Autographa californica nucleopolyhedrovirus infection results in Sf9 cell cycle arrest at G2/M phase
Autographa californica nucleopolyhedrovirus infection results in Sf9 cell cycle arrest at G2/M phase
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DOI：
10.1006/viro.1998.9097
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发表时间：
1998-04-25
期刊：
影响因子：
3.7
通讯作者：
Summers, MD
中科院分区：
文献类型：
--
作者：
Braunagel, SC;Parr, R;Summers, MD
杆状病毒感染导致宿主细胞核质内膜结构的诱导。这些膜的来源尚不清楚；但是，使用细胞膜的正常动力学和核膜作为模型，细胞周期可能在这些核内膜的形成过程中发挥作用。因此，本研究的目的之一是探讨杆状病毒感染对Sf9宿主细胞细胞周期的影响。由于关于昆虫细胞周期的数据很少，因此第一个任务是确定Sf9细胞周期动力学。悬浮培养的SF9细胞周期分布均匀(G(1)期细胞占29%，S期细胞占33%，G(2)/M期细胞占36%)，G(1)期和S期各约为6h，G(2)/M期合计约为8h。在病毒诱导的G(2)/M期停滞的同时，检测到高水平的CDC2相关组蛋白H1激酶活性和细胞周期蛋白B。到24小时P.I.不再检测到细胞周期蛋白B；然而，cdc2相关的组蛋白H1激酶活性在整个感染过程中保持不变。这些数据提示，在感染早期，细胞周期蛋白B/CDC2复合体可能被用来调节G(2)期向M期的转变，但长时间的停滞可能是由于AcMNPV编码的一种蛋白(S)所致。DNA杂交分析表明，病毒DNA复制速率最高出现在G(2)/M期停止前。我们注意到，病毒DNA复制仍然发生在感染后期，大多数细胞被阻止在G(2)/M期。由于细胞DNA复制通常不发生在G(2)期或M期，因此设计了实验，以确定即使宿主细胞DNA复制被阻止，病毒DNA复制是否也可以发生。用5-氟-2‘-脱氧尿嘧啶核苷(FdUrd)处理Sf9细胞，使其滞留在G(1)/S期交界处，然后感染AcMNPV。在被阻断的、受感染的细胞中，检测到病毒DNA复制；然而，细胞DNA保持在稳定水平。这些结果表明，细胞DNA复制不是病毒DNA复制所必需的，并且FdUrd处理对病毒DNA复制没有明显的抑制作用。当宿主细胞被冷冻在G(1)/S期时，检测到病毒DNA的复制是令人惊讶的。我们想要确定病毒感染是否进展到后代病毒产生的阶段。我们的数据表明，在冷冻的感染细胞中产生了子代萌发病毒(BV)和病毒诱导的核内微泡；然而，核内微泡具有不寻常的结构。在冻存的感染细胞的细胞核中可见极少量的包膜核衣壳，Western印迹分析未检测到包膜衍生的病毒囊膜蛋白ODV-E66和ODV-EC27。由于细胞在本实验期间一直处于G(1)期和S期的交界处，核内被包膜的微囊泡数量的减少可能是由于几个因素，包括晚期基因表达水平的降低，异常的微囊泡，或者感染细胞需要G(2)/M期才能有效地产生和成熟核内微囊泡。这些数据表明，AcMNPV感染导致细胞周期停滞在G(2)/M期，这种停滞可能是由于具有CDC2相关激酶活性的病毒编码蛋白(S)所致。我们注意到，即使当感染细胞被药物处理(FdUrd)滞留在G(1)和S期的交界处时，也会产生子代BV，这表明BV的成熟可能不需要G(2)/M期的阻止。在FDUrd滞留的细胞中也产生了核内微泡；然而，它们具有不寻常的外观，这可能是G(2)/M滞留对于ODV的最佳成熟和组装是重要的。(C)1998年学术出版社。
Baculovirus infection results in the induction of membrane structures within the nucleoplasm of the host cells. The source of these membranes is unclear; however, using the normal dynamics of cellular membranes and the nuclear envelope as a model, it is possible that the cell cycle might play a role in the regulation of formation of these intranuclear membranes. Therefore, one goal of this study was to investigate the effect of baculovirus infection on the cell cycle of Sf9 host cells. Since few data are available on the cell cycle of insect cells, the first task was to define Sf9 cell cycle kinetics. The cell cycle phase distribution of Sf9 cells grown in suspension culture was determined to be evenly distributed (29% of the cells in G(1), 33% in S, and 36% in G(2)/M phase), with the duration of G(1) and S phases both being about 6 h and the combined duration of G(2)/M phase being about 8 h. When Sf9 cells were infected with AcMNPV (Autographa californica nuclear polyhedrosis virus), approximately 84% of the cells were arrested in G(2)/M phase by 18-24 h p.i. Concomitant with the viral-induced arrest in G(2)/M phase, high levels of both cdc2-associated histone H1 kinase activity and cyclin B protein were detected. By 24 h p.i. cyclin B was no longer detected; however, cdc2-associated histone H1 kinase activity remained throughout the infection. These data suggested that early in infection, cyclin B/cdc2 complex may be used to regulate the transition from G(2) to M phase, but prolonged arrest may be due to a protein(s) encoded by AcMNPV. DNA hybridization analysis showed that the maximal rate of viral DNA replication occurred before G(2)/M arrest. We noted that viral DNA replication still occurred late in infection, when the majority of the cells were arrested in G(2)/M phase. Since cellular DNA replication normally does not occur during G(2) or M phase, experiments were designed to determine if viral DNA replication could occur even when host cell DNA replication was arrested. Sf9 cells were arrested and "frozen" at the boundary of G(1)/S phase using 5-fluoro-2'deoxyuridine (FdUrd) treatment and then infected with AcMNPV. In the blocked, infected cells, viral DNA replication was detected; however, cellular DNA remained at steady-state levels. These results suggested that cellular DNA replication was not necessary for viral DNA replication and show that viral DNA replication was not significantly inhibited by FdUrd treatment. It was a surprise to detect viral DNA replication when the host cells were "frozen" at G(1)/S phase. We wanted to determine if the viral infection was progressing to the stage of progeny virus production. Our data showed that progeny budded virus (BV) and virus-induced intranuclear microvesicles were produced in the frozen, infected cells; however, the intranuclear microvesicles had an unusual structure. They were irregular in shape and thickened compared to those observed in a normal infection.Very few enveloped nucleocapsids were visible in the nucleus of the frozen, infected cells and the occluded-derived virus envelope proteins, ODV-E66 and ODV-EC27, were not detected by Western blot analyses, Since the cells were sustained at the boundary of G(1) and S phases for the duration of this experiment, the decreased amount of enveloped ODV in the nucleus could be due to several factors, including decreased levels of proteins expressed from late genes, aberrant microvesicles, or the necessity of G(2)/M phasing of the infected cell for efficient production and maturation of intranuclear microvesicles. These data indicate that AcMNPV infection results in cell cycle arrest in G(2)/M phase and this arrest may be due to a viral-encoded protein(s) that has cdc2-associated kinase activity. We note that progeny BV are produced even when infected cells are arrested at the border of G(1) and S phases by drug treatment (FdUrd), indicating that arrest in G(2)/M may not be required for the maturation of BV. Intranuclear microvesicles are also produced in FDUrd-arrested cells; however, they have an unusual appearance and it may be that the G(2)/M arrest is important for optimal maturation and assembly of ODV. (C) 1998 Academic Press.