Genetic Structure Of Murine Retroviruses

该项目的一个主要目标是阐明混合逆转录病毒感染对诱导疾病的影响。近年来，我们对混合逆转录病毒感染的研究主要集中在共接种嗜性和多嗜性混合mulv的小鼠身上。多嗜性mulv是由亲生态mulv与近交系小鼠基因组中存在的内源性包膜序列重组而成，表现出改变的感染宿主范围，并利用与亲生态mulv不同的细胞表面受体。在一些情况下，多性mulv直接涉及发病机制，包括诱导增殖性、免疫和神经系统疾病。我们已经观察到，在共接种的小鼠中，对多性病毒的传染性传播产生了深远的影响，同时伴随着非常迅速的神经系统疾病的诱导，而单独接种任何一种病毒都没有观察到。最初的结果是在NFS/N小鼠与一种生态性MuLV和一种已知在IRW中具有神经致病性的多性MuLV共同接种后观察到的，而非NFS/N小鼠。这些研究已经扩展到证明NFS/N和IRW小鼠的神经系统疾病是由以前未观察到的神经致病性的其他多性病毒诱导的。混合感染导致神经系统疾病的一个常见影响是多性病毒在中枢神经系统（CNS）外周组织中的传播大大增强。这种现象是由嗜生态病毒颗粒内多性病毒基因组的假分型介导的。最初在中枢神经系统中检测到的多性mulv也是假型的，然而随后病毒在中枢神经系统中的快速传播似乎是通过非假型的多性病毒粒子进行的。这种快速传播几乎与神经症状的出现同时发生。在混合感染中不表现出增强外周复制的多性病毒表现出较小程度的中枢神经系统感染，并且未观察到诱导神经系统疾病。总之，这些研究表明，神经致病性可能是多性mulv的一个普遍特性；侵袭中枢神经系统需要外周复制的阈值，多性病毒通过多性受体结合蛋白与中枢神经系统细胞上受体的相互作用进行传播可能是诱导神经病理的必要条件。我们研究的另一个方面涉及逆转录病毒感染细胞的机制的检查。包膜病毒的感染性侵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，并通过内吞途径中防止囊泡酸化的药物的短暂处理被抑制，而ph非依赖性病毒不受这些药物的抑制，并被认为通过与质膜直接融合进入细胞。几乎所有的逆转录病毒，包括两性性小鼠白血病病毒（MuLV）和人类免疫缺陷病毒1型，都被归类为pH无关。然而，嗜生态MuLV被认为是一种ph依赖性病毒。我们检查了嗜生态性和两性性mulv的感染进入，发现它们同样受到NH4Cl和阻断囊泡酸化的巴霉素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剂也能阻止囊泡运输。因此，mulv的部分抑制可能是该途径中病毒在阻滞期间失活的结果。为了支持这一观点，我们发现，在用药物治疗靶细胞期间，mulv的传染性丧失与在同一时间内37℃自发失活的活性丧失密切相关。此外，在感染持续时间很短以尽量减少自发失活的影响的条件下，这些药物对感染的效率没有影响。这些结果表明，在药物短暂作用下，嗜性和两性性mulv的感染过程都被阻止而不是中止。进一步的实验表明，这种阻滞发生在细胞内，因此两性性和生态性mulv的感染过程都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不能抑制传染性。对药物缺乏敏感性被认为是通过与质膜直接融合而感染的证据，但这可能只是反映了病毒在治疗过程中的稳定性。一个恰当的例子可能是HIV-1的传染性进入。如果HIV-1是通过内体途径而不是通过直接与质膜融合进入的，那么考虑到其半衰期比药物治疗时间长6- 10倍，它将不会在溶酶促体药物的标准检测中反映出来。在上述研究的基础上，构建了将葡萄球菌蛋白A的fc结合域整合到病毒SU蛋白中的靶向逆转录病毒载体。以前的靶向逆转录病毒结合了单链抗体或其他结合特定细胞表面分子的配体。这些研究的目的是得到一个单一的载体，可以结合不同的抗体针对不同的细胞类型。构建了多个载体，其中一个载体在小鼠和人源细胞系中表现出抗体依赖的转导活性。与迄今为止报道的所有靶向逆转录病毒载体一样，转导活性需要改变后的SU蛋白与野生型MuLV包膜蛋白共同表达。受MuLV感染的小鼠细胞在其表面表达高水平的MuLV包膜蛋白，并且由于病毒干扰而对同一类型病毒的感染难以抵抗。M-MuLV感染的细胞包被针对M-MuLV SU蛋白的IgG抗体后，被protein a /M-MuLV载体成功诱导，而未处理的细胞或SU蛋白的IgM抗体处理的细胞则无法成功诱导。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，而不太可能依赖于野生型包膜蛋白与生态受体的结合。另一系列实验表明，卡波西肉瘤细胞在血管内皮生长因子（VEGF）的作用下增殖成功。将结合VEGF受体（KDR/Flk-1）的抗体包裹在卡波西肉瘤细胞后，实现了细胞的转导。这些结果表明，igg结合的逆转录病毒载体可以提供一种高效的多用途基因传递载体，用于靶向靶细胞表面表达的特异性抗原。

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.