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Alphavirus nsP2 interacts with host pathways for viral minus-strand synthesis and replication complex stability.

机译:甲病毒nsP2与宿主途径相互作用,以减少病毒的负链合成和复制复合物的稳定性。

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摘要

Alphavirus nsP2 has been described to play a critical role in regulating host responses. To investigate the function of P23 proteins in host-cell interaction, SIN2V, a Sindbis virus (SIN) mutant unable to cleave P23 to nsP2 and nsP3, was constructed and found to bear two phenotypes: continuous minus strand synthesis and unstable RTC activity (Gorchakov et al., 2008). This finding allowed me to test the hypothesis that only mature nsPs, not P23 can activate and control host responses. SIN2V was employed to probe the fate of SIN2V minus strands, the conditions needed to rescue its phenotypes and the mechanism of RTC inactivation. It was found that unlike wt SIN, SIN2V RTC containing P23 was not able to switch between 26S mRNA synthesis and 49S plus strand synthesis and did not alter the ratios of 49S:26S plus-strand RNA during infection. Ethidium bromide staining of the RTC double-stranded (RF) RNA cores and 3H-uridine radiolabeling of the RFs were used to quantify RTC levels and replication activity. It was shown that wt SIN accumulated ∼ 27,000 RFs per infected BHK21 cell. SIN2V RFs were accumulated to 112% of wt SIN levels although SIN2V theoretically produced ∼260% of wildtype minus strand amounts. This meant that those RTC complexes in excess of wt levels must have been degraded. The SIN2V minus strands made early or late functioned as authentic templates and their template activity was similarly susceptible to inactivation. Alphavirus nsP proteins introduced through superinfection with Semliki Forest virus rescued the P23 defective function needed to induce minus strand synthesis cessation. The loss of SIN2V RTC activity was not due to the physical degradation, but rather to the proteasome action. Inhibition of unbiquitin activating enzyme E1 activity also rescued SIN2V RTC activity, suggesting that the mature nsP2 and possibly nsP3 proteins block this ubiquitin-proteasome pathway. Taken together, these findings demonstrated that mature nsP2 and possibly nsP3 but not P23 activates a host response to shut off minus strand synthesis and blocks a host response to protect RTC activity.
机译:阿尔法病毒nsP2已被描述在调节宿主反应中起关键作用。为了研究P23蛋白在宿主细胞相互作用中的功能,构建了SIN2V(一种无法将P23裂解为nsP2和nsP3的Sindbis病毒(SIN)突变体),发现其具有两种表型:连续负链合成和不稳定的RTC活性(Gorchakov等人,2008)。这一发现使我能够检验以下假设:只有成熟的nsP,而不是P23才能激活和控制主机响应。 SIN2V被用来探测SIN2V负链的命运,挽救其表型所需的条件以及RTC失活的机制。发现与wt SIN不​​同,含有P23的SIN2V RTC不能在26S mRNA合成和49S加链合成之间切换,并且在感染过程中不会改变49S∶26S加链RNA的比例。 RTC双链(RF)RNA核心的溴乙锭染色和RF的3H-尿苷放射性标记用于定量RTC水平和复制活性。结果表明,wt SIN每个感染的BHK21细胞积累了约27,000个RF。尽管理论上SIN2V产生的野生型负链数量约为260%,但SIN2V的RF积累到wt SIN水平的112%。这意味着那些超过wt水平的RTC复合物必须已经降解。早晚制造的SIN2V负链可作为真实模板,它们的模板活性同样容易失活。通过用Semliki Forest病毒进行超级感染而引入的Alphavirus nsP蛋白挽救了诱导负链合成停止所需的P23缺陷功能。 SIN2V RTC活性的丧失不是由于物理降解,而是由于蛋白酶体的作用。抑制泛素激活酶E1的活性也挽救了SIN2V RTC的活性,表明成熟的nsP2和可能的nsP3蛋白阻止了这种泛素-蛋白酶体途径。综上所述,这些发现表明,成熟的nsP2和可能的nsP3而不是P23激活宿主反应以关闭负链合成,并阻断宿主反应以保护RTC活性。

著录项

  • 作者

    Mai, Junbo.;

  • 作者单位

    The University of Toledo.;

  • 授予单位 The University of Toledo.;
  • 学科 Biology Virology.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 192 p.
  • 总页数 192
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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