Genetic Structure Of Murine Retroviruses

该项目的主要目标是阐明混合逆转录病毒感染对疾病诱导的影响。最近，我们对混合逆转录病毒感染的研究主要集中在共接种共嗜性和多向性 MuLV 混合物的小鼠上。多向性 MuLV 是由亲向性 MuLV 与近交小鼠品系基因组中存在的内源包膜序列重组形成的，并表现出改变的感染宿主范围，并利用与亲向性 MuLV 使用的受体不同的细胞表面受体。在一些情况下，多向性 MuLV 直接参与发病机制，包括诱导增殖、免疫和神经系统疾病。我们观察到多向病毒在共同接种的小鼠中的感染性传播产生了深远的影响，同时非常快速地诱导了神经系统疾病，这是单独接种任何一种病毒后都没有观察到的。初步结果是在 NFS/N 小鼠与亲嗜性 MuLV 和多向性 MuLV 共同接种后观察到的，已知这两种 MuLV 在 IRW 中具有神经致病性，但在 NFS/N 小鼠中则不然。这些研究已扩展到证明其他多向性病毒可在 NFS/N 和 IRW 小鼠中诱导神经系统疾病，而此前尚未观察到这些病毒具有神经致病性。混合感染导致神经系统疾病的一个常见效应是大大增强多向病毒在中枢神经系统 (CNS) 周围组织中的传播。这种现象是由共嗜性病毒颗粒内多向性病毒基因组的假型介导的。最初在中枢神经系统中检测到的多向性 MuLV 也是假型的，但随后病毒在中枢神经系统中的快速传播似乎是由非假型的多向性病毒体进行的。这种快速传播几乎与神经系统症状的出现同时发生。在混合感染中未表现出外周复制增强的多向性病毒表现出较低程度的中枢神经系统感染，并且尚未观察到诱发神经系统疾病。总体而言，这些研究表明神经致病性可能是多向性 MuLV 的一般特性。入侵中枢神经系统需要外周复制的阈值，并且多向病毒通过多向受体结合蛋白与中枢神经系统细胞上的受体相互作用而传播可能是诱导神经病理学的必要条件。 我们研究的另一个方面涉及检查逆转录病毒感染细胞的机制。有包膜病毒的感染性进入被认为是通过两种机制之一进行的。 pH依赖性病毒通过受体介导的内吞作用进入细胞，并通过使用防止内吞途径中囊泡酸化的试剂进行短暂处理来抑制，而pH非依赖性病毒则不受此类试剂的抑制，并且被认为通过与质膜直接融合进入细胞。几乎所有逆转录病毒，包括双嗜性鼠白血病病毒 (MuLV) 和 1 型人类免疫缺陷病毒，都被归类为与 pH 无关。然而，生态性 MuLV 被认为是一种 pH 依赖性病毒。我们检查了亲嗜性和兼嗜性 MuLV 的感染性进入，发现它们同样受到阻止囊泡酸化的 NH4Cl 和巴弗洛霉素 A 的抑制。在实验过程中，这些药物仅部分抑制了这两种病毒。阻断内吞囊泡酸化的药物也能阻止囊泡运输。因此，MuLV 的部分抑制可能是该途径停滞期间病毒失活的结果。为了支持这一论点，我们发现，在用药物治疗靶细胞期间，MuLV 感染性的丧失与同一时间段内 37 摄氏度自发失活导致的活性丧失密切相关。此外，在感染持续时间非常短以尽量减少自发失活的影响的情况下，药物对感染效率没有影响。这些结果表明，亲嗜性和兼嗜性 MuLV 的感染过程都被药物短暂处理所抑制，而不是中止。进一步的实验表明，停滞发生在细胞内隔室中，因此兼嗜性和亲嗜性 MuLV 的感染过程很可能涉及内吞作用。这些研究的一个重要方面涉及对实验的解释，在这些实验中，阻断内吞酸化的药物无法抑制感染性。对药物缺乏敏感性已被视为通过与质膜直接融合而感染的证据，但这可能只是反映了治疗过程中病毒的稳定性。 HIV-1 的传染性进入就是一个典型的例子。如果 HIV-1 的感染性进入是通过内体途径而不是通过与质膜直接融合，则考虑到其半衰期比药物治疗持续时间长 6 至 10 倍，则它不会反映在使用溶酶体药物的标准测定中。 除了上述研究之外，还构建了靶向逆转录病毒载体，其将葡萄球菌蛋白A的Fc结合域整合到病毒的SU蛋白中。以前的靶向逆转录病毒已经掺入了单链抗体或结合特定细胞表面分子的其他配体。这些研究的目的是衍生出一种可以通过与不同抗体结合来靶向多种细胞类型的单一载体。构建了许多载体，其中一种在鼠类和人源细胞系中表现出抗体依赖性转导活性。与迄今为止报道的所有靶向逆转录病毒载体的情况一样，转导活性需要改变的 SU 蛋白与野生型 MuLV 包膜蛋白的共表达。感染 MuLV 的小鼠细胞表面表达高水平的 MuLV 包膜蛋白，并且由于病毒干扰而不易被相同病毒类型感染。已用针对 M-MuLV SU 蛋白的 IgG 抗体包被的 M-MuLV 感染细胞可以通过 Protein A/M-MuLV 载体成功转导，而未处理的细胞或用针对 SU 蛋白的 IgM 抗体处理的细胞则不能。这些结果表明转导途径依赖于抗体与其细胞表面配体的结合，不太可能依赖于野生型包膜蛋白与亲嗜性受体的结合。另一系列实验证明了卡波西肉瘤细胞的成功转导，该细胞响应血管内皮生长因子（VEGF）而增殖。用结合 VEGF 受体 (KDR/Flk-1) 的抗体包被细胞后，即可实现卡波西肉瘤细胞的转导。这些结果表明，Ig 结合逆转录病毒载体可以提供一种有效的多功能基因递送载体，用于靶向靶细胞表面表达的特定抗原。

A primary goal of this project is to elucidate the effect of mixed retrovirus infections on the induction of disease. Recently our studies of mixed retrovirus infection have focused on mice co-inoculated with mixtures of ecotropic and polytropic MuLVs. Polytropic MuLVs are formed by recombination of ecotropic MuLVs with endogenous envelope sequences present in the genomes of inbred mouse strains and exhibit an altered infectious host range and utilize a cell surface receptor distinct from the receptor utilized by ecotropic MuLVs. In several instances polytropic MuLVs have been directly implicated in pathogenesis, including the induction of proliferative, immunological, and neurological disorders. We have observed profound effects on the infectious spread of the polytropic virus in co-inoculated mice, concomitant with a very rapid induction of neurological disease not observed after inoculation with either virus alone. The initial results were observed after co-inoculation of NFS/N mice with an ecotropic MuLV and a polytropic MuLV known to be neuropathogenic in IRW but not NFS/N mice. These studies have been extended to demonstrate the induction of neurological disease in NFS/N and IRW mice by other polytropic viruses that had not been previously observed to be neuropathogenic. A common effect of mixed infections resulting in neurological disease is a greatly enhanced spread of the polytropic virus in tissues peripheral to the central nervous system (CNS). This phenomenon is mediated by pseudotyping of polytropic viral genomes within ecotropic virus particles. Polytropic MuLVs initially detected in the CNS are also pseudotyped, however a subsequent rapid spread of the virus in the CNS appears to proceed by polytropic virions that are not pseudotyped. This rapid spread is nearly coincident with the onset of neurological symptoms. Polytropic viruses that do not exhibit enhanced peripheral replication in mixed infections exhibit a lesser degree of CNS infection and have not been observed to induce neurological disease. Overall, these studies suggest that neuropathogenicity may be a general property of polytropic MuLVs; that a threshold of peripheral replication is required for invasion of the CNS, and that spread of the polytropic virus through interaction of the polytropic receptor-binding protein with receptors on CNS cells may be a requirement for the induction of neuropathology. Another aspect of our studies involves examination of the mechanism of infection of cells by retroviruses. Infectious entry of enveloped viruses is thought to proceed by one of two mechanisms. pH-dependent viruses enter the cells by receptor-mediated endocytosis and are inhibited by transient treatment with agents that prevent acidification of vesicles in the endocytic pathway, while pH-independent viruses are not inhibited by such agents and are thought to enter the cell by direct fusion with the plasma membrane. Nearly all retroviruses, including amphotropic murine leukemia virus (MuLV) and human immunodeficiency virus type 1, are classified as pH independent. However, ecotropic MuLV is considered to be a pH-dependent virus. We have examined the infectious entry of ecotropic and amphotropic MuLVs and found that they were equally inhibited by NH4Cl and bafilomycin A that block vescicular acidification. These agents inhibited both viruses only partially over the course of the experiments. Agents that block the acidification of endocytic vesicles also arrest vesicular trafficking. Thus, partial inhibition of the MuLVs could be the result of virus inactivation during arrest in this pathway. In support of this contention, we found that that the loss of infectivity of the MuLVs during treatment of target cells with the drugs closely corresponded to the loss of activity due to spontaneous inactivation at 37 degrees C in the same period of time. Furthermore, the drugs had no effect on the efficiency of infection under conditions in which the duration of infection was held to a very short period to minimize the effects of spontaneous inactivation. These results indicate that the infectious processes of both ecotropic and amphotropic MuLVs were arrested rather than aborted by transient treatment of the cells with the drugs. Further experiments indicated that the arrest occurred in an intracellular compartment, thus the infectious process of both the amphotropic and ecotropic MuLVs very likely involved endocytosis. An important aspect of these studies pertains to the interpretation of experiments in which agents that block endocytic acidification fail to inhibit infectivity. A lack of sensitivity to the drugs has been taken as evidence for infection via direct fusion with the plasma membrane, however it may simply reflect virus stability over the course of the treatment. A case in point may be the infectious entry of HIV-1. If the infectious entry of HIV-1 is by an endosomal route rather than by direct fusion with plasma membrane, it would not be reflected in standard assays with lysosomotropic agents, considering that its half-life is 6- to 10-fold longer than the duration of the treatment with the agents. In addition to the above studies, targeted retrovirus vectors were constructed which incorporate the Fc-binding domain of Staphylococcal Protein A into the SU protein of the virus. Previous targeted retroviruses have incorporated single chain antibodies or other ligands which bind specific cell-surface molecules. The aim of these studies is to derive a single vector that can be targeted to various cell types by binding to different antibodies. A number of vectors were constructed, one of which exhibited antibody-dependent tranducing activity in cell lines of murine and of human origin. As is the case with all targeted retrovirus vectors reported to date, transduction activity required co-expression of the altered SU protein with the wild-type MuLV envelope protein. MuLV-infected murine cells express high levels of the MuLV envelope protein on their surface and are refractory to infection by the same virus type due to viral interference. M-MuLV-infected cells which had been coated with an IgG antibody directed at the M-MuLV SU protein were successfully tranduced by a Protein A/M-MuLV vector, whereas untreated cells or cells treated with an IgM antibody to the SU protein were not. These results indicate that the route of transduction was dependent on the binding of the antibody to it's cell-surface ligand and unlikely to be dependent on binding of the wild-type envelope protein to the ecotropic receptor. Another series of experiments demonstrated the successful transduction of Kaposi sarcoma cells which proliferate in response to vascular endothelial growth factor (VEGF). Transduction of Kaposi sarcoma cells was achieved after coating the cells with an antibody that binds a receptor for VEGF (KDR/Flk-1). These results suggest that Ig-binding retrovirus vectors could provide an efficient versatile gene delivery vehicle for targeting to specific antigens expressed on the surface of target cells.