Evidence for segment-nonspecific packaging of the influenza A virus genome
Evidence for segment-nonspecific packaging of the influenza A virus genome
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DOI：
10.1128/jvi.76.14.7133-7139.2002
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发表时间：
2002-07-01
影响因子：
5.4
通讯作者：
Parslow, TG
中科院分区：
文献类型：
--
作者：
Bancroft, CT;Parslow, TG
甲型流感病毒基因组由8个负链RNA片段（称为vRNAs）组成，所有这些片段都必须被包装才能产生有感染性的病毒粒子。然而，目前尚不清楚单个vRNAs是特异性包装还是随机包装，而且病毒粒子的总vRNA容纳量也不清楚。我们构建了病毒核蛋白（NP）、神经氨酸酶（NA）和非结构（NS）vRNAs的修饰形式，它们编码绿色或黄色荧光蛋白，并利用基于质粒的甲型流感病毒组装系统研究了这些蛋白被包装的效率。通过在流式细胞仪的单轮感染性测定中对荧光标记的转导进行评分，对包装进行了精确和定量的评估。我们发现，在病毒粒子有限的条件下，成对的带有不同标记的vRNAs会竞争包装，但这种竞争是非特异性的。代表不同vRNAs的报告基因并非如特异性包装所预期的那样累加包装，而是似乎在病毒粒子中竞争一个共同的位置。此外，发现3% - 5%具有转导能力的病毒会包含两个不同的报告基因，无论这些报告基因代表相同还是不同的vRNAs——这一发现与随机包装相符，而与特异性包装不符。概率估计表明，为了仅靠偶然达到这种双重转导水平，每个甲型流感病毒粒子必须平均包装9 - 11个vRNAs。
The influenza A virus genome is composed of eight negative-sense RNA segments (called vRNAs), all of which must be packaged to produce an infectious virion. It is not clear whether individual vRNAs are packaged specifically or at random, however, and the total vRNA capacity of the virion is unknown. We have created modified forms of the viral nucleoprotein (NP), neuraminidase (NA), and nonstructural (NS) vRNAs that encode green or yellow fluorescent proteins and studied the efficiency with which these are packaged by using a plasmid-based influenza A virus assembly system. Packaging was assessed precisely and quantitatively by scoring transduction of the fluorescent markers in a single-round infectivity assay with a How cytometer. We found that, under conditions in which virions are limiting, pairs of alternatively tagged vRNAs compete for packaging but do so in a nonspecific manner. Reporters representing different vRNAs were not packaged additively, as would be expected under specific packaging, but instead appeared to compete for a common niche in the virion. Moreover, 3 to 5% of transduction-competent viruses were found to incorporate two alternative reporters, regardless of whether those reporters represented the same or different vRNAs - a finding compatible with random, but not with specific, packaging. Probabilistic estimates suggest that in order to achieve this level of dual transduction by chance alone, each influenza A virus virion must package an average of 9 to 11 vRNAs.