Exploitation of nucleic acid packaging signals to generate a novel influenza virus-based vector stably expressing two foreign genes
Exploitation of nucleic acid packaging signals to generate a novel influenza virus-based vector stably expressing two foreign genes
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DOI：
10.1128/jvi.77.19.10575-10583.2003
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发表时间：
2003-10-01
影响因子：
5.4
通讯作者：
Kawaoka, Y
中科院分区：
文献类型：
--
作者：
Watanabe, T;Watanabe, S;Kawaoka, Y
在病毒复制的最后一步，病毒基因组必须被整合到后代病毒粒子中，然而在包括流感病毒在内的RNA病毒中，这一过程所需的基因组区域基本上是未知的。最近，有报道称，神经氨酸酶(NA)编码区的两端对于将该vRNA片段整合到流感病毒粒子中至关重要(Y.Fujii，H.Goto，T.Watanabe，T.Yoshida，和Y.Kawaoka，Proc。娜塔莉。阿卡德。SCI。美国100：2002-2007,2003)。为了确定其病毒粒子掺入所需的血凝素(RA)vRNA中的信号，我们对该片段进行了一系列缺失构建。随后的分析表明，编码区3‘端的9个核苷酸和5’端的80个核苷酸足以有效地将病毒粒子掺入RA vRNA。通过产生一种病毒，其RA和NA vRNA编码区分别被水疱性口炎病毒糖蛋白(VSVG)和绿色荧光蛋白(GFP)取代，同时保留这些片段的病毒粒子掺入信号，评估了这些信息在流感病毒中稳定表达的有效性。尽管缺乏RA和NA蛋白，但病毒粒子表面只有VSVG的病毒仍然活着，即使在反复传代后也能在细胞内产生表达GFP的空斑，这表明两个外源基因可以稳定地整合到甲型流感病毒中。这些发现可以作为构建甲型流感病毒的模型，该病毒旨在表达和/或传递外来基因。
At the final step in viral replication, the viral genome must be incorporated into progeny virions, yet the genomic regions required for this process are largely unknown in RNA viruses, including influenza virus. Recently, it was reported that both ends of the neuraminidase (NA) coding region are critically important for incorporation of this vRNA segment into influenza virions (Y. Fujii, H. Goto, T. Watanabe, T. Yoshida, and Y. Kawaoka, Proc. Natl. Acad. Sci. USA 100:2002-2007,2003). To determine the signals in the hemagglutinin (RA) vRNA required for its virion incorporation, we made a series of deletion constructs of this segment. Subsequent analysis showed that 9 nucleotides at the 3' end of the coding region and 80 nucleotides at the 5' end are sufficient for efficient virion incorporation of the RA vRNA. The utility of this information for stable expression of foreign genes in influenza viruses was assessed by generating a virus whose RA and NA vRNA coding regions were replaced with those of vesicular stomatitis virus glycoprotein (VSVG) and green fluorescent protein (GFP), respectively, while retaining virion incorporation signals for these segments. Despite the lack of RA and NA proteins, the resultant virus, which possessed only VSVG on the virion surface, was viable and produced GFP-expressing plaques in cells even after repeated passages, demonstrating that two foreign genes can be incorporated and maintained stably in influenza A virus. These findings could serve as a model for the construction of influenza A viruses designed to express and/or deliver foreign genes.