Moloney Murine Leukemia Virus Infects Cells of the Developing Hair Follicle after Neonatal Subcutaneous Inoculation in Mice

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1 JOURNAL OF VIROLOGY, Mar. 1999, p Vol. 73, No X/99/$ Copyright 1999, American Society for Microbiology. All Rights Reserved. Moloney Murine Leukemia Virus Infects Cells of the Developing Hair Follicle after Neonatal Subcutaneous Inoculation in Mice MICHAEL A. OKIMOTO AND HUNG FAN* Department of Molecular Biology and Biochemistry and Cancer Research Institute, University of California, Irvine, California Received 25 August 1998/Accepted 3 November 1998 The nature of Moloney murine leukemia virus (M-MuLV) infection after a subcutaneous (s.c.) inoculation was studied. We have previously shown that an enhancer variant of M-MuLV, Mo PyF101 M-MuLV, is poorly leukemogenic when used to inoculate mice s.c., but not when inoculated intraperitoneally. This attenuation of leukemogenesis correlated with an inability of Mo PyF101 M-MuLV to establish infection in the bone marrow of mice at early times postinfection. These results suggested that a cell type(s) is infected in the skin by wild-type but not Mo PyF101 M-MuLV after s.c. inoculation and that this infection is important for the delivery of infection to the bone marrow, as well as for efficient leukemogenesis. To determine the nature of the cell types infected by M-MuLV and Mo PyF101 M-MuLV in the skin after a s.c. inoculation, immunohistochemistry with an anti-m-mulv CA antibody was performed. Cells of developing hair follicles, specifically cells of the outer root sheath (ORS), were extensively infected by M-MuLV after s.c. inoculation. The Mo PyF101 M-MuLV variant also infected cells of the ORS but the level of infection was lower. By Western blot analysis, the level of infection in skin by Mo PyF101 M-MuLV was approximately 4- to 10-fold less than that of wild-type M-MuLV. Similar results were seen when a mouse keratinocyte line was infected in vitro with both viruses. Cells of the ORS are a primary target of infection in vivo, since a replication defective M-MuLVbased vector expressing -galactosidase also infected these cells after a s.c. inoculation. Moloney murine leukemia virus (M-MuLV) is a simple replication-competent retrovirus that induces T-cell lymphomas in susceptible strains of mice. M-MuLV-induced leukemogenesis has been studied extensively (9). It is a multistep process that includes well-characterized molecular events such as insertional activation of proto-oncogenes (5, 11) and the formation of recombinant polytropic (i.e., mink cell focus-forming) viruses (10). End-stage tumors appear with a mean latency of 3 to 4 months after neonatal inoculation. In our studies of M-MuLV pathogenesis, we have made use of an enhancer variant of M-MuLV, Mo PyF101 M-MuLV. Mo PyF101 M-MuLV contains enhancer sequences from the F101 strain of polyomavirus inserted directly downstream of the M-MuLV enhancer sequences in the U3 region of the viral long terminal repeat (LTR) (14). We have previously shown that this M-MuLV variant is poorly leukemogenic when used to inoculate neonatal NIH Swiss mice subcutaneously (s.c.) despite the fact that it replicates well in vivo (7). Mo PyF101 M-MuLV also does not appear to induce many of the preleukemic changes normally associated with M-MuLV pathogenesis (2). Interestingly, if Mo PyF101 M-MuLV is injected intraperitoneally (i.p.), mice develop disease with wild-type kinetics (1). There is a delay in the appearance of infectious virus in the bone marrow of mice infected with Mo PyF101 M-MuLV s.c. compared to mice infected i.p., which suggested that efficient leukemogenesis requires high-level infection in the bone marrow at early times postinfection (1). This work also suggested that there is a cell type(s) present in the skin that is restricted for expression of Mo PyF101 M-MuLV but not for wild-type M-MuLV. Such a cell would efficiently deliver * Corresponding author. Mailing address: Department of Molecular Biology and Biochemistry, Cancer Research Institute, University of California, Biological Sciences II, Rm. 3221, Irvine, CA Phone: (949) Fax: (949) hyfan@uci.edu. wild-type M-MuLV (but not Mo PyF101 M-MuLV) infection from the skin to the bone marrow after a s.c. inoculation. In this study, immunohistochemistry was used to identify cells that are infected in the skin after an s.c. inoculation with M-MuLV and Mo PyF101 M-MuLV. The results indicated that cells of developing hair follicles are the predominant site for M-MuLV infection in the skin and that these cells are not efficiently infected by Mo PyF101 M-MuLV. In addition, cells of the developing hair follicle appeared to be primary targets of infection, since the same cells were infected after s.c. inoculations with a replication-defective M-MuLV based vector. MATERIALS AND METHODS Cell culture and viral inoculations. The M-MuLV and Mo PyF101 M-MuLV producer cells lines (43D and 25-3, respectively [7, 12]) and the NIH 3T3 cell line were all grown in Dulbecco s modified Eagle medium (DMEM) supplemented with 10% calf serum. The Pam212 cell line (23) was grown in DMEM supplemented with 10% fetal bovine serum. For virus stocks, supernatants from 43D and 25-3 cells were harvested from cultures at 70% confluency after 48 h and clarified by low-speed centrifugation (1,200 g for 10 min). Aliquots of viral supernatants were stored at 70 C until use and thawed only once. The titers of the viral stocks were determined by a focal immunofluorescence assay described below. Viral stocks were used to inoculate neonatal NIH Swiss mice subcutaneously at 1 to 2 days of age. The BAG vector stock was obtained from Psi-2 cells expressing the BAG vector as above and as described previously (17, 19). Neonatal (1 to 2 days old) NIH Swiss mice were inoculated s.c. with 200 l ofbag vector stock (ca to BAG infectious units [IU]/ml). Preparation of skin protein extracts. Mice were sacrificed by cervical dislocation or CO 2 asphyxiation and depilated by using a chemical depilatory agent (Nair; Carter Products, New York, N.Y.). Skin from around the site of inoculation was removed, weighed, and placed in a 10-fold excess of a solution containing 2% sodium dodecyl sulfate (SDS), 100 mm dithiothreitol, and 60 mm Tris (ph 6.8). The samples were agitated at room temperature for 2 to 4 h and boiled for 5 min. Large particulate matter was removed by low-speed centrifugation (400 g for 3 min). Chromosomal DNA was sheared by passing the sample repeatedly through a 20-gauge needle. The sample was then subjected to centrifugation at 10,000 g, and the supernatant was removed and stored at 20 C until use. Immunohistochemistry. Skin samples were fixed in 10% neutral buffered formalin (Sigma) overnight. Samples were embedded in paraffin and sectioned by a commercial histology laboratory. These sections were then deparaffinized, and 2509

2 2510 OKIMOTO AND FAN J. VIROL. endogenous peroxidase activity was neutralized by incubation in 2% H 2 O 2 for 5 min at room temperature. Slides were then incubated in Dako Target Retrieval Solution (Dako, Carpinteria, Calif.) for 30 min at 90 to 95 C to unmask antigens that may have been affected by the paraffin embedding. After the slides were washed for 5 min in phosphate-buffered saline (PBS), they were blocked by incubation with 10% normal goat serum (NGS) in PBS for 2 to 4hatroom temperature. The slides were then incubated with an anti-m-mulv capsid antigen (CA) rabbit polyclonal antibody (16) at a dilution of 1:10,000 in PBS plus 3% NGS overnight at 4 C. Slides were washed twice with PBS supplemented with 1% NGS for 10 min and incubated with a peroxidase-conjugated goat anti-rabbit antibody (Vector Laboratories, Burlingame, Calif.) at a dilution of 1:200 in PBS supplemented with 3% NGS for 1 h at room temperature. The slides were washed as described above and incubated with a peroxidase substrate (ABC; Vector Laboratories), and infected cells were visualized by light microscopy. For the detection of cells infected by the BAG vector, skin was fixed and paraffin embedded and deparaffinized as described above. To detect infected cells an anti- -galactosidase antibody (5 33 ; Boulder, Co.) was used in conjunction with the Dako Catalyzed Signal Amplification System (Dako) according to the manufacturer s instructions. Western blot analysis. Ten to 15 g of protein extract from skin samples were separated by electrophoresis on a SDS 10% polyacrylamide gel electrophoresis gel and transferred to nitrocellulose by electroblotting. To test if transfer of protein had occurred, the nitrocellulose membranes were stained with Pounceau red (Sigma, St. Louis, Mo.) immediately after transfer. Membranes were blocked for 1 h in 5% nonfat dry milk (Carnation, Glendale, Calif.) and then washed twice for 5 min and once for 15 min in PBS containing 0.05% Tween 20. The nitrocellulose membranes were then incubated for 1 h under constant agitation with a 1:5,000 dilution of the rabbit anti-m-mulv CA antibody (see above) in PBS containing 0.05% Tween 20. The blots were then washed as before and incubated for 1 h with a 1:20,000 dilution of a peroxidase-conjugated donkey anti-rabbit antibody (Amersham, Arlington Heights, Ill.). The membranes were washed as before and incubated with the SuperSignal Chemiluminescent Substrate (Pierce, Rockford, Ill.) for 2 min before being exposed to X-ray film. Infectivity assay. Infectivity assays were performed by seeding 10 5 NIH 3T3 fibroblasts or Pam212 cells onto replicate 5-cm tissue culture dishes followed by infection with 1 ml of serial dilutions of M-MuLV or Mo PyF101 M-MuLV. The cells were allowed to grow to confluence and were then fixed with 50% methanol for 5 min and washed with PBS for 5 min. Cells were then incubated for 30 min with the supernatant from the 548 hybridoma cell line that produces an anti-gag monoclonal antibody that reacts with M-MuLV-infected cells (4). Cells were washed twice with PBS containing 1% calf serum. A fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibody (1:200 dilution; Pierce) was then overlaid for 30 min. Cells were washed as before, and infected foci were visualized by fluorescence microscopy with an FITC filter. RESULTS Identification of M-MuLV-infected cells in the skin. To identify cells in the skin that were infected by M-MuLV after an s.c. inoculation, neonatal NIH Swiss mice were inoculated with ca IU and sacrificed at various times postinfection (1 to 10 weeks). Skin at the site of inoculation was removed, fixed, embedded, sectioned, and processed for immunohistochemical staining with a rabbit polyclonal antibody to M-MuLV CA protein as described in Materials and Methods. Microscopy of the stained sections was used to visualize the infected cells. The predominant M-MuLV-infected cells in the skin were those in the hair follicles (Fig. 1C, E, and G). The outer root sheath (ORS) of the hair follicle frequently showed the highest levels of infection. The ORS consists primarily of keratinocytes and is a site of high mitotic activity in developing hair follicles (8). M-MuLV is a simple retrovirus that can only infect cells that are undergoing mitosis (21); thus, it was reasonable that ORS cells would show high levels of M-MuLV infection. In addition to the ORS, cells of the sebaceous glands in the hair follicle were also infected (see below). During development, cells of the sebaceous gland form as an outgrowth of the ORS, so it is possible that they could have resulted from the same originally infected cells. It was noteworthy that in hair follicles that showed infection, most of the ORS cells were CA antigenpositive (Fig. 1G). This might indicate efficient spread of infection between the hair follicle cells. Alternatively, this might reflect infection of progenitor cells in the hair follicle that divided and differentiated. In addition to cells of the hair follicle, other individual M-MuLV-infected cells were detected in the skin (see the arrow in Fig. 1G). These cells were relatively uncommon: when multiple microscope fields were counted, the number of infected single cells was less than 1% of the number of infected hair follicles. The nature of the individual infected cells is still being investigated. In general, they did not stain for the cell surface markers Thy-1.2 or mac-1, which suggested that they were not T lymphocytes or macrophages. The low numbers of these individually infected cells could reflect their frequency in the skin, the fact that they are only transiently present in the skin, or a low efficiency of infection. Low efficiency of skin infection by the Mo PyF101 M-MuLV variant. As described in the introduction, the motivation for studying M-MuLV infection in the skin came from experiments with the poorly leukemogenic Mo PyF101 M-MuLV variant. Figure 2A shows the differences between the wild-type and Mo PyF101 M-MuLV LTRs. Our previous experiments had indicated that Mo PyF101 M-MuLV does not efficiently deliver infection from the site of an s.c. inoculation to the bone marrow. This in turn suggested that Mo PyF101 M-MuLV does not efficiently infect some cells in the skin. Thus, it was of interest to examine the skin from Mo PyF101 M-MuLVinfected mice. As shown in Fig. 1D, when mice were infected s.c. with equivalent amounts of this virus, the frequency of hair follicle infection was lower than for mice infected with wild-type M-MuLV. When multiple microscopic fields were counted, approximately 50% of the hair follicles of mice inoculated with wild-type M-MuLV were infected compared to 27% of the hair follicles infected in Mo PyF101 M-MuLVinfected mice. Moreover, the intensity of CA protein staining in the Mo PyF101 M-MuLV-infected hair follicles was generally less than that observed for infection by wild-type virus (more easily visible at higher magnification [compare Fig. 1E and F and Fig. 1G and H). Individual infected cells outside of the hair follicles could also be detected in Mo PyF101 M-MuLV-infected mice, and their frequency was equivalent to that of wild-type M-MuLV-infected animals. The lower frequency and intensity of CA antigen-staining in mice inoculated s.c. with Mo PyF101 M-MuLV supported the notion that this virus is less efficient at infecting and expressing in cells of the skin. While the immunohistochemical staining indicated that Mo PyF101 M-MuLV was less efficient than wild-type M-MuLV at establishing infection in the skin after an s.c. inoculation, an independent assessment was desirable. Therefore, Western blot analysis for CA antigen of skin samples from mice infected s.c. by either wild-type or Mo PyF101 M-MuLV was carried out as shown in Fig. 3. Skin samples from mice of different ages were analyzed and equal amounts of protein were loaded onto the SDS-polyacrylamide gel. In the Western blots, both the Pr65 gag polyprotein precursor and the mature CA protein (p30) were evident; the latter presumably reflected mature virions in the skin samples. As shown in the figure, at all ages skin samples from wild-type M-MuLVinfected mice consistently showed higher levels of infection than equivalent samples from Mo PyF101 M-MuLV-infected animals. These results were consistent with the immunohistochemical staining patterns. To further quantify the differences in levels of infection, Western blot analysis was used to compare, on the same blot of protein, 10 g ofmo PyF101 M-MuLV-infected skin extract to serial dilutions of extract made from age-matched wild-type M-MuLV-infected skin. As shown in Fig. 4, the dilution of M-MuLV-infected skin that gave an equivalent Western blot signal suggested that there was between 4- and 10-fold less CA

3 VOL. 73, 1999 M-MuLV INFECTION IN THE SKIN 2511 FIG. 1. M-MuLV infection in the skin. Neonatal NIH Swiss mice were inoculated s.c. with ca fluorescence immunoassay PFU (see Materials and Methods). Animals were sacrificed at various times postinfection, and skin from around the site of inoculation was harvested, paraffin embedded, and sectioned. Immunohistochemistry was then performed on these sections to identify infected cells. The sections were first incubated with an antibody to the M-MuLV CA protein. This was followed by incubation with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. The tissue was then incubated with a horseradish peroxidase substrate (that gives a purple color after reaction), and infected cells were visualized by light microscopy. Examples of this are shown here. Panels A and B show skin samples from control uninoculated mice that were stained with the anti-ca antibody. Panels C, E, and G show skin samples from mice that were infected with wild-type M-MuLV and sacrificed 4 weeks postinfection. Longitudinal (E) and transverse (C and G) cross-sections of hair follicles are shown; panel G is at higher magnification. Note the intense signal from cells that make up the outer region of the hair follicles (the ORS), indicating extensive infection. Panels D, F, and H show skin samples from mice that were infected with Mo PyF101 M-MuLV which were also sacrificed 4 weeks postinfection. The regions that are infected with Mo PyF101 M-MuLV are the same as those infected with wild-type M-MuLV (cells of the ORS), but the intensity of the signal is far less. Small cells that were not associated with hair follicles were also found to be infected (arrow in panel G) with both M-MuLV and Mo PyF101 M-MuLV, but these cells were not common. Bar, 10 m.

4 2512 OKIMOTO AND FAN J. VIROL. FIG. 2. The M-MuLV and Mo PyF101 LTRs. (A) M-MuLV LTR. The enhancer sequences of M-MuLV lie within two 75-bp direct repeats in the U3 region of the LTR. Each direct repeat contains consensus binding sites for a number of well-characterized transcription factors. The Mo PyF101 LTR is shown in the lower half of panel A. The enhancer region of the polyomavirus strain F101 has been inserted directly downstream of the M-MuLV direct repeats. Binding motifs in the PyF101 enhancers include two copies of a BPV-like enhancer core (B core) and three copies of a polyoma enhancer core (C 1 and C 2 ) (7). None of the viral structural proteins are altered in Mo PyF101 M-MuLV. (B) BAG and Mo PyBAG vectors. The BAG vector has been previously described (19). It contains the wild-type M-MuLV LTR driving the transcription of the bacterial lacz gene. It also contains the Neo r gene as a selectable marker. The Mo PyBAG vector is similar to the BAG vector except that it has the LTR from Mo PyF101 M-MuLV regulating lacz expression. Both vectors were obtained from transfected Psi-2 cells as described in Materials and Methods.

5 VOL. 73, 1999 M-MuLV INFECTION IN THE SKIN 2513 FIG. 3. M-MuLV and Mo PyF101 M-MuLV infection in the skin. Protein extracts were made from the skin of mice that had been infected s.c. with wild-type M-MuLV and Mo PyF101 M-MuLV and sacrificed at various times postinfection. Western blot analysis was then performed on these extracts as described in Materials and Methods. Equal amounts of skin extract (10 g of protein) were analyzed in each lane. With the anti-ca antibody, both the viral p30 and the Pr65 gag proteins were detected. As shown here, protein extracts from mice that had been infected with wild-type M-MuLV had significantly higher levels of both viral proteins than equivalent amounts of protein extract from age-matched mice infected with Mo PyF101 M-MuLV. Downloaded from protein in Mo PyF101 M-MuLV-infected skin that in skin infected by wild-type M-MuLV. Mo PyF101 M-MuLV shows less efficient infection of a keratinocyte line in vitro. The immunohistochemistry indicated that ORS cells of the hair follicle are a predominant site of infection by M-MuLV in the skin. Since the Mo PyF101 M-MuLV-infected mice showed less infection of these cells, this suggested that cells of the ORS may support less efficient infection of the latter virus. To test this, since the ORS consists predominantly of keratinocytes, in vitro infection of the Pam212 mouse keratinocyte line was carried out. Table 1 shows infection of wild-type and Mo PyF101 M-MuLV on NIH 3T3 and Pam212 cells, as measured in a focal immunofluorescence assay. The results indicated that wild-type M-MuLV infected the two cell lines with similar efficiencies; in contrast, Mo PyF101 M-MuLV showed less infection when measured on Pam212 cells. After correction for the relative efficiency of wild-type M-MuLV infection on Pam212 cells versus NIH 3T3 cells, the relative infectivity for Mo PyF101 M-MuLV on Pam212 cells was approximately fivefold less than for wild-type M-MuLV. This was in general agreement with the estimated decrease in infection in the skin for this virus as seen in the immunohistochemistry and Western blot analyses. Hair follicle cells are the initial targets of infection in the skin. While the preceding experiments indicated that hair follicle cells were the predominant infected cells in the skin after s.c. inoculation, there were two possible explanations for their infection. Hair follicle cells (or their progenitors) could have been initially infected by M-MuLV during the s.c. inoculation. Alternatively, infection could have spread from other cells to them. To distinguish between these possibilities, we employed s.c. infection with a helper-free replication-defective M-MuLVbased vector expressing the bacterial -galactosidase gene (BAG; Fig. 2B). When the BAG vector is produced by transfection of Psi-2 packaging cells (that express M-MuLV virion proteins [15]), the resulting vector particles will infect the same cells that M-MuLV does, but they will not spread by infection to neighboring cells. We have previously used in vivo infection of BAG vector to identify the first cells infected in the bone marrow of i.p.-inoculated mice (17). Newborn NIH Swiss mice were inoculated s.c. with helperfree BAG vector stocks (ca IU) and sacrificed 1 week postinfection. Skin samples from the region of inoculation were processed as described above, but immunohistochemical staining was performed with an anti- -galactosidase antibody. The results are shown in Fig. 5. The staining patterns from BAGinfected skin were very similar to those of M-MuLV infected skin. Cells of the ORS, as well as of the sebaceous gland, were infected by the BAG vector (Fig. 5C and D), indicating that these cells are targets for the initial infection by M-MuLV. Overall, less than 0.5% of hair follicles in BAG-infected skin stained positive for the BAG vector. Typically, only single follicles within a microscope field stained positive, but within such follicles multiple infected cells were observed (ca. 5 to 20 infected cells per cross-section). The field shown in Fig. 5C is unusual in that several adjacent follicles showed infection. Individual cells not associated with hair follicles were not infected by the BAG vector. In addition to the BAG vector, another replication-defective vector was studied, Mo Py BAG. Mo Py BAG is based on the BAG vector but contains the Mo PyF101 LTR driving -galactosidase expression (Fig. 2B) (3). This vector was also transfected into the Psi-2 cell line, and the resulting supernatant was used to infect neonatal mice s.c. (ca IU per on September 21, 2018 by guest

6 2514 OKIMOTO AND FAN J. VIROL. cells to support expression from the Mo PyF101 LTR was lower than that for the wild-type M-MuLV LTR. Such a conclusion was consistent with the reduced efficiency of skin infection and expression shown by Mo PyF101 M-MuLV described above. DISCUSSION FIG. 4. Quantification of Mo PyF101 M-MuLV infection in the skin. To quantify the differences in the amount of virus present in the skin, serial dilutions of a wild-type M-MuLV-infected skin extract were compared to an extract from Mo PyF101 M-MuLV-infected skin on the same Western blot. Panel A shows protein extract from mice sacrificed 4 weeks postinfection. A serial dilution of 10, 7.5, 5.0, 2.5, and 1.0 g of M-MuLV-infected skin extract was compared to 10 g of protein extract from an age-matched Mo PyF101 M-MuLV-infected mouse. The viral p30 protein is shown. The intensity of the signal from 10 g ofthe Mo PyF101 M-MuLV skin extract was comparable to the signal seen from 1.0 g of M-MuLV skin extract, indicating that there was approximately 10-fold less viral protein. A similar analysis was performed in panel B with mice that had been sacrificed 6 weeks postinfection. In this case, the Mo PyF101 M-MuLV skin extract contained about fourfold less viral protein. animal). Skin sections were processed as mentioned above. When immunohistochemistry was performed on skin from mice infected with Mo Py BAG, no infected hair follicles were observed. This suggested that the ability of hair follicle Virus In these experiments, we studied the cell types infected by M-MuLV in the skin after a neonatal subcutaneous inoculation. The results indicated that the great majority of infected cells were in hair follicles. Indeed, by 4 to 6 weeks of age, approximately 50% of the hair follicles showed extensive infection, as measured by immunohistochemistry for M-MuLV CA antigen. The predominant infected cells within the hair follicles were cells of the ORS. The ORS consists primarily of keratinocytes; cells of this region play an important role in the generation and maintenance of hair follicles (13). In addition, cells of the sebaceous gland (which produces sebum for the hair follicle) were also infected; this was plausible since sebaceous gland cells are derived from the ORS during development (22). The finding that hair follicles are extensively infected by M-MuLV in the skin was plausible since cells in the hair follicles have high mitotic activity even in adult mice. Productive infection by simple retroviruses such as M-MuLV requires passage of cells through mitosis (21); moreover, even in cells that have acquired M-MuLV proviruses, virus production is more efficient if the cells are cycling (18). In addition to the hair follicles, M-MuLV infection was also detected in single cells within the skin of animals inoculated s.c. These cells were much less frequently detected, i.e., the frequency of individually infected cells was approximately 1% of the frequency of infected hair follicles; moreover, if each infected cell in a hair follicle were counted, then the difference would have been even greater. As described in Results, these infected cells did not stain with markers for hematopoietic cells such as Thy-1 and mac-1. Thus, they do not appear to be T lymphocytes or macrophages, two cell types that are known to be readily infectable by M-MuLV later in the course of infection. One cell type that would be of interest is the Langerhans cell, by analogy to infection by lentiviruses (20). However, Langerhans cells are terminally differentiated, so it seems un- TABLE 1. Relative infectivity of M-MuLV and Mo PyF101 M-MuLV on NIH 3T3 and Pam212 cell lines Dilution No. of infected cells a (dilution 1, dilution 2 [avg]) with: 3T3 cells Pam212 cells Infectivity ratio b M-MuLV 10 3 TMTC, TMTC (TMTC) TMTC, TMTC (TMTC) , 862 (898) 298, 424 (361) , 98 (94) 56, 78 (67) , 8 (9) 6, 8 (7) 10 7 NC, NC (NC) NC, NC (NC) Titer (IU/ml, 10 6 ) Mo PyF101 M-MuLV 10 3 TMTC, TMTC (TMTC) 657, 567 (612) , 532 (539) 74, 66 (70) , 74 (88) 18, 6 (12) , 8 (8) 1, 0 (0.5) 10 7 NC, NC (NC) NC, NC (NC) Titer (IU/ml, 10 6 ) a The number of infected cells was determined by fluorescence immunoassay as discussed in Materials and Methods. Each dilution of viral supernatant was infected on target cells in duplicate; the average is given in parentheses. TMTC, too many to count; NC, not counted. Infectivity assays were performed three times with similar results. A representative assay is shown here. b The infectivity ratio is defined as follows: (viral titer on NIH 3T3)/(viral titer on Pam212).

7 VOL. 73, 1999 M-MuLV INFECTION IN THE SKIN 2515 Downloaded from FIG. 5. Infection by BAG vector in the skin. Neonatal NIH Swiss mice were inoculated with ca BAG vector particles. Mice were sacrificed 1 week postinfection, and skin was removed from around the site of inoculation and prepared as described above. Infected cells were visualized by immunohistochemistry with an anti- -galactosidase antibody as described in Materials and Methods. In this protocol, a specific antibody reaction results in deposition of a brown stain. Panels A and B show skin samples from age-matched uninoculated mice stained with the anti- -galactosidase antibody. Panels C and D show skin samples from BAG-infected mice. Note that in panel C cells in the ORS of the hair follicle are infected by the BAG vector and in panel D cells of the sebaceous gland are infected. Since the BAG vector is replication defective, these cells must be primary targets for M-MuLV infection. Bar, 10 m. likely that they could be efficiently infected with a simple retrovirus such as M-MuLV. While extensive hair follicle infection was identified after s.c. inoculation, experiments with wild-type M-MuLV could not distinguish between hair follicles being primary targets for infection or alternatively becoming infected by some other initially infected cell in the skin. In order to address this question, infection with the helper-free, replication-defective M- MuLV-derived BAG retroviral vector was carried out. Infection resulted in the appearance of BAG-infected hair follicles, albeit at a lower frequency than in mice infected with replication-competent M-MuLV. Thus, hair follicles are primary (and possibly secondary) targets for M-MuLV infection after s.c. inoculation of neonates. It was very noteworthy that BAGinfected hair follicles often showed the same high frequency of infected cells within an infected hair follicle as observed in wild-type M-MuLV-infected mice. Since the BAG vector is replication defective, this suggests that most of the infected cells in a hair follicle arose by division of an infected cell, as opposed to spread of the infection within the hair follicle. Indeed, it seems likely that the initial cells infected in the neonatal animal after s.c. inoculation are hair follicle progenitors. Interestingly, others have reported that an area of the ORS known as the Wuste or bulge region may contain follicular stem cells capable of regenerating an entire hair follicle (6). As described in the introduction and in Results, the motivation behind these experiments was to identify the cells in the skin responsible for delivering M-MuLV infection to the bone marrow. Since the hair follicles contain the predominant cells infected by M-MuLV in the skin, they are the likely candidates for delivering infection to the bone marrow. Experiments with the Mo PyF101 M-MuLV variant supported this suggestion. Mo PyF101 M-MuLV does not efficiently establish early bone marrow infection after s.c. inoculation (1), and we found that the skin from mice inoculated s.c. with this virus showed substantially lower levels of infection. Both the number of infected hair follicles and the intensity of antigen staining in the infected cells was lower. Quantification of the levels of CA antigen in skin of Mo PyF101 M-MuLV-infected animals also revealed lower levels of infection (ca. 4- to 10-fold) than for animals infected with wild-type virus. Finally, in vitro infection of a mouse keratinocyte line also showed less efficient infection than for wild-type M-MuLV. If, as the results suggest, hair follicle cells are responsible for delivery of virus to the bone marrow, the mechanism by which on September 21, 2018 by guest

8 2516 OKIMOTO AND FAN J. VIROL. this takes place must still be elucidated. One possibility is that the hair follicle cells act as centers of virus production; the resulting virus could then enter the circulation and infect bone marrow cells. Another possibility is that the hair follicle cells might infect other cells that deliver infection to the bone marrow. For instance, the singly infected cells detected in the skin might be mobile cells that traffic to the bone marrow. The finding of infected hair follicle cells also has potential implications for spread of infection from animal to animal. It seems likely that infectious virus could be present in the skin or on the hair released from infected animals. This virus might be a source of natural spread to an uninfected animal, as a result of grooming, biting, or nursing. It should be noted that, in these experiments, we examined the skin for infection. However, during s.c. inoculation, tissues under the skin also come into direct contact with the inoculated virus, e.g., the underlying musculature and the membranes separating the muscle and the skin. It is possible that cells of these other tissues are also involved in the delivery of infection from the site of an s.c. inoculation to the bone marrow. In future experiments, it will be important to address this issue. ACKNOWLEDGMENTS This work was supported by grant CA32455 from the National Cancer Institute. M.A.O. was supported by NIH training grant number 5 T32 AI The support of the UCI Cancer Research Institute and the Chao Family Comprehensive Cancer Center is also gratefully acknowledged. REFERENCES 1. Belli, B., and H. Fan The leukemogenic potential of an enhancer variant of Moloney murine leukemia virus varies with the route of inoculation. J. Virol. 68: Brightman, B. K., B. R. Davis, and H. 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