Second-strand genome conversion of adeno-associated virus type 2 (AAV-2) and AAV-5 is not rate limiting following apical infection of polarized human airway epithelia
Second-strand genome conversion of adeno-associated virus type 2 (AAV-2) and AAV-5 is not rate limiting following apical infection of polarized human airway epithelia
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DOI：
10.1128/jvi.77.13.7361-7366.2003
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发表时间：
2003-07-01
影响因子：
5.4
通讯作者：
Engelhardt, JF
中科院分区：
文献类型：
--
作者：
Ding, W;Yan, ZY;Engelhardt, JF
已知重组腺相关病毒5型（MAV-5）通过顶端感染有效地感染气道上皮。相比之下，rAAV-2已显示在从顶端表面转导气道上皮细胞方面固有地无效。然而，三肽蛋白酶体抑制剂（如LLnL）可以通过调节病毒的细胞内运输和加工来显著增强rAAV-2从人极化气道上皮细胞的顶端表面的转导。为了进一步研究rAAV-2和rAAV-5之间的潜在差异，这可能解释它们改变从顶膜分离气道上皮细胞的能力，我们研究了这两种rAAV血清型的泛素/蛋白酶体途径的功能参与和第二链合成的限速方面。为此，我们进行了研究，通过使用荧光素酶和增强型绿色荧光蛋白（EGFP）报告载体，比较LLnL改变rAAV-2和rAAV-2/5转导效率的程度。我们的结果表明，LLnL的病毒感染时的共同管理显着增强rAAV-2/5和rAAV-2从气道上皮细胞的顶端表面的转导。虽然rAAV-2/5在从顶膜转导上皮细胞方面稍微更有效，但在蛋白酶体抑制剂存在下，rAAV-2转导上级rAAV-2/5。有趣的是，两种血清型的基底外侧膜进入途径均未受到LLnL添加的显著影响，这表明顶端和基底外侧感染途径具有rAAV-2和rAAV-5的独特细胞内加工途径。比较短自身互补（scAAV）与全长常规AAV EGFP载体的转导的研究表明，rAAV基因组的第二链合成对于任一血清型都不是速率限制性的，或者在极化气道上皮细胞的顶端感染后被蛋白酶体抑制剂改变。这些发现表明，rAAV-2和rAAV-5共享类似的细胞内病毒加工障碍，涉及泛素/蛋白酶体系统，但似乎不涉及第二链合成。
Recombinant adeno-associated virus type 5 (MAV-5) is known to efficiently transduce airway epithelia via apical infection. In contrast, rAAV-2 has been shown to be inherently ineffective at transducing airway epithelia from the apical surface. However, tripeptide proteasome inhibitors (such as LLnL) can dramatically enhance rAAV-2 transduction from the apical surface of human polarized airway epithelia by modulating the intracellular trafficking and processing of the virus. To further investigate potential differences, between rAAV-2 and rAAV-5 that might explain their altered ability to transduce airway epithelia from the apical membrane, we examined the functional involvement of the ubiquitin/proteasome pathway and rate-limiting aspects of second-strand synthesis for these two rAAV serotypes. To this end, we conducted studies to compare the extent to which LLnL alters transduction efficiencies with both rAAV-2 and rAAV-2/5 by using luciferase and enhanced green fluorescent protein (EGFP) reporter vectors. Our results demonstrate that the coadministration of LLnL at the time of viral infection significantly enhanced transduction of both rAAV-2/5 and rAAV-2 from the apical surface of airway epithelia. Although rAAV-2/5 was slightly more effective at transducing epithelia from the apical membrane, rAAV-2 transduction was superior to that of rAAV-2/5 in the presence of proteasome inhibitors. Interestingly, the basolateral membrane entry pathways for both serotypes were not significantly affected by the addition of LLnL, which suggests that apical and basolateral infectious pathways possess distinctive intracellular processing pathways for both rAAV-2 and rAAV-5. Studies comparing the transduction of short self-complementary (scAAV) to full-length conventional AAV EGFP vectors suggested that second-strand synthesis of rAAV genomes was not rate limiting for either serotype or altered by proteasome inhibitors following apical infection of polarized airway epithelia. These findings suggest that both rAAV-2 and rAAV-5 share similar intracellular viral processing barriers that involve the ubiquitin/proteasome system, but do not appear to involve second-strand synthesis.