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.
