]构建了编码西尼罗河病毒（WNV）、登革病毒（DENV）或黄热病病毒报告基因复制子RNA的cDNA。我们实验室构建的用于WNV的复制子质粒[9]以及这里描述的DENV4复制子，其排列顺序为5'-非翻译区（UTR）、衣壳（C）的N端编码序列、带有翻译终止密码子的海肾荧光素酶（Rluc）报告基因，以及来自脑心肌炎病毒（EMCV）的内部核糖体进入位点（IRES）元件，用于下游编码多聚蛋白前体CterE-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5的ORF的不依赖帽的翻译，随后是3'-UTR。在第二个DENV4复制子中，Rluc基因依次融合在20个氨基酸（aa）的口蹄疫病毒2A蛋白酶编码序列、新霉素抗性基因（Neo(r)）、一个终止密码子以及EMCV前导序列的下游，随后是与第一个复制子相同的多聚蛋白编码序列和3'-UTR。第一个复制子可用于通过瞬时转染实验研究参与病毒RNA复制的顺式作用元件和反式作用因子。第二个DENV4复制子用于通过转染复制子RNA并在新霉素类似物G418存在下进行筛选，建立稳定的猴肾（Vero）细胞系。这个复制子可用于筛选和鉴定作为病毒复制潜在抑制剂的抗病毒化合物。
