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.     

A viral non-coding region determining neuropathogenicity of murine leukemia virus A8 is responsible for envelope protein expression in the rat brain

Friend murine leukemia virus clone A8 causes spongiform neurodegeneration in the rat brain. A 0.3-kb fragment containing the R-U5-5' leader sequence of A8 is required in addition to the A8-env gene to induce spongiosis. The A8-env gene is a primary determinant of neuropathogenicity. Comparative studies of the neuropathogenic virus R7f, which carries the 0.3-kb fragment of A8 and A8-env on the background of the non-neuropathogenic clone 57, and the non-neuropathogenic virus Rec5, which carries A8-env on the background of 57, showed that the 0.3-kb fragment of A8 was responsible for increasing the ratio of Env/Gag expression in the brain, but not in the spleen.     

Identification of genetic determinants that regulate tumorigenicity of Friend murine leukemia virus in rats

A neuropathogenic variant of Friend murine leukemia virus (FrMLV), clone A8, has been shown to cause thymoma and infiltration of leukemic cells to organs at 7-8 weeks post-infection in rats with a more rapid progression than clone 57. We have previously reported that the determinant for induction of aggressive leukemia in rats is located in the ClaI-AatII fragment containing the long terminal repeat (LTR) and the 5' half of the 5' leader sequence of A8 virus. Further studies of chimeric viruses restricted the determinant for the induction of thymoma to only the 0.6-kb ClaI-KpnI fragment of A8. This fragment contains a 0.1 kb region of the 3' terminus of the env gene, the intergenic region, the U3, and the 5' half of the R region in the LTR. Major differences in the fragment between A8 and 57 viruses were found in the U3 region, especially in the enhancer motifs. These results indicate that the enhancer region of A8-LTR contributes to the manifestation of thymoma with rapid progression in rats.     

Kinetic studies of the effect of a 17‐nucleotide difference in the 0.3‐kb region containing the R‐U5‐5′ leader sequence of Friend murine leukemia virus on viral gene expression

In addition to the env gene, a 0.3‐kb fragment containing the R‐U5‐5′ leader sequence is essential for the induction of spongiform neurodegeneration by Friend murine leukemia virus (Fr‐MLV) clone A8 and it also influences expression of the Env protein. Kinetic studies were carried out using two recombinant viruses, R7f, carrying the A8 0.3‐kb fragment, and Rec5, carrying the 0.3‐kb fragment of the non‐neuropathogenic Fr‐MLV clone 57. These analyses suggested that the 0.3‐kb fragment influenced the expression level of the Env protein by regulating the amount of spliced env‐mRNA rather than the amount of total viral mRNA or viral production.     

Chimeric influenza virus hemagglutinin proteins containing large domains of the Bacillus anthracis protective antigen: protein characterization, incorporation into infectious influenza viruses, and antigenicity

Large polypeptides of the Bacillus anthracis protective antigen (PA) were inserted into an influenza A virus hemagglutinin glycoprotein (HA), and the chimeric proteins were functionally characterized and incorporated into infectious influenza viruses. PA domain 1', the region responsible for binding to the other toxin components, the lethal factor and edema factor, and domain 4, the receptor binding domain (RBD), were inserted at the C-terminal flank of the HA signal peptide and incorporated into the HA1 subunit of HA. The chimeric proteins, designated as LEF/HA (90 amino acid insertion) and RBD/HA (140 amino acid insertion), were initially analyzed following expression using recombinant vaccinia viruses. Both chimeric proteins were shown to display functional phenotypes similar to that of the wild-type HA. They transport to the cell surface, can be cleaved into the HA1 and HA2 subunits by trypsin to activate membrane fusion potential, are able to undergo the low-pH-induced conformational changes required for fusion, and are capable of inducing the fusion process. We were also able to generate recombinant influenza viruses containing the chimeric RBD/HA and LEF/HA genes, and the inserted PA domains were maintained in the HA gene segments following several passages in MDCK cells or embryonated chicken eggs. Furthermore, DNA immunization of mice with plasmids that express the chimeric RBD/HA and LEF/HA proteins, and the recombinant viruses containing them, induced antibody responses against both the HA and PA components of the protein. These approaches may provide useful tools for vaccines against anthrax and other diseases.     

Characterization of chimeras between the ecotropic Moloney murine leukemia virus and the amphotropic 4070A envelope proteins.

A series of 22 chimeric envelope (env) genes were generated between the ecotropic Moloney murine leukemia virus and the amphotropic 4070A isolate. The chimeric envelopes were expressed within the complete, replication-competent provirus and tested for virus viability by transient expression assays. Eleven of the 22 viruses were viable. Five of these chimeric viruses showed an ecotropic host range, and six exhibited an amphotropic host range and viral interference. The host range determinants map to the first half of the surface (SU) protein. The N-terminal 72 amino acids of 4070A (42 of processed SU) are not required for amphotropic receptor usage. Ecotropic and amphotropic viruses differ in their ability to form large, multinucleated syncytia when cocultured with the rat XC cell line. Ecotropic murine leukemia virus forms large syncytia with XC cells, whereas no syncytia are reported for amphotropic virus. All chimeras which contained the N-terminal half of the ecotropic SU protein, encoding the receptor binding domain, formed the large multinucleated syncytia with XC cells.     

Neurodegenerative disease induced by the wild mouse ecotropic retrovirus is markedly accelerated by long terminal repeat and gag-pol sequences from nondefective Friend murine leukemia virus.

The wild mouse ecotropic retrovirus (WM-E) induces a spongiform neurodegenerative disease in mice after a variable incubation period of 2 months to as long as 1 year. We isolated a molecular clone of WM-E (15-1) which was weakly neurovirulent (incidence, 8%) but was highly leukemogenic (incidence, 45%). Both lymphoid and granulocytic leukemias were observed, and these leukemias were often neuroinvasive. A chimeric virus was constructed containing the env and 3' pol sequences of 15-1 and long terminal repeat (LTR), gag, and 5' pol sequences from a clone of Friend murine leukemia virus (FB29). FB29 has been shown previously to replicate to high levels in the central nervous system (CNS) but is not itself neurovirulent. This finding was confirmed at the DNA level in the current study. Surprisingly, intraperitoneal inoculation of neonatal IRW mice with the chimeric virus (FrCasE) caused an accelerated neurodegenerative disease with an incubation period of only 16 days and was uniformly fatal by 23 days postinoculation. Introduction of the LTR of 15-1 into the FrCasE genome yielded a virus (FrCasEL) with a degree of neurovirulence intermediate between those of 15-1 and FrCasE. No differences were found in the levels of viremia or the relative levels of viral DNA in the spleens of mice inoculated with 15-1, FrCasE, or FrCasEL. However, the levels of viral DNA in the CNS correlated with the relative degrees of neurovirulence of the respective viruses (FrCasE greater than FrCasEL greater than 15-1). Thus, the env and 3' pol sequences of WM-E (15-1) were required for neurovirulence, but elements within the LTR and gag-pol regions of FB29 had a profound influence on the level of CNS infection and the rate of development of neurodegeneration.     

Late virus replication events in microglia are required for neurovirulent retrovirus-induced spongiform neurodegeneration: evidence from neural progenitor-derived chimeric mouse brains.

CasBrE is a neurovirulent murine retrovirus which induces a spongiform myeloencephalopathy in susceptible mice. Genetic mapping studies have indicated that sequences responsible for neurovirulence reside within the env gene. To address the question of direct envelope protein neuroxicity in the central nervous system (CNS), we have generated chimeric mice expressing the CasBrE envelope protein in cells of neuroectodermal origin. Specifically, the multipotent neural progenitor cell line C17.2 was engineered to express the CasBrE env gene as either gp70/p15E (CasE) or gp70 alone (CasES). CasE expression in these cells resulted in complete (>10(5)) interference of superinfection with Friend murine leukemia virus clone FB29, whereas CasES expression resulted in a 1.8-log-unit decrease in FB29 titer. Introduction of these envelope-expressing C17.2 cells into the brains of highly susceptible IRW mice resulted in significant engraftment as integral cytoarchitecturally correct components of the CNS. Despite high-level envelope protein expression from the engrafted cells, no evidence of spongiform neurodegeneration was observed. To examine whether early virus replication events were necessary for pathogenesis, C17.2 cells expressing whole virus were transplanted into mice in which virus replication in the host was specifically restricted by Fv-1 to preintegration events. Again, significant C17.2 cell engraftment and infectious virus expression failed to precipitate spongiform lesions. In contrast, transplantation of virus-expressing C17.2 progenitor cells in the absence of the Fv-1 restriction resulted in extensive spongiform neurodegeneration by 2 weeks postengraftment. Cytological examination indicated that infection had spread beyond the engrafted cells, and in particular to host microglia. Spongiform neuropathology in these animals was directly correlated with CasBrE env expression in microglia rather than expression from neural progenitor cells. These results suggest that the envelope protein of CasBrE is not itself neurotoxic but that virus infectious events beyond binding and fusion in microglia are necessary for the induction of CNS disease.     

Human immunodeficiency virus type 1 tropism for brain microglial cells is determined by a region of the env glycoprotein that also controls macrophage tropism.

Human immunodeficiency virus type 1 (HIV-1), the agent of AIDS, frequently infects the central nervous system. We inoculated adult human brain cultures with chimeric viruses containing parts of the env gene of a cloned primary isolate from brain tissue, HIV-1 JRFl, inserted into the cloned DNA of a T-cell-tropic strain. A chimeric virus containing the carboxy-terminal portion of HIV-1 JRFl env did not replicate in these brain tissue cultures, while a chimera expressing an env-encoded protein containing 158 amino acids of HIV-1 JRFl gp120, including the V3 loop, replicated well in brain microglial cells, as it does in blood macrophages. Infection of brain microglial cells with such a chimera was blocked by an antibody to the V3 loop of gp 120. Thus, env determinants in the region of gp120, outside the CD4-binding site and comprising the V3 loop, are critical for efficient viral binding to and/or entry into human brain microglia.     

Nucleotide sequencing of an apparent proviral copy of env mRNA defines determinants of expression of the mouse mammary tumor virus env gene.

To extend our understanding of the organization and expression of the mouse mammary tumor virus genome, we determined the nucleotide sequence of large regions of a cloned mouse mammary tumor virus strain C3H provirus that appears to be a DNA copy of env mRNA. In conjunction with analysis of several additional clones of integrated and unintegrated mouse mammary tumor virus DNAs, we came to the following conclusions: (i) the mRNA for env is generated by splicing mechanisms that recognize conventional eucaryotic signals at donor and acceptor sites with a leader of at least 289 bases in length; (ii) the first of three possible initiation codons for translation of env follows the splice junction by a single nucleotide and produces a signal peptide of 98 amino acids; (iii) the amino terminal sequence of the major virion glycoprotein gp52env is confirmed by nucleotide sequencing and is encoded by a sequence beginning 584 nucleotides from the 5' end of env mRNA; (iv) the final 17 amino acids at the carboxyl terminus of the primary product of env are encoded within the long terminal repeat by the 51 bases at the 5' end of the U3 domain; and (v) bases 2 through 4 at the 5' end of the long terminal repeat constitute an initiation codon that commences an open reading frame capable of directing the synthesis of a 36-kilodalton protein.     

Neural Stem Cells as Engraftable Packaging Lines Can Mediate Gene Delivery to Microglia: Evidence from Studying Retroviral env-Related Neurodegeneration

The induction of spongiform myeloencephalopathy by murine leukemia viruses is mediated primarily by infection of central nervous system (CNS) microglia. In this regard, we have previously shown that CasBrE-induced disease requires late, rather than early, virus replication events in microglial cells (W. P. Lynch et al., J. Virol. 70:8896-8907, 1996). Furthermore, neurodegeneration requires the presence of unique sequences within the viral env gene. Thus, the neurodegeneration-inducing events could result from microglial expression of retroviral envelope protein alone or from the interaction of envelope protein with other viral structural proteins in the virus assembly and maturation process. To distinguish between these possible mechanisms of disease induction, we engineered the engraftable neural stem cell line C17-2 into packaging/producer cells in order to deliver the neurovirulent CasBrE env gene to endogenous CNS cells. This strategy resulted in significant CasBrE env expression within CNS microglia without the appearance of replication competent virus. CasBrE envelope expression within microglia was accompanied by increased expression of activation markers F4/80 and Mac-1 (CD11b) but failed to induce spongiform neurodegenerative changes. These results suggest that envelope expression alone within microglia is not sufficient to induce neurodegeneration. Rather, microglia-mediated disease appears to require neurovirulent Env protein interaction with other viral proteins during assembly or maturation. More broadly, the results presented here prove the efficacy of a novel method by which neural stem cell biology may be harnessed for genetically manipulating the CNS, not only for studying neurodegeneration but also as a paradigm for the disseminated distribution of retroviral vector-transduced genes.     

Purification of DNA complementary to the env gene of avian sarcoma virus and analysis of relationships among the env genes of avian leukosis-sarcoma viruses.

The env gene of avian leukosis-sarcoma viruses encodes a glycoprotein that determines the host range and surface antigenicitiy of virions. We have purified radioactive DNA (cDNAgp) complementary to at least a portion of the env gene for viral subgroups A and C; complementary DNA was synthesized with purified virions of wild-type avian sarcoma virus, and RNA from a mutant with a deletion in env was used to select DNA specific to env by molecular hybridization. The genetic complexity of cDNAgp for subgroup A (ca. 2,000 nucleotides) was sufficient to represent the entire deletion and most or all of the env cistron. The deletions in env in two independently isolated strains of virus (Bryan and rdNY8SR) overlap, and cDNAgp represents nucleotide sequences common to both deletions. By contrast, we could detect no overlap between deletions in env and deletions in the adjacent viral gene src. Laboratory stocks of viral subgroups A, B, C, D and E do not contain detectable amounts of env deletions when tested by molecular hybridization; hence, segregation of deletions in env is a less frequent event that the segregation of deletions in the viral transforming gene src (Vogt, 1971). We found extensive homology among the nucleotide sequences encoding the env genes of virus strains indigenous to chickens (subgroups A, B, C, D, and E) although subgorups B, D and E appear to differ slightly from subgroups A and C at the env locus. By contrast, viruses obtained from pheasant cells (subgroups F and G) have env genes with little or no relationship to env genes of chikcen viruses. According to available data, viruses of subgroup F arose by recombination between an avarian sarcoma virus and viral genes in the genome of ring-necked pheasants, whereas subgroup G viruses may be entirely endogenous to golden pheasants.     

Activation of oncogenicity of the c-rel proto-oncogene.

Reticuloendotheliosis virus strain T (Rev-T) induces a lethal lymphoma in young birds and transforms avian lymphoid cells in vitro. The transforming gene of Rev-T, v-rel, was derived from the turkey proto-oncogene c-rel. Comparison of the nucleotide sequences of v-rel and c-rel indicates that in addition to several internal amino acid changes relative to c-rel, p59v-rel has amino acid sequences at both ends derived from the reticuloendotheliosis virus strain A-related virus env gene (K. C. Wilhelmsen, K. Eggleton, and H. M. Temin, J. Virol. 52:172-182, 1984). In this report, the v-rel sequences important for transformation were defined by constructing recombinant retroviruses in which c-rel sequences replaced the analogous v-rel sequences. These recombinant viruses expressing chimeric proteins were tested for their ability to transform spleen cells in vitro and to induce tumors in young chickens. Activation of the oncogenicity of c-rel in Rev-T required alteration of the amino terminus and the central region of the protein. Deletion of the noncoding sequences 3' to c-rel and of most of the helper virus-related env sequences was necessary for the formation of Rev-T.     

Differential glycosylation of the Cas-Br-E env protein is associated with retrovirus-induced spongiform neurodegeneration

The wild mouse ecotropic retrovirus, Cas-Br-E, induces progressive, noninflammatory spongiform neurodegenerative disease in susceptible mice. Functional genetic analysis of the Cas-Br-E genome indicates that neurovirulence maps to the env gene, which encodes the surface glycoprotein responsible for binding and fusion of virus to host cells. To understand how the envelope protein might be involved in the induction of disease, we examined the regional and temporal expression of Cas-Br-E Env protein in the central nervous systems (CNS) of mice infected with the highly neurovirulent chimeric virus FrCas(E). We observed that multiple isoforms of Cas-Br-E Env were expressed in the CNS, with different brain regions exhibiting unique patterns of processed Env glycoprotein. Specifically, the expression of gp70 correlated with regions showing microglial infection and spongiform neurodegeneration. In contrast, regions high in neuronal infection and without neurodegenerative changes (the cerebellum and olfactory bulb) were characterized by a gp65 Env protein isoform. Sedimentation analysis of brain region extracts indicated that gp65 rather than gp70 was incorporated into virions. Biochemical analysis of the Cas-Br-E Env isoforms indicated that they result from differential processing of N-linked sugars. Taken together, these results indicate that differential posttranslational modification of the Cas-Br-E Env is associated with a failure to incorporate certain Env isoforms into virions in vivo, suggesting that defective viral assembly may be associated with the induction of spongiform neurodegeneration.     

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.