Gammaretroviruses: Vectors and Viruses

我们的实验室研究伽马逆转录病毒。这些逆转录病毒是人类病原体人类免疫缺陷病毒的更简单的近亲--它们在遗传结构和复制周期方面不那么复杂。对伽玛逆转录病毒的研究对生物制品的调控非常重要，原因有几个：它们被用来创建用于基因治疗临床试验的复制缺陷载体，它们是几种生物制品中的潜在污染物，它们很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)的研究上，PERV是在所有猪的基因组中发现的一种遗传遗传元件。我们发现，传染性病毒可以从猪血浆中分离出来，也可以从活化的猪血细胞中分离出来。我们开发了用于检测PERV的细胞培养试验，以及在细胞培养、动物和人类组织样本中检测病毒核酸的灵敏和定量方法。最近，我们一直在分析传染性的病毒和细胞决定因素，以便更好地了解病毒可能发生复制的条件。 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埃博拉病毒包膜糖蛋白的进入决定因素。 生物制品中的伽马逆转录病毒。使用猪组织或细胞的临床试验正在进行中。由于所有猪的基因组中都携带猪内源性逆转录病毒(PERV)，这些异种移植程序存在暴露和传播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。LTrs位于病毒DNA的两端，携带顺式作用的转录调控元件。在本研究中，我们能够从功能上确定PERV LTR中的几个结构元件：1)增强子重复数影响人293细胞的表达水平，但不影响其他细胞系的表达水平；2)假定的“Ataa”框的缺失取消了所有的表达活性；以及3)R和U5的3‘半部分中的一个元件抑制转录活性。此外，我们还完成了一项旨在评估豚鼠作为动物模型在体内研究PERV复制的实用性的研究。我们将豚鼠暴露在三种不同的PERV条件下：1)腹腔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；3)用完全弗氏佐剂复合PERV预处理。在所有病例中，我们只观察到有限的病毒感染证据，在实验期间，病毒感染要么保持在低水平，要么是短暂的。这些结果表明，豚鼠不能提供一个合适的模型来研究PERV。 识别进入细胞所需的丝状病毒糖蛋白上的关键氨基酸残基。引起丝状病毒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突变技术，在编码丝状病毒马尔堡株和埃博拉株囊膜糖蛋白GP1的cDNAs中引入突变。通过瞬时转染293T细胞，获得含有对照或突变丝状病毒糖蛋白的逆转录病毒假型，以研究每种突变对病毒趋向性和进入的影响。这项研究的目的是确定丝状病毒GP上的一个表位，该表位是促进病毒进入所必需的。鉴定这样的结构域可能会允许随后合理设计可以抑制丝状病毒感染的试剂。到目前为止，分析已经确定了埃博拉病毒GP上似乎对传染性至关重要的一个区域，因为到目前为止在这个领域引入的所有突变都使病毒不具传染性。 本项目包含2002财年项目1Z01BM002004-09和1Z01BM002007-01。

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.