Gammaretroviruses: Vectors and Viruses

我们的实验室研究γ逆转录病毒。 这些逆转录病毒是人类病原体人类免疫缺陷病毒的简单表亲-它们的遗传结构和复制周期不那么复杂。 γ逆转录病毒的研究对于生物制剂的监管非常重要，原因如下：它们已被用于创建基因治疗临床试验的复制缺陷型载体，它们是几种生物制品中的潜在污染物，它们可能存在于异种移植产品中，并有可能传播给这些产品的接受者。 我们的研究集中在猪内源性逆转录病毒（PERV）的研究，这是一种在所有猪的基因组中发现的遗传因子。 我们已经发现感染性病毒可以从猪血浆中分离，也可以从活化的猪血细胞中分离。 我们已经开发了用于检测PERV的细胞培养测定法，以及用于检测细胞培养物、动物和人体组织样品中的病毒核酸的灵敏和定量方法。 最近，我们一直在分析感染性的病毒和细胞决定因素，以更好地了解病毒复制可能发生的条件。 最近，我们在逆转录病毒方面的专业知识已经扩展到使用逆转录病毒载体技术来研究进入埃博拉病毒包膜糖蛋白的决定因素。 生物制品中的γ逆转录病毒。使用猪组织或细胞的临床试验正在进行中。由于所有的猪在其基因组中携带猪内源性逆转录病毒（PERV），这些异种移植程序具有暴露于PERV和传播PERV的风险。今年，我们分析了PERV的长末端重复序列（LTR）的功能元件。 LTR存在于病毒DNA的每一端，并携带顺式作用转录调控元件。 在本研究中，我们能够在功能上鉴定PERV LTR中的几个结构元件：1）增强子重复序列的数量影响人293细胞中的表达水平，但不影响所检查的其他细胞系; 2）缺失推定的“ATAA”盒消除了所有表达活性;和3）在R和U 5的3'半中发现的元件抑制转录活性。 此外，我们完成了一项旨在评估豚鼠作为研究PERV体内复制的动物模型的实用性的研究。 我们在三种不同条件下将豚鼠暴露于PERV：1）腹膜内注射无细胞或细胞相关的PERV; 2）用烯丙醇预处理以诱导肝细胞增殖;和3）用完全弗氏佐剂复合的PERV预处理。 在所有情况下，我们只观察到有限的病毒感染证据，这些病毒感染在实验期间保持在低水平，或者是短暂的。 这些结果表明，豚鼠不会提供研究PERV的合适模型。 丝状病毒糖蛋白进入细胞所需的关键氨基酸残基的鉴定。已显示负责起始丝状病毒的病毒感染的病毒蛋白是糖蛋白（GP）。 我们正在使用丙氨酸扫描诱变在编码马尔堡和埃博拉丝状病毒株的包膜糖蛋白GP 1的cDNA中引入突变。 携带对照或突变丝状病毒糖蛋白（GP）的逆转录病毒假型通过瞬时转染293 T细胞来获得，以研究每种突变对病毒嗜性和进入的影响。 本研究的目的是鉴定丝状病毒GP上促进病毒进入所必需的表位。 然后，这种结构域的鉴定可以允许随后合理设计可以抑制丝状病毒感染的试剂。 迄今为止的分析已经确定了埃博拉病毒GP上的一个区域，该区域似乎对感染性至关重要，因为迄今为止在该领域引入的所有突变都使病毒不具有感染性。 本项目包含2002财年项目1 Z 01 BM 002004 -09和1 Z 01 BM 002007 -01。

Our laboratory studies gammaretroviruses. These retroviruses are the simpler cousins of the human pathogen human immunodeficiency virus - they are less complex in their genetic structure and replication cycle. Study of gammaretroviruses is important to the regulation of biologics for several reasons: they have been used to create replication-defective vectors for gene therapy clinical trials, they are potential contaminants in several biological products, and they are likely to be present in xenotransplantation products, with the potential for transmission to recipients of these products. Our research has focused on the study of porcine endogenous retroviruses (PERV), an inherited genetic element found in the genome of all pigs. We have found that infectious virus can be isolated from pig plasma, as well as from activated pig blood cells. We have developed cell culture assays for detection of PERV, as well as sensitive and quantitative methods for detection of viral nucleic acids in cell culture, and animal and human tissue samples. Recently, we have been analyzing the viral and cellular determinants of infectivity in order to understand better the conditions where viral replication are likely to occur. More recently, our expertise with retroviruses has been extended to use the retroviral vector technology to study the determinants of entry on the envelope glycoprotein of Ebola virus. Gammaretroviruses in Biologics. Clinical trials using porcine tissues or cells are underway. Since all pigs carry in their genome porcine endogenous retroviruses (PERV), these xenotransplantation procedures carry the risk of exposure to and transmission of PERV. This year, we analyzed the functional elements of the long terminal repeats (LTR) of PERV. The LTRs are found at each end of the viral DNA and carry the cis-acting transcriptional regulatory elements. We were able to functionally identify several structural elements in the PERV LTR in this study: 1) the number of enhancer repeats influenced expression level in human 293 cells, but not other cell lines examined; 2) deletion of the putative "ATAA" box abrogates all expression activity; and 3) an element found in the 3' half of R and U5 suppresses transcriptional activity. In addition, we completed a study aimed toward evaluating the utility of the guinea pig as an animal model to study PERV replication in vivo. We exposed guinea pigs to PERV under three different conditions: 1) intra-peritoneal injection of cell-free or cell-associated PERV; 2) pre-treatment with allyl alcohol to induce hepatocyte proliferation; and 3) pre-treatment with Complete Freund's Adjuvant-complexed PERV. In all cases, we observed only limited evidence for viral infection which remained either at low levels for the duration of the experiment, or was transient. These results demonstrate that the guinea pig will not provide a suitable model to study PERV. Identification of Critical Amino Acid Residues on Filovirus Glycoproteins Required for Cell Entry. The viral protein responsible for initiating viral infection of filoviruses has been shown to be the glycoprotein (GP). We are using alanine-scanning mutagenesis to introduce mutations in cDNA encoding the envelope glycoprotein, GP1, for both the Marburg and Ebola strains of filovirus. Retroviral pseudotypes bearing control or mutated filovirus glycoproteins (GPs) are derived by transient transfection of 293T cells to study the effect of each mutation on viral tropism and entry. The goal of this study is to identify an epitope on the filovirus GP that is necessary to facilitate viral entry. Identification of such a domain may then allow for subsequent rational design of agents that may inhibit filovirus infection. Analysis to date has identified one region on Ebola virus GP that appears to be critical for infectivity, since all mutations introduced thus far in this domain render virus non-infectious. This project incorporates FY2002 projects 1Z01BM002004-09 and 1Z01BM002007-01.