PVC-211 Murine Leukemia Virus Research Articles

Contribution of anti-ferritin antibodies to the diagnosis of giant cell arteritis

The neuronal pathology caused by neonatal infection of rats with the PVC-211 murine leukemia virus (PVC-211 MuLV) and its underlying mechanisms are not well defined even though a loss of neurons and spongiform neurodegeneration has been reported to accompany the disease. Here we sought to identify sites of neurodegeneration using microglial reactivity as an indirect marker and to characterize microglial activation during disease progression. Using a panel of microglial antibodies including Iba1, OX-42, ED1, and anti-ferritin, we have studied the response of microglial cells to neonatal CNS infection with PVC-211 at post-infection survival times 7, 14, 21, and 28 days. We found that microglial activation occurred primarily in the spinal cord and brainstem where it gradually increased in intensity over the time course of this study. Other brain areas were relatively unremarkable in their microglial reaction to viral infection within this time frame. However, the presence of activated microglial cells was not correlated directly with the presence of viral glycoprotein (gp70), which was expressed in endothelial cells throughout the CNS. Although double-labeling of microglia with Iba1 and ED1 revealed numerous actively phagocytic microglia during disease progression, not all activated microglia were ED1-positive. In addition to the intense microglial activation, we found increased ferritin expression sporadically throughout the virus-infected brain. The ferritin-positive cells were mostly microglia that exhibited dystrophic changes and likely represented a degenerating subpopulation of microglial cells. Thus, activated microglia can co-exist with degenerating microglia in the same brain region. We attempted to localize degenerating neurons or neurites using Fluoro-Jade, anti-tau, and anti-alpha synuclein staining, but none of these procedures yielded results to indicate obvious neuronal pathology. We conclude that the visualization of microglial activation is a more sensitive measure of neuronal perturbations than direct detection of neuronal pathology which may be subtle and not produce overt degenerative changes.

Importance of macrophage inflammatory protein-1α and splenic macrophages in neurodegeneration induced by PVC-211 murine leukemia virus

We recently reported that infection of rats with the neurodegenerative disease-causing retrovirus PVC-211 MuLV results in elevated levels of the chemokine MIP-1α followed by the accumulation of activated microglia in the brain. To investigate the importance of MIP-1α in recruitment of microglia to the brain, we treated rats with MIP-1α antibodies before and after PVC-211 MuLV infection. This caused a delay in the development of paralysis which was associated with a decrease in activated microglia without affecting virus expression. To determine the source of activated microglia, rats were splenectomized 4 days after virus infection. Splenectomized rats showed a delay in disease development that was associated with decreased numbers of activated microglia without affecting virus expression. Together, these results suggest that MIP-1α is directly involved in the neurodegeneration induced in rats by PVC-211 MuLV by recruiting macrophages/microglia from the periphery into regions of the brain that eventually become diseased.

Microglial response to murine leukemia virus-induced encephalopathy is a good indicator of neuronal perturbations

The neuronal pathology caused by neonatal infection of rats with the PVC-211 murine leukemia virus (PVC-211 MuLV) and its underlying mechanisms are not well defined even though a loss of neurons and spongiform neurodegeneration has been reported to accompany the disease. Here we sought to identify sites of neurodegeneration using microglial reactivity as an indirect marker and to characterize microglial activation during disease progression. Using a panel of microglial antibodies including Iba1, OX-42, ED1, and anti-ferritin, we have studied the response of microglial cells to neonatal CNS infection with PVC-211 at post-infection survival times 7, 14, 21, and 28 days. We found that microglial activation occurred primarily in the spinal cord and brainstem where it gradually increased in intensity over the time course of this study. Other brain areas were relatively unremarkable in their microglial reaction to viral infection within this time frame. However, the presence of activated microglial cells was not correlated directly with the presence of viral glycoprotein (gp70), which was expressed in endothelial cells throughout the CNS. Although double-labeling of microglia with Iba1 and ED1 revealed numerous actively phagocytic microglia during disease progression, not all activated microglia were ED1-positive. In addition to the intense microglial activation, we found increased ferritin expression sporadically throughout the virus-infected brain. The ferritin-positive cells were mostly microglia that exhibited dystrophic changes and likely represented a degenerating subpopulation of microglial cells. Thus, activated microglia can co-exist with degenerating microglia in the same brain region. We attempted to localize degenerating neurons or neurites using Fluoro-Jade, anti-tau, and anti-alpha synuclein staining, but none of these procedures yielded results to indicate obvious neuronal pathology. We conclude that the visualization of microglial activation is a more sensitive measure of neuronal perturbations than direct detection of neuronal pathology which may be subtle and not produce overt degenerative changes.

Neurodegeneration Induced by PVC-211 Murine Leukemia Virus Is Associated with Increased Levels of Vascular Endothelial Growth Factor and Macrophage Inflammatory Protein 1α and Is Inhibited by Blocking Activation of Microglia

PVC-211 murine leukemia virus (MuLV) is a neuropathogenic retrovirus that has undergone genetic changes from its nonneuropathogenic parent, Friend MuLV, that allow it to efficiently infect rat brain capillary endothelial cells (BCEC). To clarify the mechanism by which PVC-211 MuLV expression in BCEC induces neurological disease, we examined virus-infected rats at various times during neurological disease progression for vascular and inflammatory changes. As early as 2 weeks after virus infection and before any marked appearance of spongiform neurodegeneration, we detected vessel leakage and an increase in size and number of vessels in the areas of the brain that eventually become diseased. Consistent with these findings, the amount of vascular endothelial growth factor (VEGF) increased in the brain as early as 1 to 2 weeks postinfection. Also detected at this early disease stage was an increased level of macrophage inflammatory protein 1 alpha (MIP-1 alpha), a cytokine involved in recruitment of microglia to the brain. This was followed at 3 weeks postinfection by a marked accumulation of activated microglia in the spongiform areas of the brain accompanied by an increase in tissue plasminogen activator, a product of microglia implicated in neurodegeneration. Pathological observations at the end stage of the disease included loss of neurons, decreased myelination, and mild muscle atrophy. Treatment of PVC-211 MuLV-infected rats with clodronate-containing liposomes, which specifically kill microglia, significantly blocked neurodegeneration. Together, these results suggest that PVC-211 MuLV infection of BCEC results in the production of VEGF and MIP-1 alpha, leading to the vascular changes and microglial activation necessary to cause neurodegeneration.

Expression of inducible nitric oxide synthase and elevation of tyrosine nitration of a 32-kilodalton cellular protein in brain capillary endothelial cells from rats infected with a neuropathogenic murine leukemia virus.

PVC-211 murine leukemia virus (MuLV) is a neuropathogenic variant of Friend MuLV (F-MuLV) which causes a rapidly progressive spongiform neurodegenerative disease in rodents. The primary target of PVC-211 MuLV infection in the brain is the brain capillary endothelial cell (BCEC), which is resistant to F-MuLV infection. Previous studies have shown that changes in the envelope gene of PVC-211 MuLV confer BCEC tropism to the virus. However, little is known about how infection of BCECs by PVC-211 MuLV induces neurological disease. Previous results suggest that nitric oxide (NO), which has been implicated as a potential neurotoxin, is involved in PVC-211 MuLV-induced neurodegeneration. In this study, we show that expression of inducible nitric oxide synthase (iNOS), which produces NO from L-arginine, is induced in BCECs from PVC-211 MuLV-infected rats. Furthermore, elevated levels of a 32-kDa cellular protein modified by 3-nitrotyrosine, which is a hallmark of NO production, were observed in virus-infected BCECs. BCECs from rats infected with BCEC-tropic but nonneuropathogenic PVF-e5 MuLV, which is a chimeric virus between PVC-211 MuLV and F-MuLV, fail to induce either iNOS expression or elevation of tyrosine nitration of a 32-kDa protein. These results suggest that expression of iNOS and nitration of tyrosine residues of a 32-kDa protein in PVC-211 MuLV-infected BCECs may play an important role in neurological disease induction.

A unique heparin-binding domain in the envelope protein of the neuropathogenic PVC-211 murine leukemia virus may contribute to its brain capillary endothelial cell tropism.

Previous studies from our laboratory demonstrated that PVC-211 murine leukemia virus (MuLV), a neuropathogenic variant of Friend MuLV (F-MuLV), had undergone genetic changes which allowed it to efficiently infect rat brain capillary endothelial cells (BCEC) in vivo and in vitro. Two amino acid changes from F-MuLV in the putative receptor binding domain (RBD) of the envelope surface protein of PVC-211 MuLV (Glu-116 to Gly and Glu-129 to Lys) were shown to be sufficient for conferring BCEC tropism on PVC-211 MuLV. Recent examination of the unique RBD of PVC-211 MuLV revealed that the substitution of Lys for Glu at position 129 created a new heparin-binding domain that overlapped a heparin-binding domain common to ecotropic MuLVs. In this study we used heparin-Sepharose columns to demonstrate that PVC-211 MuLV, but not F-MuLV, can bind efficiently to heparin and that one or both of the amino acids in the RBD of PVC-211 MuLV that are associated with BCEC tropism are responsible. We further showed that heparin can enhance or inhibit MuLV infection and that the mode of action is dependent on heparin concentration, sulfation of heparin, and the affinity of the virus for heparin. Our results suggest that the amino acid changes that occurred in the envelope surface protein of PVC-211 MuLV may allow the virus to bind strongly to the surface of BCEC via heparin-like molecules, increasing the probability that the virus will bind to its cell surface receptor and efficiently infect these cells.

Evidence for oxidative damage in a murine leukemia virus‐induced neurodegeneration

Vacuolation in cellular organelles within the central nervous system is a common manifestation of oxidative injury. We found that the spongiform vacuolation observed in PVC-211 murine leukemia virus (PVC-MuLV) neurodegeneration was associated with oxidative damage as detected by immunoreactivity for 3-nitrotyrosine and protein carbonyl groups. This oxidative injury was present in brain before or concomitant with the appearance of activated microglia, vacuolation, and gliosis that characterize PVC-MuLV neuropathology. Treatment of infected F344 rat pups with the antioxidant vitamin E transiently protected and prolonged the latency of PVC-MuLV neurodegeneration. Taken together, these findings implicate oxidative damage and lipid peroxidation in the pathogenesis of PVC-MuLV neurodegeneration. This animal model may be useful for studies of mechanisms and potential therapies for progressive neurodegeneration following a well-defined insult. J. Neurosci. Res. 62:440–450, 2000. Published 2000 Wiley-Liss, Inc.

Evidence for oxidative damage in a murine leukemia virus-induced neurodegeneration.

Vacuolation in cellular organelles within the central nervous system is a common manifestation of oxidative injury. We found that the spongiform vacuolation observed in PVC-211 murine leukemia virus (PVC-MuLV) neurodegeneration was associated with oxidative damage as detected by immunoreactivity for 3-nitrotyrosine and protein carbonyl groups. This oxidative injury was present in brain before or concomitant with the appearance of activated microglia, vacuolation, and gliosis that characterize PVC-MuLV neuropathology. Treatment of infected F344 rat pups with the antioxidant vitamin E transiently protected and prolonged the latency of PVC-MuLV neurodegeneration. Taken together, these findings implicate oxidative damage and lipid peroxidation in the pathogenesis of PVC-MuLV neurodegeneration. This animal model may be useful for studies of mechanisms and potential therapies for progressive neurodegeneration following a well-defined insult.

Analysis of receptor usage by ecotropic murine retroviruses, using green fluorescent protein-tagged cationic amino acid transporters.

Entry of ecotropic murine leukemia virus (MuLV) into host cells is initiated by interaction between the receptor-binding domain of the viral SU protein and the third extracellular domain (TED) of the receptor, cationic amino acid transporter 1 (CAT1). To study the molecular basis for the retrovirus-receptor interaction, mouse CAT1 (mCAT1) was expressed in human 293 cells as a fusion protein with jellyfish green fluorescent protein (GFP). Easily detected by fluorescence microscopy and immunoblot analysis with anti-GFP antibodies, the mCAT1-GFP fusion protein was expressed in an N-glycosylated form on the cell surface and in the Golgi apparatus, retaining the ecotropic receptor function. The system was applied to compare Friend MuLV (F-MuLV) and its neuropathogenic variant, PVC-211 MuLV, which exhibits a unique cellular tropism and host range, for the ability to use various CAT family members as a receptor. The results indicated that F-MuLV and PVC-211 MuLV could infect the cells expressing wild-type mCAT1 at comparable efficiencies and that rat CAT3, but not mCAT2, conferred a low but detectable level of susceptibility to F-MuLV and PVC-211 MuLV. The data also suggested that CAT proteins might be expressed in an oligomeric form. Further application of the system developed in this study may provide useful insights into the entry mechanism of ecotropic MuLV.

The entire nucleotide sequence of friend-related and paralysis-inducing PVC-441 murine leukemia virus (MuLV) and its comparison with those of PVC-211 MuLV and Friend MuLV.

PVC-441 murine leukemia virus (MuLV) is a member of the PVC group of Friend MuLV (F-MuLV)-derived neuropathogenic retroviruses. In order to determine the molecular basis for the difference in neuropathogenicity between PVC-441 and the previously characterized PVC-211 MuLVs, the entire nucleotide sequence of PVC-441 MuLV was determined and compared with those of PVC-211 and F-MuLV. The results suggest that PVC-441 and PVC-211 MuLVs were formed as a result of random mutations of F-MuLV and developed differently. The distinct pathogenicities of PVC-441 and PVC-211 MuLVs were maintained in the viruses regenerated from their molecular clones, and the sequences responsible for the pathological differences observed can be localized to the env gene. The amino acid sequence of PVC-441 deduced from its nucleotide sequence revealed a number of differences from PVC-211, the most striking of which was a difference at position 129 of the SU proteins in the two viruses. Host range studies with a brain capillary endothelial cell line (RTEC-6) and Chinese hamster ovary cells (CHO-K1) revealed that PVC-441, like PVC-211, could infect these cells but its efficiency of infection was lower than that of PVC-211. These results may account for the difference in neuropathogenicity between PVC-441 and PVC-211.

Capillary endothelial cell tropism of PVC-211 murine leukemia virus and its application for gene transduction.

PVC-211 murine leukemia virus (MuLV) causes neurodegenerative disease following inoculation of neonatal, but not adult, mice and rats. It was previously shown that tropism for brain capillary endothelial cells (CEC) was a determinant of the viral neuropathogenicity. In this study, we demonstrate that host age-dependent replication of PVC-211 MuLV in vivo occurs in CEC in the brain as well as in other organs, such as the liver, kidney, and heart. In contrast, primary explant cultures of CEC derived from brains and livers of adult and neonatal rats could be infected by PVC-211 MuLV, suggesting that the age-dependent susceptibility was abrogated in vitro. Although CEC were generally less susceptible to MuLV-mediated gene transduction than fibroblasts, treatment of CEC with 2-deoxyglucose followed by inoculation of a PVC-211 MuLV-pseudotyped vector in the absence of heparin improved the transduction efficiency. These observations support the possibility that PVC-211 MuLV may be useful for establishing models of CEC gene transduction.

Analysis of the unique hamster cell tropism of ecotropic murine leukemia virus PVC-211.

PVC-211 murine leukemia virus (MuLV) is a neuropathogenic variant of Friend MuLV (F-MuLV). Previous studies from our laboratory demonstrated that unlike the parental F-MuLV, PVC-211 MuLV can infect rat brain capillary endothelial cells efficiently and that it has acquired genetic changes responsible for its expanded cellular tropism. To determine if PVC-211 MuLV also has expanded its host range, we tested its infectivity on Chinese hamster ovary-derived CHO-K1 cells, which are generally resistant to ecotropic MuLV. The results indicated that PVC-211 MuLV, but not F-MuLV, was highly infectious for CHO-K1 cells. Studies using glycosylation inhibitors and glycosylation mutants of CHO-K1 cells, as well as interference studies, suggested that PVC-211 MuLV has acquired the ability to interact with the ecotropic MuLV receptor on CHO-K1 cells that has undergone glycosylation-dependent modification. Using chimeric viruses between PVC-211 MuLV and F-MuLV, we were able to localize the viral genetic element crucial for CHO-K1 cell tropism within the env gene of PVC-211 MuLV and show that glycine at position 116 and lysine at position 129 of the envelope glycoprotein SU were important. These viral determinants also appear to confer tropism for other hamster cells resistant to ordinary ecotropic MuLVs. Further studies on the interaction between PVC-211 MuLV and the receptor on hamster cells may provide novel insights into the molecular mechanisms for receptor recognition and binding by viral envelope glycoproteins.

Effects of Subtle Changes in the SU Protein of Ecotropic Murine Leukemia Virus on Its Brain Capillary Endothelial Cell Tropism and Interference Properties

PVC-211 murine leukemia virus (MuLV) is a neuropathogenic variant of Friend MuLV (F-MuLV) that causes a rapidly progressive neurodegenerative disease in susceptible rodents. PVC-211 MuLV, but not the parental F-MuLV, can infect rat brain capillary endothelial cells (BCEC) efficiently, and the major determinant for BCEC tropism of PVC-211 MuLV is localized within theXbaI–BamHI fragment of the viral genome containing the 5′ half of theenvgene. To further dissect theXbaI–BamHI region for its effects on BCEC tropism, we constructed recombinant viruses between PVC-211 MuLV and F-MuLV and tested their infectivity on a cell line established from rat BCEC. Our results indicated that Glu116-to-Gly and Glu129-to-Lys substitutions in the background of the F-MuLV envelope SU protein were sufficient for conferring BCEC tropism on the virus. Interference studies of these viruses on Rat-1 fibroblastic cells showed that the structure of the SU protein encoded by theXbaI–BamHI region also has significant effects on their affinity for the rat ecotropic MuLV receptor. These results support the possibility that structural elements I and II of the SU protein are important determinants for virus–receptor interaction.

Immunogenic determinants of a neuropathogenic murine leukemia virus.

Previous studies of Cas-Br-M murine leukemia virus (MuLV) (Cas-MuLV) infection demonstrated that cytotoxic T cells (CTL) of the CD8+ phenotype play a role in resistance to the neuropathogenic effects of the virus in NFS/N mice. In the current study, we sought to identify the Cas-MuLV epitopes that are immunogenic for the CTL response. Infection of adult NFS/N mice with a well-characterized neuropathogenic variant of Friend MuLV, PVC-211 MuLV (PVC-MuLV), was not immunogenic for MuLV-specific CTL. Therefore, we constructed chimeric viruses between Cas-MuLV and PVC-MuLV. Infectious chimeras contained the Cas-MuLV env gene on a PVC-MuLV background (PVC-CasenvMuLV) and the PVC-MuLV env gene on a Cas-MuLV background (Cas-PVCenvMuLV). Cas-MuLV-specific CTL were found following inoculation of both the chimeric viruses and the parental Cas-MuLV but not the parental PVC-MuLV, despite evidence of antibody responses to both parental and chimeric MuLV. CTL generated in response to infection with PVC-CasenvMuLV and Cas-PVCenvMuLV were exclusively of the CD8+ phenotype. These results indicate that both the env and gag-pol regions of Cas-MuLV express epitopes that are immunogenic for CTL.

Viral determinants that control the neuropathogenicity of PVC-211 murine leukemia virus in vivo determine brain capillary endothelial cell tropism of the virus in vitro

PVC-211 murine leukemia virus (MuLV) is a neuropathogenic, weakly leukemogenic variant of the nonneuropathogenic, highly leukemogenic Friend MuLV (F-MuLV). Chimeric viruses constructed from PVC-211 MuLV clone 3d and F-MuLV clone 57 indicate that the env gene of PVC-211 MuLV contains the determinant(s) responsible for pathological changes in the central nervous system. However, sequences within the 5' one-third (AatII-EcoRI region) of the PVC-211 MuLV genome, which include the 5' leader sequence, the gag gene, and the 5' quarter of the pol gene, are also needed in conjunction with the env gene determinant(s) to cause clinically evident neurological disease in the majority of virus-infected animals after a short latency. In the presence of the AatII-EcoRI region of the PVC-211 MuLV genome, the PVC-211 MuLV env gene sequences encoding the amino-terminal half of the SU protein, which contains the receptor-binding region of the protein, were sufficient to cause rapidly progressive neurological disease. When PVC-211 MuLV, F-MuLV, and various chimeric viruses were tested for their ability to replicate in cultured brain capillary endothelial cells (BCEC), the primary site of PVC-211 MuLV replication within the central nervous system, there was a direct correlation between the replication efficiency of a virus in BCEC in vitro and its ability to cause neurological disease in vivo. This observation indicates that the sequences in PVC-211 MuLV that render it neuropathogenic affect its replication in BCEC and suggests that rapid and efficient replication of the virus in BCEC is crucial for the pathological changes in the central nervous system that result in development of neurological disease.

Molecular characterization of a neuropathogenic and nonerythroleukemogenic variant of Friend murine leukemia virus PVC-211.

PVC-211 murine leukemia virus (MuLV) is a replication-competent, ecotropic type C retrovirus that was isolated after passage of the Friend virus complex through F344 rats. Unlike viruses in the Friend virus complex, it does not cause erythroleukemia but causes a rapidly progressive hind limb paralysis when injected into newborn rats and mice. We have isolated an infectious DNA clone (clone 3d) of this virus which causes neurological disease in animals as efficiently as parental PVC-211 MuLV. The restriction map of clone 3d is very similar to that of the nonneuropathogenic, erythroleukemogenic Friend murine leukemia virus (F-MuLV), suggesting that PVC-211 MuLV is a variant of F-MuLV and that no major structural alteration was involved in its derivation. Studies with chimeric viruses between PVC-211 MuLV clone 3d and wild-type F-MuLV clone 57 indicate that at least one determinant for neuropathogenicity resides in the 2.1-kb XbaI-ClaI fragment containing the gp70 coding region of PVC-211 MuLV. Compared with nonneuropathogenic ecotropic MuLVs, the env gene of PVC-211 MuLV encodes four unique amino acids in the gp70 protein. Nucleotide sequence analysis also revealed a deletion in the U3 region of the long terminal repeat (LTR) of PVC-211 MuLV clone 3d compared with F-MuLV clone 57. In contrast to the env gene of PVC-211 MuLV, particular sequences within the U3 region of the viral LTR do not appear to be required for neuropathogenicity. However, the changes in the LTR of PVC-211 MuLV may be responsible for the failure of this virus to cause erythroleukemia, because chimeric viruses containing the U3 region of F-MuLV clone 57 were erythroleukemogenic whereas those with the U3 of PVC-211 MuLV clone 3d were not.