Early events of in vitro KSHV infection

描述(由申请人提供)：在我们对动物病毒如何与细胞表面受体相互作用促进生产性感染的理解上存在着根本的差距。病毒，如疱疹病毒，可在40℃以不依赖于能量的方式与细胞表面结合。相反，进入细胞并随后将衣壳转移到细胞核附近是一种活跃的、依赖于能量的现象，因此必须需要宿主细胞信号通路。我们的中心假设是，KSHV已经进化到利用它与受体的相互作用来操纵宿主先前存在的信号级联，从而介导靶细胞的进入和感染。我们正在进行的研究支持这一假设。我们发现，在内皮细胞(HMVEC-d)感染过程中，KSHV的初始附着通过硫酸乙酰肝素(HS)发生，随后与整合素(？3？1、？V？3和？V？5)和XCT分子发生短暂的相互作用。KSHV结合导致FAK、Src、PI3-K、Rho-GTP酶、Dia-2、Ezrin、PKC-β、ERK1/2和NF-βB信号分子的激活。这些分子在KSHV进入(FAK、Src、PI3-K、Rho-GTP酶)、微管(MT)乙酰化(RhoA-GTP酶)以及病毒基因表达(ERK1/2和NF-？B)中发挥作用。虽然脂筏(LR)的破坏导致了Src的激活和病毒侵入的增加，但我们观察到病毒基因表达、PI3K、RhoA、Dia-2和NF？B的激活、MT乙酰化和病毒DNA的核递送被抑制，这表明LRs在进入细胞和调节选定的信号分子方面起作用。我们还证明了c-Cbl在KSHV巨噬细胞吞噬中起作用。为了进一步验证我们的假设，我们制定了三个相互关联的、有针对性的主要目标，这三个目标将破译c-Cbl促进KSHV进入的机制，主要接头分子在KSHV感染中的作用，以及c-Cbl如何决定含有KSHV的大管胞的命运。这些研究具有重要意义，因为对KSHV感染早期事件的全面了解将为阻断KSHV和TE相关疾病启动靶细胞感染提供新的靶点。

DESCRIPTION (provided by applicant): There is a fundamental gap in our understanding of how animal virus interactions with cell surface receptors facilitate a productive infection. Bindin of viruses, such as herpesviruses, to the cell surface can occur at 40C in an energy independent manner. In contrast, entry into cells and subsequent transfer of capsids to the vicinity of the nucleus are an active, energy dependent phenomenon, and thus must require host-cell signaling pathways. Our central hypothesis is that KSHV has evolved to utilize its interactions with receptors to manipulate the host's pre-existing signal cascades to mediate entry and infection of target cells. Our ongoing studies support this hypothesis. We have shown that during infection of endothelial (HMVEC-d) cells, the initial attachment of KSHV occurs via heparan sulfate (HS) followed by a temporal interaction with integrins (?3?1,??V??3, and ?V??5) and xCT molecules. KSHV binding resulted in the activation of FAK, Src, PI3-K, Rho-GTPases, Dia-2, Ezrin, PKC-?, ERK1/2 and NF-?B signal molecules. These molecules play roles in KSHV entry (FAK, Src, PI3-K, Rho-GTPases), acetylation of microtubules (MT) (RhoA-GTPase) that facilitates the transport of KSHV capsid toward the nucleus via dynein motors, and viral gene expression (ERK1/2 and NF-?B). Although lipid raft (LR) disruption resulted in increased Src activation and virus entry, we observed the inhibition of viral gene expression, PI3K, RhoA, Dia-2 and NF?B activation, MT acetylation and nuclear delivery of viral DNA which suggested that LRs play roles in entry in cells and modulates selected signal molecules. We have also demonstrated that c-Cbl play a role in KSHV macropinocytosis. To further test our hypothesis, we have formulated three major interlinked, focused specific aims which will decipher the mechanisms by which c-Cbl facilitate KSHV entry, the role of major adaptor molecules in KSHV infection and decipher how c-Cbl dictates the fate of macropinosomes containing KSHV. These studies are significant since such comprehensive understanding of early events of KSHV infection will provide novel targets to block the initiation of target cell infection by KSHV and te associated diseases.