Neurologic Disease Induced by Polytropic Murine Retroviruses: Neurovirulence Determined by Efﬁciency of Spread to Microglial Cells

SHELLY J. ROBERTSON, KIM J. HASENKRUG, BRUCE CHESEBRO, AND JOHN L. PORTIS* Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840

Received 10 May 1996/Accepted 26 March 1997

Several murine leukemia viruses (MuLV) induce neurologic disease in susceptible mice. To identify features of central nervous system (CNS) infection that correlate with neurovirulence, we compared two neurovirulent MuLV, Fr98 and Fr98/SE, with a nonneurovirulent MuLV, Fr54. All three viruses utilize the polytropic receptor and are coisogenic, each containing a different envelope gene within a common genetic background. Both Fr98 and Fr98/SE induce a clinical neurologic disease characterized by hyperexcitability and ataxia yet differ in incubation period, 16 to 30 and 30 to 60 days, respectively. Fr54 infects the CNS but fails to induce clinical signs of neurologic disease. In this study, we compared the histopathology, regional virus distribution, and cell tropism in the brain, as well as the relative CNS viral burdens. All three viruses induced similar histopathologic effects, characterized by intense reactive astrogliosis and microglial activation associated with minimal vacuolar degeneration. The infected target cells for each virus consisted primarily of endothelial and microglial cells, with rare oligodendrocytes. Infection localized predominantly in white matter tracts of the cerebellum, internal capsule, and corpus callosum. The only feature that correlated with relative neurovirulence was viral burden as measured by both viral CA protein expression in cerebellar homogenates and quantification of infected cells. Interestingly, Fr54 (nonneurovirulent) and Fr98/SE (slow disease) had similar viral burdens at 3 weeks postinoculation, suggesting that they entered the brain with comparable efficiencies. However, spread of Fr98/SE within the brain thereafter exceeded that of Fr54, reaching levels of viral burden comparable to that seen for Fr98 (rapid disease) at 3 weeks. These results suggest that the determinants of neurovirulence in the envelope gene may influence the efficiency of virus spread within the brain and that a critical number of infected cells may be required for induction of clinical neurologic disease.

Retroviruses are important human and animal pathogens is different from the paralytic diseases induced by the ecotropic associated with a number of neoplastic, immunodeficiency, and MuLV (39). chronic degenerative disorders. They are also the etiologic Similar to the ecotropic viruses (12), the determinants for agents of certain diseases affecting the nervous system. Retro- neurovirulence in FMCF98 reside within the envelope gene. viruses, including primate and animal lentiviruses (8, 22, 31, 32, This was demonstrated by the construction of a chimeric virus, 34, 36, 37, 40), human T-cell leukemia virus type 1 (29), and Fr98 (formerly named Fr98E) (39), which contains the 3Ј pol several murine leukemia viruses (MuLV) (reviewed in refer- and env genes of FMCF98 in the background of FB29, a non- ence 51), induce a spectrum of neurologic deficits affecting neurovirulent strain of Friend MuLV (38). Fr98 induces a behavior, cognitive, and motor functions in their respective clinical disease similar to that induced by FMCF98, but with a hosts. shortened incubation period (39). In contrast, the chimeric Several different types of neurologic disease are induced by polytropic virus clone Fr54, which contains the env gene of a MuLV. Ecotropic Friend strain TR1.3 cause acute fatal cere- nonneurovirulent polytropic isolate, FMCF54, infects the cen- brovascular hemorrhage (35). Other ecotropic viruses, such as tral nervous system (CNS) but causes no neurologic deficits the wild mouse MuLV (CasBrE) (14), the Moloney MuLV (19). (tsMoBA-1) (5, 52), and the Friend MuLV (PVC-211) (20, 23) In the course of mapping studies to identify envelope se- cause more chronic disease manifested by tremor and hindlimb quences that influence neuropathogenicity, we constructed a paralysis. These viruses induce a noninflammatory spongiform series of polytropic viruses containing chimeric envelope genes neurodegeneration involving grey and sometimes white matter composed of segments from the neurovirulent polytropic virus of the neocortex, hindbrain, and spinal cord. Recently, a FMCF98 and the nonneurovirulent virus FMCF54. Like Fr98, Friend mink cell focus-forming virus, FMCF98, has also been all of these viruses had the same FB29 genetic background. shown to be neurovirulent in susceptible mice (6, 39). FMCF98 These studies demonstrated that the viral envelope gene influ- is unique among neurovirulent MuLV because it has a poly- ences relative viral burden in the brain and that, with one tropic receptor specificity (7, 42) and induces a clinical syn- exception, viral burden correlated with relative neurovirulence drome characterized by hyperexcitability and imbalance which (19). Interestingly, the exception was the nonneurovirulent virus Fr54, which exhibited a viral burden, at 2 and 3 weeks after inoculation, comparable to that of one of the neuroviru- * Corresponding author. Mailing address: Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of lent viruses, Fr98/SE, which contained a chimeric envelope Allergy and Infectious Diseases, National Institutes of Health, 903 S. gene. These results raised the intriguing possibility that enve- 4th St., Hamilton, MT 59840. Phone: (406) 363-9339. Fax: (406) 363- lope sequences that influence neuroinvasiveness (i.e., the abil- 9204. E-mail: [email protected]. ity to infect the brain) may be separable from those determin-

5287 5288 ROBERTSON ET AL. J. VIROL. ing neurovirulence (i.e., the ability to induce clinical neurologic antibody was a biotinylated goat anti-rat immunoglobulin (Vector Laboratories, disease). This is a particularly relevant issue in view of the Inc.). This was followed by an incubation with horseradish peroxidase-conjugated conflicting reports on the importance of viral burden in neu- streptavidin (BioGenex) for 15 min at room temperature. Sections were washed in PBS (three times for 10 min each), developed with AEC as described above, ropathogenicity of both human immunodeficiency virus (1, 2, and counterstained with Mayer’s hematoxylin. 17, 21) and simian immunodeficiency virus (41, 43, 46, 48). In Immunohistochemical staining of glial fibrillary acidic protein (GFAP) was this study, we examined in more detail properties of the CNS performed on paraffin-embedded sections (4 ␮m). Sections were deparaffinized, infection by two of the neurovirulent viruses, Fr98 and Fr98/ rehydrated, and washed in PBS prior to incubation with rabbit anti-bovine GFAP SE, and the nonneurovirulent virus Fr54 in an effort to identify antiserum (Dakopatts) for1hat37°C. Sections were incubated with a biotinylated goat anti-rabbit immunoglobulin (Vector) for 30 min at 37°C, followed by features of these infections that correlate with neurovirulence. an incubation with horseradish peroxidase-conjugated streptavidin (BioGenex) Surprisingly, no differences were found between the neuroviru- for 15 min at room temperature. Sections were developed with AEC as described lent and nonneurovirulent viruses with respect to the appear- above and counterstained with Mayer’s hematoxylin. ance of vacuolar degeneration, activation of astrocytes and Cell counts. Fresh, frozen brain tissue was sectioned (5 ␮m), and every eighth microglial cells, regional distribution of virus, or the cell types section was stained for viral envelope protein by using a polyclonal goat anti-gp70 infected in the brain. However, application of more quantita- antiserum as described above. The number of gp70-positive cells in the cerebella was determined in a total of five sections. From these five sections, the mean Ϯ tive measurements of viral protein expression in the brain standard deviation was determined for four mice per virus strain: Fr98/SE (52 to revealed viral burden to be a consistent correlate of neuroviru- 75 days postinoculation) and Fr54 (52 to 75 days postinoculation). The means Ϯ lence in this model. The results suggest that the neurovirulent standard errors of these two groups were compared by using the unpaired viruses differed from the nonneurovirulent virus in the extent two-tailed Student t test. of their spread among microglial cells after initial virus entry Double-label indirect immunofluorescence. Mice were anesthetized by inhalation of methoxyfluorane and perfused with PBS followed by 0.5% paraformal- into the brain. dehyde-lysine-periodate fixative (PLP) (30). Vibratome sections (50 ␮m) of PLP-fixed brain were pretreated with 0.1% Triton X-100 for 5 min and then blocked with 5% bovine serum albumin for1hatroom temperature. Viral MATERIALS AND METHODS envelope protein and cell-specific markers were labeled sequentially. Sections were incubated with primary antibodies overnight at 4°C and then with secondary Viruses, animals, and clinical disease. The chimeric viruses were generated as antibodies at room temperature for 1 to 2 h. The sections were washed (three Ј previously described (19). Briefly, Fr98 and Fr54 contain the 3 pol and env times for 10 min each) in PBS after each antibody incubation step. For localiza- sequences (SphI-to-ClaI fragment) of FMCF98 (6) and FMCF54 (33), respec- tion of viral protein in astrocytes, sections were incubated with rabbit anti-bovine tively, within the background of FB29, a nonneurovirulent strain of Friend GFAP antiserum that was detected with a tetramethylrhodamine-5-(and 6) iso- MuLV (45). Fr98/SE contains the SphI-to-EcoRI fragment of FMCF98 env thiocyanate (TRITC)-conjugated sheep anti-rabbit immunoglobulin (Cappel). combined with the EcoRI-to-ClaI fragment of FMCF54 env that was also placed Sections were then incubated with a polyclonal goat anti-MuLV gp70 antiserum within the background of FB29 (19). Virus stocks were prepared from superna- tants of confluently infected Mus dunni fibroblast cells as previously described that was detected with fluorescein isothiocyanate (FITC)-conjugated donkey ϫ 6 ϫ anti-goat immunoglobulin (ICN). For localization of viral protein in oligoden- (38). Virus titers (in focus-forming units/milliliter) were 1.3 10 for Fr98, 3.6 ␮ 106 for Fr54, and 4.2 ϫ 106 for Fr98/SE, as determined by a previously described drocytes, PLP-fixed vibratome sections (50 m) were incubated with a mouse Ј Ј Ј focal immunoassay (44) on M. dunni cells. monoclonal antibody to 2 ,3 -cyclic nucleotide 3 -phosphodiesterase (CNP) (50) Inbred Rocky Mountain White (IRW) mice were bred and raised at Rocky (kindly provided by Bruce Trapp, Cleveland Clinic, Cleveland, Ohio) that was Mountain Laboratories animal facilities. Neonatal mice (Ͻ24 h old) were inoc- detected with TRITC-conjugated goat anti-mouse immunoglobulin (Pierce). ulated intraperitoneally with approximately 3 ϫ 104 focus-forming units of virus Then sections were incubated with a polyclonal rabbit anti-MuLV gp70 that was per mouse. Mice were observed daily for clinical disease and compared with detected with an FITC-conjugated goat anti-rabbit immunoglobulin G (IgG) age-matched uninoculated control mice. The first signs of neurologic dysfunction (Cappel). Viral protein localization in microglia was performed either on PLP- were hyperactivity and bilateral imbalance when climbing up the side of the box fixed vibratome sections (50 ␮m) or on frozen sections (4 to 6 ␮m) fixed with (19, 39). Clinical disease progressed to hindlimb weakness, ataxia, and reflex 3.7% formaldehyde in PBS. Microglia were labeled with a rat monoclonal anti- abduction of the hindlimbs, at which time the mice were euthanized. All in vivo body to Mac-1 detected with Texas red-conjugated goat anti-rat IgG (Southern experiments were done in accordance with the standards established by the Biotechnology Associates, Inc.) followed by a polyclonal rabbit anti-MuLV gp70 Rocky Mountain Laboratories Animal Care and Use Committee and the Na- (kindly provided by Roland Friedrich) detected with an FITC-conjugated goat tional Institutes of Health. anti-rabbit IgG (Cappel). For all dual-labeling schemes, each secondary antibody Histopathology. Animals were anesthetized by inhalation of methoxyfluorane was shown not to bind in the absence of primary antibody or cross-react with and perfused through the left ventricle of the heart with phosphate-buffered primary antibodies of different species. Double-labeling immunofluorescence saline (PBS; 10 mM Na HPO ,10mMNaHPO , 130 mM NaCl [pH 7.4]) 2 4 2 4 was performed on brains from at least three mice per virus strain: Fr98 (20 to 34 followed by 4% paraformaldehyde in phosphate buffer (0.1 M Na HPO , 0.1 M 2 4 days postinoculation), Fr98/SE (48 to 73 days postinoculation), Fr54 (48 to 73 NaH2PO4 [pH 7.4]). Brains were removed and fixed in 4% paraformaldehyde- phosphate buffer for an additional 24 to 48 h, after which time they were days postinoculation), and uninoculated controls (20 to 73 days of age). Immun- dehydrated through graded ethanols and embedded in paraffin (Polysciences, ofluorescence-labeled sections were analyzed by using a Nikon Microphot SA Inc.). Coronal or sagittal sections (4 ␮m) were stained with hematoxylin and fluorescence microscope. eosin by using standard histology techniques. Brain sections were evaluated for Immunoblotting. Mice were anesthetized by inhalation of methoxyfluorane neuropathology in four mice inoculated with Fr98 (21 days postinoculation), five and exsanguinated by axillary vessel incision. Brains were taken from infected mice inoculated with Fr98/SE (57 to 74 days postinoculation), eight mice inoc- mice at 3 and 8 weeks postinoculation with age-matched uninoculated mice. ulated with Fr54 (55 to 159 days postinoculation), and seven uninoculated con- Extracts from mouse cerebellum were prepared by making 10% (wt/vol) homog- trols (21 to 106 days of age). enates in 0.5% Nonidet P-40 in TNE (0.01 M Tris base, 0.15 M NaCl, and 0.001 Immunohistochemistry. Immunohistochemical staining was performed on M EDTA [pH 7.4]) and were clarified by centrifugation as described elsewhere fresh frozen sections (4 to 6 ␮m), collected on glass slides, and fixed in 3.7% (39). Immunoblots were performed as previously described (11). Briefly, proteins formaldehyde in PBS for 5 min at room temperature. Sections were blocked for were separated by sodium dodecyl sulfate–12% polyacrylamide gel electrophore- 30 min at 37°C with either 5% bovine serum albumin or 3% normal serum of the sis (SDS-PAGE) and transferred to Immobilon P membranes (Millipore). Cap- same species as the secondary antibody. Sections were washed (three times for 10 sid protein (p30) was detected with rabbit anti-p30gag antiserum (26) followed by min each) in PBS after each antibody incubation. For detection of viral protein horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad Laboratories) and determination of virus distribution, sagittal sections were incubated with a and developed with the ECL (enhanced chemiluminescence) system (Amer- polyclonal goat anti-MuLV gp70 antiserum (kindly provided by Roland sham). Friedrich, Institute of Medical Virology, Geissen, Germany) for1hat37°C and The relative amounts of viral protein were determined by immunoblot anal- then incubated with horseradish peroxidase-conjugated rabbit anti-goat immunoglobulin (ICN Biochemicals Inc.) for 30 min at room temperature. Sections yses of twofold serial dilutions of cerebellar homogenates. Samples from three were developed with 3-amino-9-ethylcarbazole (AEC) in 0.1 M acetate buffer mice per virus strain per time point were analyzed in pairs on the same immu- (pH 5.0) and were counterstained with Mayer’s hematoxylin. noblot as follows: (i) Fr98 compared with Fr54, both at 3 weeks postinoculation; For detection of activated microglia/pericytes, the same immunohistochemical (ii) Fr98/SE compared with Fr54, both at 3 weeks postinoculation; (iii) Fr98/SE staining procedure was used except that the primary antibody was a rat mono- compared with Fr54, both at 8 weeks postinoculation; and (iv) Fr98 at 3 weeks clonal antibody directed to Mac-1 (CD11b complement receptor) (47). Mac-1 is compared with Fr98/SE at 8 weeks postinoculation. The comparisons of the a specific marker of microglia/macrophages in the brain, and upregulation of this relative viral protein content were made only between paired samples analyzed protein is associated with microglia/macrophage activation (27). The secondary on the same immunoblot. VOL. 71, 1997 CORRELATES OF POLYTROPIC MuLV NEUROVIRULENCE 5289

RESULTS an intense astrogliosis throughout the brain, as indicated by the increased expression of the astrocytic marker GFAP (39). Histopathological features of Fr98- and Fr54-infected As noted earlier, Fr54 infects the CNS but fails to induce brains. Neonatal IRW mice inoculated with Fr98 exhibit clin- clinical neurologic disease in neonatally inoculated IRW mice ical signs of neurologic disease 16 to 30 days postinoculation (19). To determine the relationship of glial activation and (19, 39). As previously reported, the histopathology associated vacuolar degeneration to neurologic disease, we analyzed the with Fr98 infection was characterized by mild vacuolar lesions pathological changes associated with Fr54 infection compared most prevalent in the lateral cerebellar nuclei (dentate nu- with that induced by the neurovirulent viruses, Fr98 and Fr98/ cleus) and thalamus (39), with no evidence of inﬂammatory SE. Interestingly, immunohistochemical staining of Fr54-in- inﬁltrates. The predominant pathologic feature, however, was fected brain for GFAP revealed an intense astrogliosis in a

FIG. 1. Reactive astrogliosis and microglial activation induced by Fr98 and Fr54. (A to C) Immunohistochemical staining for astrocytes on paraffin sections, using anti-GFAP antiserum. Panels show the lateral cerebellar nuclei (dentate nucleus) in brains taken from uninoculated (A), Fr54-inoculated (B), and Fr98-inoculated (C) mice. Notice the dark staining astrocytes in panels B and C not seen in panel A. Reactive astrogliosis was a consistent finding in all infected mice analyzed: Fr98 (five mice, 35 to 45 days postinoculation), Fr54 (eight mice, 55 to 159 days postinoculation), and Fr98/SE (five mice, 57 to 74 days postinoculation [not shown]). (D to F) Immunohistochemical staining for microglia/pericytes on cryostat sections of brain, using anti-Mac-1 monoclonal antibody. Panels show Mac-1-positive cells (arrows) in white matter tracts of the cerebella of brains taken from uninoculated (D), Fr54-inoculated (E), and Fr98-inoculated (F) mice. Microglial activation was a consistent finding in brains of all infected mice examined: Fr98 (six mice, 25 to 34 days postinoculation), Fr54 (three mice, 26 to 73 days postinoculation), and Fr98/SE (three mice, 48 to 73 days postinoculation [not shown]). The Fr98-inoculated mice analyzed exhibited severe clinical signs of neurologic disease. Fr54-inoculated mice were clinically indistinguishable from age-matched uninoculated controls. Sections were viewed by differential interference microscopy on a Nikon Microphot SA microscope. Bar, 80 ␮m. 5290 ROBERTSON ET AL. J. VIROL.

FIG. 2. Localization and morphology of infected cells in brains of Fr98-, Fr54-, and Fr98/SE-infected mice. Shown is immunohistochemical staining of frozen sections of brain for viral envelope glycoprotein with an polyclonal goat anti-gp70 antiserum. These panels show the cerebella of mice inoculated with Fr98 (33 days postinoculation) (A), Fr54 (B), and Fr98/SE (C), (both 48 days postinoculation) and uninoculated (48 days of age) (D). Note the immunohistochemical staining of cells with bipolar or highly ramified morphology (arrows and panel A, inset) as well as staining associated with blood vessels (panels B and C, arrowheads). This figure is representative of consistent findings in four mice per virus strain: Fr98 (18 to 33 days postinoculation), Fr54 (26 to 75 days postinoculation), and Fr98/SE (52 to 75 days postinoculation). Sections were viewed by differential interference microscopy. distribution similar to that previously reported for Fr98-in- clonal anti-MuLVgp70 antiserum which recognizes the enve- fected brain (Fig. 1A to C). In addition to astrogliosis, we lope proteins of all three viruses. Brains of Fr98- and Fr98/SE- looked for activation of microglia, which has been found in inoculated mice were analyzed at the time clinical disease was other retrovirus-induced neurologic diseases and may play an apparent (3 to 4 weeks and 6 to 8 weeks, respectively). Fr54- important role in neuropathogenesis (13). Activated microglia, inoculated mice were analyzed at 6 to 8 weeks postinoculation. which stained positive with anti-Mac-1 monoclonal antibody, The most predominant gp70 staining for both neurovirulent were found in both Fr98- and Fr54-infected brain and were localized primarily in the white matter tracts of the cerebellum (Fig. 1E and F, arrows), internal capsule, and corpus callosum. Focal collections of activated microglia associated with blood TABLE 1. Quantitation of cells expressing gp70 in the cerebella of vessels in the brainstem and cerebral cortex were also noted. Fr98/SE- and Fr54-infected mice at 8 weeks postinoculation Mac-1 staining was not observed in uninoculated controls (Fig. No. of infected cellsa Group mean Virus Animal 1D). Fr98/SE induced similar astrocytic and microglial activa- (mean Ϯ SD) Ϯ SEM tion (data not shown). In addition to the glial activation, mild Fr98/SE 1 192.8 Ϯ 32.5 vacuolar degeneration in the lateral cerebellar nuclei and/or Ϯ Ϯ thalamus was observed in brains of Fr98-inoculated mice (four 2 196.4 26.1 186.0 6.8 3 166.2 Ϯ 34.3 of four), Fr98/SE-inoculated mice (three of five), and Fr54- 4 188.6 Ϯ 50.4 inoculated mice (five of eight) (not shown). Thus, we were unable to detect any qualitative differences in the pathology Fr54 1 102.2 Ϯ 7.2 induced by neurovirulent and nonneurovirulent retroviruses. 2 111.8 Ϯ 19.0 104.6 Ϯ 11.6 Localization of virus and identification of virus-infected 3 74.6 Ϯ 11.6 cells in the brain. Since we were interested in finding corre- 4 130.0 Ϯ 21.3 lates of neurovirulence, it was important to determine if there a Determined by counting the number of gp70-positive cells in five 4-␮m-thick were any differences in regional distribution of these viruses in sections of the cerebella per animal. Comparison of the two groups by using the brain. Sagittal sections of brain were stained with a poly- Student’s t test gave a P value of Ͻ0.001. VOL. 71, 1997 CORRELATES OF POLYTROPIC MuLV NEUROVIRULENCE 5291

nuclei and thalamus, where parenchymal cells reminiscent of microglia were detected (see below). In the absence of any obvious differences in regional distribution of virus, we examined the specific cell types in brains infected by these viruses. The cells staining for viral envelope protein in brains of Fr98-, Fr54-, and Fr98/SE-infected mice exhibited similar morphologies (Fig. 2). Infected cells were either associated with blood vessels (endothelia and/or pericytes) or localized in the CNS parenchyma and characterized by a bipolar or highly ramified morphology typical of activated microglia (Fig. 2, inset). It was apparent that the distribution of viral envelope protein closely paralleled that of Mac-1-positive microglia (compare Fig. 2 with Fig. 1E and F). To confirm this observation and identify any other infected cell type(s), we performed dual-label immunofluorescence using the polyclonal anti-MuLV gp70 antiserum in conjunction with antibodies directed against various cell-specific markers. Since viral antigens were concentrated primarily in white matter without neuronal infection, antibodies to the glial cell markers Mac-1 (activated microglia), CNP (oligodendrocytes), and GFAP (activated astrocytes) were used. In mice inoculated with either neurovirulent or nonneurovirulent virus, viral antigen colocalized primarily with cells expressing the microglial cell marker, Mac-1 (Fig. 3A to D). In addition to microglial cells, a small number of the infected cells were identified as oligodendrocytes (Fig. 3E and F). Despite the pronounced astroglial activation, no viral antigen colocalized with GFAP-positive astrocytes in brains infected with Fr98 (Fig. 4), Fr54, or Fr98/SE. Comparisons of relative viral burden in the brain. Because the cerebellum appeared to be the primary target of infection for these viruses and because the clinical disease is consistent with cerebellar dysfunction (i.e., imbalance), quantification of viral burden focused on this region of the brain. In a previous study, Fr98, Fr54, and Fr98/SE were shown to have similar plasma viremia levels, indicating that these viruses did not differ in the ability to replicate in the periphery (19). Although viral capsid (CA) protein (p30) was first detected in the brain at 2 weeks postinoculation for all three viruses (19), immunoblot analyses suggested that the amount of viral protein in the cerebella of Fr98-infected mice was greater than that in cerebella of Fr54- and Fr98/SE-infected mice (19). To quantify this

FIG. 3. Identification of Fr98- and Fr54-infected cells in brain. Shown is dual immunofluorescent labeling of cryostat sections of brains taken from Fr98- inoculated (A and B) and Fr54-inoculated (C and D) mice. Panels A and C show immunofluorescent labeling of viral envelope protein (anti-gp70), with corresponding labeling of the microglial cell marker, anti-Mac-1, shown in panels B and D. Viral antigen colocalized with the microglial cell marker in both Fr98- and Fr54-infected brains. Localization of virus in oligodendrocytes was shown by dual immunofluorescent labeling of viral envelope protein (E) with the oligo- dendrocyte marker anti-CNP (F) in vibratome sections of brain taken from Fr98-infected mice. Analyses of Fr54- and Fr98/SE-infected brains revealed similar results (data not shown). Bars in panels A and C, 20 ␮m; bar in panel E, 15 ␮m.

and nonneurovirulent viruses was detected in the white matter of the cerebellum and consisted of both vascular and parenchymal cells. Infected vascular and parenchymal cells were observed in the corpus callosum and internal capsule but were less frequent than in the cerebellum. Focal areas of infected FIG. 4. Dual immunoﬂuorescent labeling of infected cells and astrocytes. cells were also observed within the brainstem, colliculus, cere- Vibratome sections were dual labeled for viral envelope protein (green; arrow) and GFAP (red; arrowhead). As is represented in this photo of cells in the white bral cortex, hippocampus, thalamus, and cerebellar nuclei. In- matter tract of the cerebellum, no colocalization of these two antigens was fection in grey matter areas appeared to be restricted to cells detected in brains of Fr98-infected mice. Analyses of brains from Fr54- and associated with small blood vessels except in the cerebellar Fr98/SE-inoculated mice revealed similar results. Bar, 20 ␮m. 5292 ROBERTSON ET AL. J. VIROL.

Moreover, the amount of viral protein in brains from clinically ill Fr98/SE-infected mice at 8 weeks postinoculation had achieved levels indistinguishable from those seen in clinically ill Fr98-infected mice at 3 weeks postinoculation (Fig. 5C). Together, these results suggested that entry of Fr54 and Fr98/SE into the brain occurred with comparable efﬁciencies but that the spread of Fr98/SE within the brain was more rapid than that of Fr54. Furthermore, these data suggested that induction of clinical neurologic disease may require a critical threshold of viral protein expression. The differences observed in the relative levels of viral protein expression between Fr54 and Fr98/SE at 8 weeks could have been a consequence of differences in the frequency of infected cells or in the amount of viral protein expressed on a per-cell basis. This issue was examined by counting infected cells in sections of cerebellum stained for viral envelope protein in Fr54- and Fr98/SE-inoculated mice at 8 weeks postinoculation. The results indicated that the number of infected cells in Fr98/SE-inoculated mice was 1.5- to 2-fold higher than that in Fr54-inoculated mice (Table 1). This difference is in the same range as that detected by immunoblot analysis (Fig. 5B), suggesting that the differences in viral burden were reﬂective of differences in frequency of virus-infected cells within the brain.

DISCUSSION The comparative studies of coisogenic polytropic MuLV strains differing in neurovirulence revealed that the amount of virus infection in the CNS appeared to be an important factor correlating with clinical neurologic disease. In contrast, there was no correlation between disease and the regional distribution of virus in the CNS, the types of brain cells infected, or the neuropathology induced by these viruses. FIG. 5. Western blot analyses of p30 capsid protein in Fr98-, Fr54-, and Fr98/SE-infected brains. Total cerebellar protein homogenates clarified with The regional distribution of polytropic MuLV-infected cells low-speed centrifugation (10%, wt/vol) from mice neonatally inoculated with was notably restricted in comparison to the widespread CNS Fr98, Fr54, or Fr98/SE and age-matched uninoculated controls were separated distribution of previously studied ecotropic MuLV such as by SDS-PAGE and probed with anti-p30 antiserum. Twofold serial dilutions of FrCasE (25). Cells infected by the polytropic MuLV were lo- the homogenates were analyzed to compare the relative amounts of viral protein in the brain. (A) Comparison of Fr98-, Fr54-, and Fr98/SE-infected brains at 3 calized primarily to the white matter of the cerebellum, corpus weeks postinoculation, showing that Fr98-infected mice contained two- to four- callosum, and subcortex. In contrast, neuroinvasive ecotropic fold-greater amounts of viral capsid protein than either Fr54- or Fr98/SE-in- MuLV infect cells throughout the grey and white matter of the fected mice. (B) At 8 weeks, the amount of viral capsid protein in samples from brain, brainstem, and spinal cord. The restriction of the poly- Fr98/SE-infected mice exceeded that in age-matched Fr54-infected mice and (C) tropic viruses is likely a function of the envelope gene, since reached levels equivalent to those of Fr98-infected mice at 3 weeks postinocu- E lation. These results were a consistent finding in the analysis of brain homoge- the ecotropic virus FrCas mentioned above shares long ter- nates from three mice per virus strain per time point. The clinical status of the minal repeat, gag, and pol sequences with the polytropic viruses mice at each time point is presented at the right (ϩ, clinical neurologic disease; Ϫ reported here. Since retrovirus infection requires cell division , clinically normal). (18), a possible explanation for the restricted distribution of the polytropic viruses is that they are less efficient at reaching the CNS at a time when target cells in the brain are still actively apparent difference, we compared the relative levels of viral dividing. It is also possible that the differences in virus distri- protein in a dilution series of 10% cerebellar homogenates bution reflect differential expression of the polytropic and eco- from Fr98-, Fr54-, and Fr98/SE-infected mice. Homogenates tropic receptors in the brain. were analyzed in pairs by immunoblot analysis after separation The predominant target cells in the CNS for both neuro- by SDS-PAGE, using a polyclonal anti-capsid (CA) protein virulent and nonneurovirulent viruses were vascular endothe- (p30) antiserum (Fig. 5) (26). At 3 weeks postinoculation, the lial cells/pericytes and activated microglia. Astrocytes were level of CA protein in the cerebella of Fr98-inoculated mice consistently negative for viral protein. Since the astrocyte was two- to fourfold higher than that in either Fr98/SE- or marker, GFAP, used in this analysis may identify only the Fr54-inoculated mice (Fig. 5A). At this time, Fr98-inoculated population of astrocytes that is activated, it is possible that mice were exhibiting clinical disease whereas Fr98/SE-inocu- there were productively infected GFAP-negative astrocytes. lated mice were asymptomatic. At 3 weeks, the contents of CA However, this possibility is unlikely for two reasons. First, in protein observed in Fr54- and Fr98/SE-inoculated mice were double-label immunofluorescence studies, the vast majority of indistinguishable (Fig. 5A). cells expressing viral protein were Mac-1-positive microglia. At 8 weeks postinoculation, a time when Fr98/SE-inoculated Second, the morphology of the infected cells in the CNS pa- mice were developing ataxia (19), immunoblot analysis re- renchyma was most consistent with that of activated microglia vealed that expression of viral protein in the brains of Fr98/ (Fig. 2). SE-infected mice had exceeded the viral burden in age- As has been shown for the neuropathogenic ecotropic matched Fr54-infected mice by two- to fourfold (Fig. 5B). MuLV (3, 25), human immunodeficiency virus (24, 49), and VOL. 71, 1997 CORRELATES OF POLYTROPIC MuLV NEUROVIRULENCE 5293 other animal lentiviruses (43), polytropic MuLV appear pri- ciency of spread within the microglial compartment. The coiso- marily to infect cells of the microglia/macrophage lineage in genic nature of these viruses identifies the envelope gene as an the CNS parenchyma. The importance of microglial cell infec- important determinant of this phenotype. It is noteworthy that tion in retrovirus-induced neuropathogenesis has been dem- the viral burden in the cerebella of Fr98-infected mice reached onstrated by engraftment experiments where microglial cells levels two- to fourfold higher than those in cerebella of both infected with a neurovirulent ecotropic MuLV induced focal Fr54- and Fr98/SE-infected mice at 2 (19) and 3 (Fig. 5) weeks spongiform degeneration in the brains of mice (27), while the postinoculation, suggesting that Fr98 may be more efficient at implantation of infected cells of neuroglial origin (oligoden- infecting endothelial cells in addition to microglial cells. drocytes and astrocytes) failed to induce neuropathology (28). Although the neuropathologic changes examined in this Microglial cells may mediate neuropathogenesis, in part, by the study did not correlate with neurovirulence, the results of these production of nonviral neurotoxins, the effects of which have studies were interesting in several respects. First, the neuro- been demonstrated in in vitro culture systems (15, 16). In light virulent viruses Fr98 and Fr98/SE induced only mild spongi- of these findings, it is particularly interesting that both the form degeneration in the brain, despite the dramatic clinical nonneurovirulent polytropic virus Fr54 and the neurovirulent neurologic deficits exhibited by infected mice. The finding of Fr98 and Fr98/SE exhibited microglial tropism. These results similar lesions in Fr54-inoculated mice, which appeared clini- demonstrate that microglial tropism per se was not sufficient cally normal, implies that these vacuolar lesions might not be for induction of disease. Thus, envelope gene variations may clinically relevant in this model. The lack of obvious disease- influence neurovirulence in this model by altering other qual- specific structural CNS damage suggests that the nature of the itative and/or quantitative aspects of CNS infection. insult leading to clinical disease may involve disruption of The results of this study indicated that there was a direct physiological processes in the brain which may not be detect- correlation between neurovirulence and the level of virus in- able at the light microscopic level. Second, reactive astrogliosis fection in the brain. Viral burden was measured by both the and microglial activation were induced by all three viruses amount of viral capsid protein and the number of infected regardless of differences in neurovirulence and viral burden. cells. It is noteworthy that the levels of viral protein in the Thus, the glial response appeared to correlate with virus in- cerebella of Fr98/SE- and Fr54-infected mice were indistin- fection in the CNS rather than clinical disease. However, it is guishable at 2 (19) and 3 (Fig. 5) weeks postinoculation yet possible that the glial response induced by virulent and aviru- differed significantly at 8 weeks postinoculation. In the com- lent viruses is qualitatively or quantitatively different. For ex- parison of the two neurovirulent viruses, Fr98 and Fr98/SE, ample the patterns or quantity of cytokines expressed by reac- there was a direct correlation between kinetics of viral protein tive astrocytes and activated microglia may vary among accumulation in the brain and incubation period of clinical different virus strains. To begin to address this issue, we are disease. Furthermore, the viral burden in the cerebella of Fr98- currently comparing the cytokine responses in the brains of and Fr98/SE-infected mice reached comparable levels, albeit mice infected with neurovirulent and nonneurovirulent viruses. at different times, coincident with the onset of clinical disease, suggesting that a critical threshold of viral burden may be ACKNOWLEDGMENTS required to induce neurologic disease. Similar to previous studies of the neurovirulent ecotropic virus tsMoBA (4), these We thank Richard Bessen for helpful discussions regarding this study, and we thank Bob Evans and Gary Hettrick for assistance with results suggest that the capacity of Fr98 and Fr98/SE to infect graphics. more target cells in the brain than Fr54 may alone explain their This work was supported in part by a National Institutes of Health neurovirulence. 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