disease and to investigate the relationship between BCEC tropism and neuropathogenicity. Our results suggest that the

Transcription

1 JOURNAL OF VIROLOGY, Aug. 1993, p X/93/ $02.00/0 Copyright X 1993, American Society for Microbiology Vol. 67, No. 8 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 MICHIAKI MASUDA,1 PAUL M. HOFFMAN,2 AND SANDRA K. RUSCETTI'* Laboratory of Molecular Oncology, National Cancer Institute, Frederick, Maryland ,1 and Retrovirus Research Center, Department of Veterans Affairs Medical Center, Baltimore, Maryland Received 25 February 1993/Accepted 3 May 1993 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 (4atH-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 AatH-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. PVC-211 murine leukemia virus (MuLV) is a replicationcompetent ecotropic type C retrovirus isolated after passage of the Friend virus complex through F344 rats (11). While PVC-211 MuLV causes a rapidly progressive neurodegenerative disease in susceptible rats and mice, it is much less efficient at inducing erythroleukemia than the virus from which it was derived (15). Rats infected as newborns with PVC-211 MuLV develop clinical signs of neurological disease by 3 weeks of age, and tissues from the central nervous system (CNS) of these animals show extensive evidence of perivascular astrogliosis and neuropil vacuolation without inflammation (10). By immunohistochemical staining, it was shown that the primary target of virus infection is the brain capillary endothelial cell (BCEC), whereas within the CNS, reactive astrocytes and degenerating neurons showed no evidence of virus infection (10). We recently described the isolation of an infectious molecular clone (clone 3d) of PVC-211 MuLV and demonstrated that the virus is structurally very similar to the nonneuropathogenic Friend MuLV (F-MuLV) clone 57 (15, 23). In vivo studies on neuropathogenicity with chimeric viruses constructed from PVC-211 MuLV clone 3d and F-MuLV clone 57 suggested that the env gene of PVC-211 MuLV contains the determinants that are essential but not sufficient for induction of complete neurological disease (15). In this study, we used additional PVC-211 MuLV and F-MuLV chimeric viruses to extend our analysis of the viral determinants required for the development of neurological * Corresponding author disease and to investigate the relationship between BCEC tropism and neuropathogenicity. Our results suggest that the 5' half of the SU (gp70) coding region of the env gene of PVC-211 MuLV contains the determinant(s) responsible for the induction of pathological changes in the CNS, but other determinants in the 5' one-third of the viral genome appear to influence latency, the ability of a virus to induce pathological changes in the CNS and rapidly progressive neurological disease, and its replication efficiency on BCEC in vitro. Thus, certain env and non-env sequences in PVC-211 MuLV may be essential for its rapid, high-level replication in BCEC, and this interaction between the virus and BCEC may be the basis for its neuropathogenicity in vivo. MATERIALS AND METHODS DNA analysis. Basic recombinant DNA techniques, such as plasmid preparation and hybridization analysis, were performed by standard methods (27). For hybridization analysis of integrated viral DNA, high-molecular-weight DNA was extracted from virus-infected cells. Ten micrograms of DNA was digested with BamHI, separated on an agarose gel, and transferred to a nylon membrane. The samples were then hybridized with a 32P-labeled probe prepared from an equimolar mixture of the 0.83-kb BamHI- BamHI env gene fragment from PVC-211 MuLV clone 3d and F-MuLV clone 57. For amplification of the integrated viral DNA by polymerase chain reaction (PCR), a synthetic oligonucleotide (5'-TCTGTGGACTTGGTGGCCAG-3') was used as the 5' primer, and another oligonucleotide (5'-TGAG TCGGATCCCGAAAGT-3') was used as the 3' primer. This set of primers can amplify the 0.6-kb env-specific region of

2 VOL. 67, 1993 NEUROPATHOGENICITY AND CELL TROPISM OF PVC-211 MuLV 4581 both PVC-211 MuLV and F-MuLV equally well. One microgram of genomic DNA was amplified through 25 cycles of PCR with a GeneAmp PCR kit (Perkin Elmer Cetus, Norwalk, Conn.) according to the supplier's manual, with each cycle consisting of denaturation at 94 C for 1 min, annealing at 60 C for 1 min, and elongation at 72 C for 4 min. Amplified DNA samples were subjected to agarose gel electrophoresis, transferred to a nylon membrane, and hybridized with the 32P-labeled BamHI-BamHI env gene probe described above. Construction of chimeric viral genomes. Chimeric viruses were constructed from the neuropathogenic PVC-211 MuLV clone 3d (15) and the nonneuropathogenic F-MuLV clone 57 (16). F-MuLV clone 57 was recloned into puc19 for these experiments. Plasmid DNAs of PVC-211 MuLV clone 3d and F-MuLV clone 57 were digested with the restriction enzymes specified below, and the DNA fragments were separated by agarose gel electrophoresis, purified with GeneClean (Bio 101, La Jolla, Calif.), and ligated with T4 DNA ligase (Takara Biochemical, Inc., Berkeley, Calif.). Chimeric viruses PVF-A through PVF-E have been described previously (15). For construction of PVF-G, -I, and -J, either the EcoRI-XbaI region, the HindIII-XbaI region, or the AatII-XbaI region of PVC-211 MuLV clone 3d was replaced by the corresponding region of F-MuLV clone 57, respectively. For construction of PVF-H, the EcoRI-ClaI region of PVF-A was replaced by the corresponding region of PVF-C. Thus, PVF-H has the KpnI-EcoRI region and the XbaI-ClaI region of PVC-211 MuLV clone 3d in the background of F-MuLV clone 57. For construction of PVF-L, -M, and -N, either the BamHI-ClaI region, the XbaI-BamHI region, or the XbaI-ClaI region of PVF-H was replaced by the corresponding region of F-MuLV clone 57, respectively. For construction of PVF-K, the HindIII-EcoRI region of PVF-D was substituted by the corresponding region of PVC-211 MuLV clone 3d. Chimeric virus constructions were validated by digestion with restriction enzymes that have cleavage sites unique either to PVC-211 MuLV clone 3d or to F-MuLV clone 57. Cells and viruses. Uninfected and virus-producing NIH 3T3 cells, XC cells, NRK cells, and Rat-1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Rat BCEC were prepared and grown as recently described (10). Viruses were obtained from NIH 3T3 cells transfected by the calcium phosphate method (8), with the modifications described by Andersson et al. (2), by using viral DNA prepared as described previously (15). Briefly, viral DNA was excised from the vector by digestion with EcoRI, treated with T4 ligase for 15 min to obtain a nonpermutated form of the viral genome, precipitated with ethanol, and used for transfection. Virus titer was determined by the XC cell fusion assay (25). Animals. F344 rats were obtained from Harlan Sprague Dawley, Indianapolis, Ind., and housed in the Small Animal Facility, Department of Veterans Affairs Medical Center, Baltimore, Md. Two-day-old rats were inoculated intracerebrally (i.c.) with 0.03 ml of supernatant from the virus-producing NIH 3T3 cells and observed for neurological signs as described previously (15) for up to 28 weeks postinoculation. Histopathological examination. Virus-infected animals were examined for pathological changes in the CNS during the symptomatic period or at 28 weeks postinfection if they remained asymptomatic. The brain and spinal cord were immersed in buffered Formalin, and paraffin-embedded sections were stained with hematoxylin and eosin. Multiple sections were evaluated for evidence of typical neuropathology previously described for PVC-211 MuLV-induced disease, including perivascular astrogliosis and neuropil vacuolation without inflammation (10). V'iral protein analysis. Uninfected and virus-infected cells were labeled with [ 5S]methionine for 15 min, lysates were precipitated with goat antiserum to Rauscher MuLV gp7o or p30 (National Cancer Institute, Bethesda, Md.), and precipitated proteins were analyzed by denaturing polyacrylamide gel electrophoresis as described previously (26). Immunofluorescence microscopy of virus-infected cells with anti-gp7o antibody was performed as described previously (10). RESULTS env gene of PVC-211 MuLV determines pathology within the CNS but is not sufficient for complete neurological disease expression. We recently showed, using chiineras of PVC-211 MuLV and F-MuLV (PVF-A through PVF-E), that the env gene of PVC-211 MuLV contains the determinant(s) that is essential but not sufficient for development of complete neurological disease in the virus-infected animals during the 16 weeks that they were observed (15). In this study, we observed the animals for a longer period and examined them for microscopic pathological changes in the CNS in order to further define the role of the PVC-211 MuLV env gene in neuropathogenesis (Fig. 1). As described in the previous study (15), chimera PVF-A, which contains the U3 region of the long terminal repeat (LTR) of F-MuLV in the background of PVC-211 MuLV, caused neurological disease as efficiently as PVC-211 MuLV. In addition, the pathological changes in the CNS of PVF-A-infected rats were as extensive as those in PVC-211-infected rats. However, PVF-B, which contains the F-MuLV env gene in the background of PVF-A, failed to cause neurological disease within 28 weeks postinoculation, and no abnormal changes in the CNS were detected histologically. Rats injected with PVF-C, which contains the env gene and U3 LTR sequences of PVC-211 MuLV in an F-MuLV background, did not develop clinical signs of neurological disease even when examined for up to 28 weeks after virus infection. However, when the CNS tissues from these asymptomatic rats were examined histologically for pathological changes, four of the five rats examined showed perivascular astrogliosis and neuropil vacuolation without inflammation, which is characteristic of PVC-211 MuLV-induced neurodegeneration. Although rats infected with PVF-D, which contains only the env gene from PVC-211 MuLV in an F-MuLV background, remained asymptomatic for 16 weeks after virus infection (15), three of eight PVF-D-infected animals developed abnormal neurological signs at 20 weeks postinoculation, and two of two symptomatic rats and one of two asymptomatic rats examined histologically showed neuropathological changes characteristic of PVC-211 MuLV infection. It should be noted that the vacuolation and gliosis induced by PVF-C and PVF-D in asymptomatic animals was less extensive than that induced by PVC-211 MuLV. Thus, although chimeric viruses carrying the env gene of PVC-211 MuLV can cause a distribution of pathological lesions similar to that associated with PVC-211 MuLV, these changes may not be extensive enough to overcome the compensatory function of the CNS and to cause clinical signs of neurological disease in the majority of the rats. Neither clinically evident neurological disease nor pathological changes in the CNS were induced by chimera PVF-E, which does not have the env gene of PVC-211 MuLV but has the F-MuLV-

3 4582 MASUDA ET AL. J. VIROL. PVC-2 11 cl. 3d F-MuLV cl. 57 PVF-A Xb C K Clinical Di sease U 21/21 X- H LTR poi1...,iltr env IZ 0/12 18/18 ] 0/16 0/16 Neurooathoqeni ci ty Onset Pathological (weeks) channes in the CNS 3 10/10 3 3/8 20 FIG. 1. Effect of the env gene of PVC-211 MuLV in causing pathological changes in the CNS and clinical neurological disease. The viral genomes are represented in their nonpermutated form, flanked at their ends by the LTR sequences. In the chimeric viral genomes, solid regions are sequences derived from PVC-211 MuLV clone 3d and open regions are sequences derived from F-MuLV clone 57. The positions of the gag, pol, and env genes and the LTR are shown at the bottom. Restriction sites used to construct the chimeras are shown at the top: C, ClaI; K, KjpnI; Xb, XbaI. Viruses were obtained from NIH 3T3 cells transfected with each viral DNA and inoculated intracerebrally into newborn F344 rats. Neuropathogenicity was determined as described in Materials and Methods and is represented as the number of animals showing clinical signs of neurological disease/number of virus-infected animals, median time to onset of disease postinoculation, and the number of animals showing pathological changes in the CNS/number of animals examined. Among those infected with PVF-D, two of two symptomatic rats examined showed pathological changes in the CNS at 22 weeks postinoculation and one of two asymptomatic rats showed pathological changes in the CNS at 28 weeks postinoculation. derived env gene. These observations indicate that the env gene of PVC-211 MuLV contains the primary determinant(s) responsible for the pathological changes in the CNS but is not sufficient for the development of complete neurological disease. Non-env determinants in the PVC-211 MuLV genome enhance clinical neurological disease induction. Since our data indicated that non-env sequences in PVC-211 MuLV played a role in enhancing the induction of neurological disease, we prepared another series of chimeric viruses and examined their ability to induce abnormal neurological signs and pathological changes in the CNS in order to determine which non-env sequences were important. As shown in Fig. 2, all of the chimeras bear the PVC-211 MuLV-derived XbaI-ClaI region, which includes the gp7o coding region of the env gene, and various amounts of the 5' one-third of PVC-211 PVF-G K PVF-B _71 PVF-C PVF-D PVF-E AH I E -L _ 0/10 MuLV in an F-MuLV background. When injected into rats, these chimeras were all capable of eventually causing pathological changes in the CNS, but there were differences in the level of pathological changes caused by these viruses and their potential to induce clinical neurological disease. PVF-G, which contains the 5' leader sequence, the entire gag gene, and the 5'-terminal region of the pol gene of PVC-211 MuLV, was able to induce neurological disease in rats with an incidence and latency indistinguishable from those of PVC-211 MuLV. The pathological changes in the CNS caused by PVF-G were as extensive as those induced by PVC-211 MuLV. PVF-I, whose HindIII-EcoRI region is replaced by the corresponding region from F-MuLV, caused clinical neurological disease in 10 of 12 injected rats, but the latency was longer than in rats injected with PVF-G (10 weeks versus 3 weeks). PVF-J, whose AatII-HindIII region Neurogathogen i ci tv Xb C Clinical Disease Onset (weeks) Pathological changes in the CNS 9/9 3 4/4 0/4 4/4 0/5 4/5 3/4 0/4 PVF- I 10/ /5 PVF- J 0/16 4/4 PVF-LKET LTR L- gag po1 l,iltr env Effect of non-env regions of the PVC-211 MuLV genome on manifestation of neurological disease. The genomes of chimeric FIG. 2. viruses are represented as described in the legend to Fig. 1. Restriction sites: A, AatII; C, ClaI; E, EcoRI; H, HindIII; Xb, XbaI. The neuropathogenicity of the viruses was assayed and is shown as described in the legend to Fig. 1. Three symptomatic and two asymptomatic rats infected with PVF-I and three symptomatic and one asymptomatic rat infected with PVF-K were examined for neuropathological changes. 8/9 8 4/4

4 VOL. 67, 1993 NEUROPATHOGENICITY AND CELL TROPISM OF PVC-211 MuLV 4583 PVF-H E K 1h E Xb B C K Clinical Disease n.a- j 8/8 Neuropathogeni ci tv Onset Pathological (weeks) chanaes in the CNS 3 3/3 PVF-L PVF-M E 6/6 E-M I I 0/8 PVF-N 0/4 LTF gag pol1 env Localization of the env gene determinant responsible for neuropathogenicity to the 5' half of the SU coding region. The genomes FIG. 3. of the chimeric viruses and their neuropathogenicity are represented as described in the legend to Fig. 1. Restriction sites: B, BamHI; C, ClaI; E, EcoRI; K, KpnI; Xb, XbaI. 6 3/3 0/4 was further replaced by the corresponding region from F-MuLV, failed to cause any clinical signs of neurological disease within 28 weeks after virus infection and induced less extensive pathological changes in the CNS than PVF-G. PVF-K, which is structurally similar to PVF-D (Fig. 1) but has the PVC-211 MuLV-derived HindIII-EcoRI region, was able to cause clinical neurological disease in eight of nine injected rats with a latency of 8 weeks, versus the lower incidence (three of eight rats) and the longer latency (20 weeks) of PVF-D (Fig. 1). These data suggest that both the AatII-HindIII region and the HindIII-EcoRI region of PVC- 211 MuLV contain determinants which enhance the induction of neurological disease and that they may act in a cooperative manner. When the DNA sequences between the AatII and the HindIlI sites in PVC-211 MuLV clone 3d and F-MuLV clone 57 are compared, PVC-211 MuLV contains 18 substitutions in this region, which result in seven amino acid changes in the N-terminal region of the glycosylated gag precursor protein (6, 22) and four amino acid changes in the N-terminal half of the MA (p15) protein. The HindIII-EcoRI region of PVC-211 MuLV contains 38 point mutations, which result in substitution of three amino acids in the C-terminal half of the MA protein, four amino acids in p12, six amino acids in the CA (p30) protein, one amino acid in the NC protein (plo), one amino acid in the PR protein, and five amino acids in the N-terminal region of the reverse transcriptase (RT). This region also has a 3-bp deletion, resulting in the loss of a proline residue in the MA protein of PVC-211 MuLV compared with the MA protein of F-MuLV clone 57. 5' half of SU coding region of PVC-211 MuLV env contains the determinant for induction of pathological changes and neurological disease. To further localize the determinant in the env gene of PVC-211 MuLV that is essential for the induction of neurological disease, we constructed additional chimeras containing different portions of the PVC-211 MuLV env gene (Fig. 3). All of these chimeras contained equivalent amounts of PVC-211 MuLV non-env determinants in the 5' one-third (AatII-EcoRI region) of the genome. As shown in Fig. 3, chimera PVF-H (which contains the entire SU coding region of the env gene of PVC-211 MuLV) induced neurological disease as efficiently as the parental PVC-211 MuLV. PVF-L (which contains the XbaI-BamHI region corresponding to the 5' half of the SU coding region of the PVC-211 MuLV env gene) was also neuropathogenic in 100% of the injected rats, although the onset of the disease was slightly delayed compared with the onset after PVC-211 MuLV infection (6 weeks versus 3 weeks). However, PVF-M (which contains only the BamHI-ClaI region of the env gene of PVC-211 MuLV, encoding the carboxyl half of SU) was unable to induce typical spongiform neurodegenerative lesions or clinical neurological disease for up to 28 weeks after injection, although 1 of the 16 rats injected with PVF-M developed an endothelial cell-derived brain tumor 18 weeks after virus inoculation. These observations suggest that the 5' half of the SU-encoding region of the env gene of PVC-211 MuLV is essential for neuropathogenicity. Although the 3' half of the SU coding region of the env gene of PVC-211 MuLV may play a role in decreasing the latency of disease induction in the presence of the 5' half of the PVC-211 MuLV env gene, a virus carrying that region alone is not capable of causing typical neurodegenerative changes in the CNS. Level of virus replication in cultured BCEC correlates with neuropathogenicity. Previous studies indicated that PVC-211 MuLV expression occurred predominantly in BCEC and was much more extensive than nonneuropathogenic F-MuLV expression (10). We therefore designed studies to determine whether viral replication efficiency in BCEC was a crucial determinant for neuropathogenicity. Primary cultures of BCEC were prepared from 3-week-old Fisher rats and infected with PVC-211 MuLV, F-MuLV, PVF-A, or PVF-B at a multiplicity of infection of 1.0. For comparison, rat fibroblast cell lines (NRK or Rat-i) were infected with the same viruses. The level of virus infection was measured by detecting integrated viral DNA by hybridization analysis and by detecting viral protein expression via metabolic labeling and immunoprecipitation. All four viruses showed a similar level of integration in NRK cells (Fig. 4A) and in Rat-i cells (data not shown). However, in BCEC (Fig. 4B), viral DNA could be detected only in cells infected with PVC-211 MuLV (lane 2) and PVF-A (lane 4), both of which are neuropathogenic. Viral DNA could not be detected by hybridization analysis in BCEC infected with F-MuLV (Fig. 4B, lane 3) or PVF-B (Fig. 4B, lane 5) but were detectable by PCR analysis (data not shown), indicating that only a few viral integrations had occurred in these cells. By comparison with the hybridization signal obtained for a known amount of cloned viral DNA, the levels of integrated F-MuLV and PVF-B DNA were estimated to be much less than one copy per cell, even at a multiplicity of infection of 1.0. This indicates that the efficiency of the infection was reduced at an early stage in the replication cycle, before integration. The level of viral protein production detected by immunoprecipitation was comparable to the level of virus integra-

5 4584 MASUDA ET AL. A B (kb) i (kb) _awl FIG. 4. Analysis of integrated viral DNA in fibroblasts and BCEC infected with neuropathogenic and nonneuropathogenic viruses. NRK cells (A) or BCEC (B) were mock infected or infected with virus at a multiplicity of infection of 1.0. Four days after virus infection, high-molecular-weight DNA was extracted and analyzed for the presence of integrated viral DNA as described in Materials and Methods. Viruses tested were PVC-211 MuLV (lane 2), F-MuLV clone 57 (lane 3), PVF-A (lane 4), and PVF-B (lane 5). Lane 1 in each panel represents DNA from mock-infected cells. The numbers at the left of each panel indicate the positions of molecular size markers (in kilobases). tion (Fig. 5). Rat-i cells infected with each of the viruses expressed a similar level of the envelope precursor protein (Pr85e.v) (Fig. 5A) and thegag precursor protein (Pr659aE) (Fig. SB). In contrast, there were significant differences in the levels of viral proteins produced in BCEC infected with the four viruses. BCEC infected with neuropathogenic PVC-211 MuLV and PVF-A expressed much higher levels of the envelope (Fig. SC) and gag (Fig. SD) precursor proteins than BCEC infected with the nonneuropathogenic F-MuLV and PVF-B. Immunofluorescence microscopy demonstrated that greater than 90% of BCEC were infected with the neuropathogenic viruses, whereas less than 10% of the cells were infected with the nonneuropathogenic viruses (data not shown). Viral determinants that affect neuropathogenicity also determine BCEC tropism. To further investigate the relationship between the ability of a virus to replicate in cultured BCEC and neuropathogenicity, we compared the BCEC tropism of various PVC-211 MuLV/F-MuLV chimeric viruses that differ in their neuropathogenicity. Each virus was inoculated onto BCEC or control NRK cells at a multiplicity of infection of 1.0, and integrated viral DNA was detected by hybridization analysis. As shown in Fig. 6A, C, and E, viral DNA was easily detected in NRK cells infected with any of the chimeric viruses, but there were significant differences in Pr85 nv" Pr659,39 A B ~~~~~~~~~~ J. VIROL. the levels of viral DNA detected in BCEC. Chimera PVF-G, which contains both env and non-env determinants from PVC-211 MuLV and is as neuropathogenic as PVC-211 MuLV (Fig. 2), demonstrated a high level of infectivity on BCEC (Fig. 6B, lane 1). In BCEC infected with PVF-I (Fig. 6B, lane 2), which has the HindIII-EcoRI region of F-MuLV and induces clinical symptoms of neurological disease by 10 weeks postinoculation (Fig. 2), less viral DNA was detected than in PVF-G-infected BCEC. PVF-J, which lacks both non-env determinants in the AatII-HindIII region and the HindIII-EcoRI region from PVC-211 MuLV and induces neuropathology but not clinical signs (Fig. 2), showed a very low level of virus integration into BCEC (Fig. 6B, lane 3). We also compared chimeras PVF-D and PVF-K, which differ only in the HindIII-EcoRI region of the genome, for their ability to replicate on BCEC. Only a low level of viral DNA could be detected in BCEC infected with PVF-D (Fig. 6D, lane 4), which contains the HindIII-EcoRI region from F-MuLV and causes clinical signs of neurological disease in a third of inoculated rats after a 20-week latency. In contrast, relatively high levels of viral DNA could be detected in cells infected with PVF-K (Fig. 6D, lane 5), which contains the HindIII-EcoRI region from PVC-211 MuLV and causes abnormal neurological signs by 8 weeks postinoculation. Finally, we compared chimeras PVF-L and PVF-N, which differ in the XbaI-BamHI region of the env gene, for their ability to replicate on BCEC. Although these two chimeric viruses were equally infectious on NRK cells (Fig. 6E), there was a striking difference in their infectivity on BCEC. Neuropathogenic PVF-L (which contains the region of the PVC-211 MuLV env gene encoding the amino-terminal half of the SU protein) showed a high level of virus integration (Fig. 6F, lane 6), while the amount of viral DNA in BCEC infected with PVF-N (whose entire env gene is derived from F-MuLV) was below the level of detection by hybridization analysis (Fig. 6F, lane 7). Thus, there appears to be a direct correlation between the ability of a chimeric virus to replicate on BCEC and its ability to cause neuropathological changes in the CNS and clinically evident neurological disease. DISCUSSION Passage of the nonneuropathogenic F-MuLV through rats resulted in the generation of a highly neuropathogenic virus, PVC-211 MuLV (11). Our studies have been aimed at C D t*0-2c0o -g f.r.~~~4 FIG. 5. Immunoprecipitation of viral proteins from fibroblasts and BCEC infected with neuropathogenic and nonneuropathogenic viruses. Rat-i cells (A and B) and BCEC (C and D) were labeled with [35S]methionine for 15 min at 7 days after virus infection (multiplicity of infection, 1.0), and cell lysates were immunoprecipitated with either goat anti-rauscher virus gp7o serum (A and C) or goat anti-rauscher virus p30 serum (B and D). Viruses tested were PVC-211 MuLV (lanes 2), F-MuLV clone 57 (lanes 3), PVF-A (lanes 4), and PVF-B (lanes 5). Lane 1 in each panel represents uninfected cells. Positions of the envelope precursor Pr85e"v and the gag precursor Pr65gag are shown on the left. The signals above 65 kda are compatible with glycosylated gag proteins (6). The positions of molecular size markers (in kilodaltons) are shown on the right.

6 VOL. 67, 1993 NEUROPATHOGENICITY AND CELL TROPISM OF PVC-211 MuLV 4585 A B C D E F K- FIG. 6. Effects of the viral determinants responsible for neuropathogenicity on BCEC tropism. NRK cells (A, C, and E) and BCEC (B, D, and F) were infected with PVF-G (lanes 1), PVF-I (lanes 2), PVF-J (lanes 3), PVF-D (lanes 4), PVF-K (lanes 5), PVF-L (lanes 6), or PVF-N (lanes 7) at a multiplicity of infection of 1.0. Integrated viral DNA was detected by hybridization analysis as described in Materials and Methods. Numbers on the left of each panel show the positions of molecular size markers (in kilobases). determining what genetic elements acquired by PVC-211 MuLV make it capable of causing a rapidly progressive neurodegenerative disease and understanding how they function. Molecular cloning and sequencing of the virus revealed that PVC-211 MuLV is closely related in structure to the nonneuropathogenic F-MuLV clone 57 (15, 23), and this similarity has made it feasible to prepare chimeras from the two viruses and attempt to fine map the determinants in PVC-211 MuLV responsible for its neuropathogenicity. We previously showed, by using chimeric viruses, that the env gene of PVC-211 MuLV was essential but not sufficient for the virus to cause neurological disease (15). This suggested that the env gene of PVC-211 MuLV plays a key role in causing neurological disease but that the virus also contains an additional determinant(s) in the non-env region of its genome, which is absent in F-MuLV, that is responsible for complete neuropathogenicity. In this study, we demonstrated that env gene sequences from PVC-211 MuLV are sufficient for causing microscopic pathological changes in the CNS of infected rats and that certain non-env sequences as well as env gene sequences in PVC-211 MuLV regulate the neuropathogenicity of the virus in vivo by determining its replication efficiency in BCEC, a primary site of neuropathogenic PVC-211 MuLV replication. We observed that the majority of asymptomatic rats infected with chimeric viruses (PVF-C, -D, and -J) containing only env gene sequences from PVC-211 MuLV exhibited neuropathological changes qualitatively comparable to but quantitatively less extensive than those caused by wild-type PVC-211 MuLV. This indicates that the expression of the env gene of PVC-211 MuLV in the CNS is sufficient for the induction of pathological changes but that an additional non-env determinant(s) regulates the level of pathological changes and the expression of abnormal neurological signs. Because the chimeras bearing the env gene of PVC-211 MuLV failed to cause clinically overt neurological disease as efficiently as the parental PVC-211 MuLV unless they contained sequences from the 5' one-third (AatII-EcoRI region) of the PVC-211 MuLV genome, the latter region of PVC-211 MuLV appeared to contain the non-env determinant(s) necessary for the rapid and complete induction of neurological disease in the presence of the env gene determinant. In an attempt to further identify the non-env determinant(s) within theaatal-ecori region, we prepared chimeric viruses containing env sequences from PVC-211 MuLV and various amounts of the 5' half of the PVC-211 MuLV genome. Our data indicated that at least two separate determinants exist within this region, one within the AatII- HindIII region and another within the HindIII-EcoRI region, since viruses which carry only one of the two can cause clinical neurological disease. The time to onset of neurological signs, however, is longer with viruses carrying only one of the two regions than with viruses that carry both regions (8 to 10 weeks versus 3 weeks). Therefore, these two determinants appear to function cooperatively. The env gene determinant for neuropathogenicity was further localized within the XbaI-BamHI region of PVC-211 MuLV, which includes the 5' half of the SU protein-coding region of the env gene. In the presence of the PVC-211 MuLV-derived AatII-EcoRI region, this region of the env gene of PVC-211 MuLV in the context of nonneuropathogenic F-MuLV sequences was sufficient to confer neuropathogenicity on a virus. The major determinants for the pathogenicity of two other neuropathogenic viruses, Cas- Br-E MuLV and tsl Moloney MuLV, were also mapped within the 5' half of the SU coding region (17, 28). The primary target of PVC-211 MuLV infection within the CNS is the BCEC (11), which appears to be a site of infection for other neuropathogenic MuLVs (12, 20, 24, 29). Thus, virus infection of BCEC may be an essential early step in neuropathogenesis by MuLVs, and viral determinants that regulate neuropathogenicity may also control the BCEC tropism of the virus. In order to test this, we compared the ability of PVC-211 MuLV, F-MuLV, and various neuropathogenic and nonneuropathogenic chimeric viruses for their ability to replicate in cultured BCEC prepared from rat brains. Our results indicated a direct correlation between the ability of a virus to replicate in BCEC in vitro and the neuropathogenicity of the virus in vivo. In the presence of the PVC-211 MuLV-derived env gene, the non-env determinants in both the AatII-HindIII region and the HindIII- EcoRI region of PVC-211 MuLV enhanced the infectivity of the virus on BCEC. In contrast, in the presence of the AatII-EcoRI region of PVC-211 MuLV, which contains both non-env determinants for neuropathogenicity, the 5' half of the SU protein-coding region of the env gene from PVC-211 MuLV was essential and sufficient for conferring high replication efficiency in BCEC on a virus. These data indicate that both non-env gene determinants and the 5' half of the SU protein-coding region of the env gene are crucial for determining the ability of the virus to infect BCEC. The N-terminal half of the SU protein, which appears to be important for the BCEC tropism of PVC-211 MuLV, corresponds to the region involved in receptor binding (9). Therefore, the SU protein of PVC-211 MuLV may bind to receptors on BCEC more efficiently than that of F-MuLV. The receptor on fibroblasts used by ecotropic MuLVs has been identified at the molecular level by the isolation of a DNA clone encoding it (1). It is not known whether PVC-211 MuLV uses the same receptor molecule on BCEC, but if it does, the receptor must be altered in some way to allow PVC-211 MuLV but not F-MuLV to bind efficiently. Our preliminary hybridization analysis of mrna with an ecotropic MuLV receptor cdna probe (1) indicates that BCEC express much lower levels of the gene than Rat-1 fibroblasts (14). Thus, PVC-211 MuLV either may be able to bind to this receptor with a much higher affinity than F-MuLV or may use another receptor on BCEC encoded by a distinct gene. It is intriguing to speculate that PVC-211 MuLV may have evolved from F-MuLV into a neuropathogenic virus because it has acquired the ability to enter BCEC by binding to a receptor expressed exclusively on this cell type. Compared with F-MuLV clone 57, PVC-211 MuLV has 12 amino acid substitutions in the region of the SU protein (15) that is

7 4586 MASUDA ET AL. responsible for BCEC tropism. Studies are in progress to identify which substitutions are responsible for the significantly different BCEC tropism observed with these two viruses. It is not known how the non-env determinants of PVC-211 MuLV control the ability of this virus to replicate in BCEC. TheAatII-HindIII region of PVC-211 MuLV, compared with the corresponding region of F-MuLV clone 57, contains substitutions of 18 nucleotides. It has previously been shown that F-MuLV clone FB29 contains a 5' region, delineated by the Kjpnl site in the R region of the LTR and the PstI site upstream of the AUG codon for Pr65fag, that influences the onset of neurological disease caused by chimeras containing the env gene of the neuropathogenic Cas-Br-E MuLV (21). Since this region of F-MuLV clone FB29 shares a stretch of about 200 bp with the AatII-HindIII region of PVC-211 MuLV, it is possible that a determinant of PVC-211 MuLV necessary for the development of rapid clinical neurological disease resides within this 200-bp region. This region, delineated by the AatII and PstI sites, corresponds to the 5' leader sequence of the gag gene and has been reported to encode the N-terminal region of the glycosylated gag precursor protein (22). When the nucleotide sequences of this region are compared between PVC-211 MuLV (23) and FB29 MuLV (18), both of which appear to carry an element that reduces disease latency, and F-MuLV clone 57 (GenBank accession number X02794) and Cas-Br-E (19), both of which appear to lack this element, only three nucleotides were shared by PVC-211 MuLV and F-MuLV clone FB29 and lacking in the other two viruses. Only one of these three nucleotides results in an amino acid change in the aminoterminal region of the glycosylated gag precursor protein. Interestingly, the sizes of the glycosylated gag precursor proteins of PVC-211 MuLV, F-MuLV, PVF-A, and PVF-B appeared to differ in Rat-1 cells depending on the derivation of the 5' leader sequence encoding the amino-terminal region of the protein (Fig. SB). These differences might be due to the amino acid substitution mentioned above. Although no definite role has been attributed to the glycosylated gag precursor protein, it has been suggested that the protein may regulate virus maturation (4, 7). However, since the glycosylated gag protein is expressed on the cell surface but not incorporated into the virion (5, 13), the mechanism by which it could affect the early stage of virus replication in BCEC is unknown. The possibility that the nucleotide sequences in this region have a cis-acting function that controls viral replication should also be considered. The other 5' region of PVC-211 MuLV which appears to be necessary for the rapid induction of neurological disease, delineated by the HindIII and EcoRI sites, corresponds to the 3' three-quarters of the gag gene and the 5' terminus of the pol gene. Compared with F-MuLV clone 57, this region of PVC-211 MuLV contains 38 nucleotide changes and a 3-bp deletion. The deletion results in the loss of one proline residue in the MA (p15) protein of PVC-211 MuLV. This region of the MA protein of F-MuLV clone 57 has a stretch of three proline residues, which might constitute a hinge region to determine the protein conformation, and it is possible that the loss of one proline residue in this region has a significant effect on protein structure. Interestingly, both F-MuLV clone FB29 and Cas-Br-E MuLV, which, like PVC-211 MuLV, carry sequences in the HindIII-EcoRI region that decrease the time to onset of neurological disease (21), are also missing the same proline in their MA proteins. It has been reported that alterations in the MA protein can affect virus replication at an early stage (3). Therefore, it is J. VIROL. possible that the MA protein of F-MuLV clone 57 is inhibitory to virus replication at a step before integration into BCEC and that the changes that have occurred in the PVC-211 MuLV MA protein eliminate this inhibitory effect. Further studies are needed to determine the role of these sequences in the replication of PVC-211 MuLV in BCEC. These studies indicate that PVC-211 MuLV has become highly neuropathogenic through the acquisition of genetic changes that facilitate its replication in BCEC. Although other neuropathogenic MuLVs also appear to infect BCEC (12, 20, 24, 29), our studies with PVC-211 MuLV are the first to show that virus infection of BCEC may play a key role in the induction of neurological disease. It is possible that the BCEC play a role only as a gateway for the virus to propagate into the CNS. However, since glial cells and neurons, the only cells that show reactivity or pathological changes in the CNS after PVC-211 MuLV inoculation, are not productively infected by the virus (10), it is more likely that virus-infected BCEC actively exert direct or indirect effects on glial cells and neurons. Virus-infected BCEC may produce molecules that stimulate astrocyte proliferation or are directly neurotoxic. Studies are in progress to test these possibilities. ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance of Natalie Dugger, Charlotte Hanson, and Nimisha Vyas. We thank Nancy Lohrey for synthetic oligonucleotide primers for PCR. 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