IDENTIFYING TARGETS FOR THERAPEUTIC INTERVENTIONS USING PROTEOMIC TECHNOLOGY

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 这是太平洋西北国家实验室的一项研究，旨在表征已知致病性的定义正痘病毒颗粒的综合蛋白质组。我们关注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; Zaire株）。在这个目标中，细胞外的蛋白质组分 对致病性MPV进行了有囊膜病毒（EEV）和细胞内成熟病毒（IMV）的检测，并与VV进行了比较。在评估和比较了病毒颗粒之后，我们将注意力转移到感染后宿主细胞的改变上。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，目的是鉴定响应于致病性正痘病毒感染而合成的新型病毒和细胞蛋白。从这些研究中，我们发现，被报道为从VV感染的细胞中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，我们还鉴定了另一种病毒编码的蛋白质，其在培养基中大量分泌，没有已知的报道功能。这一发现并不令人惊讶，因为有许多注释的开放阅读框架没有已知的同源物。因此，在感染细胞的上清液中检测到这种病毒编码的蛋白质表明该蛋白质具有尚未鉴定的功能，其可能会影响周围细胞的功能。此外，我们开始对来自实验感染MPV的动物的支气管肺泡样本进行蛋白质组学分析，以鉴定促进MPV相关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.