IDENTIFYING TARGETS FOR THERAPEUTIC INTERVENTIONS USING PROTEOMIC TECHNOLOGY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这是与太平洋西北国家实验室签订的一项分包合同，以表征已知致病性的已定义正痘病毒颗粒的综合蛋白质组。我们重点研究的正痘病毒是野生型非致病性牛痘病毒(VV；Western Reserve株)和致病性人猴痘病毒(MPV；扎伊尔株)。在这个目标中，细胞外的蛋白质成分 用病毒囊膜病毒(EEV)和细胞内成熟病毒(IMV)检测MPV，并与VV进行比较。在评估和比较了病毒颗粒后，我们将重点转移到感染后宿主细胞的变化上。在这里，我们比较了VV和MPV感染的HeLa细胞的细胞内和细胞外蛋白质组，目的是鉴定新的病毒和细胞蛋白，以响应致病的正痘病毒感染。从这些研究中，我们发现据报道从VV感染的细胞分泌的特定病毒编码蛋白也从MPV感染的细胞中分泌。有趣的是，我们还发现了另一种病毒编码的蛋白，该蛋白在媒体中大量分泌，其功能未知。这一发现并不令人惊讶，因为有许多带注释的开放阅读框架，但没有已知的同源基因。因此，在感染细胞的上清中检测到这种病毒编码蛋白表明，该蛋白具有尚未确定的功能，可能会影响周围细胞的功能。此外，我们对实验感染MPV的动物的细支气管肺泡样本进行了蛋白质组学分析，以确定促进MPV相关性肺炎的病毒编码蛋白。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This was a subcontract with Pacific Northwest National Laboratories to characterize the comprehensive proteome of defined orthopoxvirus particles of known pathogenicity. The orthopoxviruses that we focused on were wild-type nonpathogenic vaccinia virus (VV; Western Reserve strain) and pathogenic human monkeypox virus (MPV; Zaire strain). In this objective, the protein components of the extracellular enveloped virus (EEV) and intracellular mature virus (IMV) were determined for pathogenic MPV, and compared to that of VV. Having evaluated and compared virus particles, we shifted our focus towards alterations to the host cell following infection. Here we compared the intracellular and extracellular proteome of VV and MPV infected HeLa cells with the goal to identify novel viral and cellular proteins that are synthesized in response to pathogenic orthopoxvirus infection. From these studies, we found that specific viral encoded proteins reported to be secreted from VV-infected cells were also secreted from MPV-infected cells. Interestingly, we also identified another viral encoded protein that has no known reported function abundantly secreted in the media. This finding is not surprising, as there are a number of annotated open reading frames with no known homologue. As such, the detection of this viral encoded protein in the supernatant of infected cells suggest the protein has a yet to be identified function that can potentially impact the function of surrounding cells. Additionally, we initiated proteomic analysis on bronchio-alveolar samples derived from animals experimentally infected with MPV to identify viral encoded proteins that facilitate MPV-associated pneumonia.