JOURNAL OF VIROLOGY, Mar. 1992, p. 1649-1654
Vol. 66, No. 3
0022-538X/92/031649-06$02.00/0 Copyright © 1992, American Society for Microbiology
Persistent Infection of Normal Mice with Human Immunodeficiency Virus CHIARA LOCARDI, PATRIZIA PUDDU, MARIA FERRANTINI, ELEONORA PARLANTI, PAOLA SESTILI, FRANCO VARANO, AND FILIPPO BELARDELLI* Laboratory of Virology, Istituto Superiore di Sanitez, Viale Regina Elena 299, 00161 Rome, Italy Received 11 July 1991/Accepted 22 November 1991
In this article, we report the establishment of persistent HIV type 1 infection of normal Swiss mice after a single intraperitoneal injection with high-producing HIV-infected U937 cells. Anti-HIV antibodies were found more than 500 days after the original injection, and p24 antigenemia was detected in approximately 50%37s of the mice. By polymerase chain reaction (PCR) techniques, HIV-specific gag and env sequences were detected in DNA samples from peripheral blood mononuclear cells (PBMC) and peritoneal cells of seropositive mice 300 to 500 days after inoculation with HIV-infected cells. These DNA samples did not contain human DNA sequences, as determined by PCR analysis using primers and the probe for the HLA-DQ at gene. Low levels of p24 and detectable human reverse transcriptase activity were found in cultures of PBMC and peritoneal macrophages. Cocultivation of PBMC, peritoneal cells, and spleen cells with human uninfected U937 or CEM (a T lymphoma cell line) cells resulted in HIV infection of the target cells, as determined by PCR analysis and/or p24 assays. The intravenous injection of untreated Swiss mice with the PBMC from PCR-positive mice resulted in the development of an increasing antibody response to HIV in the recipient animals. Together these results indicate that cells from seropositive Swiss mice were persistently infected with HIV and were capable of producing infectious virus. The development of persistent HIV infection in an immunocompetent mouse may represent the starting point for further studies aimed at defining the host mechanisms involved in the restriction of virus replication, defining the pathogenesis of HIV infection, and testing antiviral compounds and vaccines.
Human immunodeficiency virus type 1 (HIV-1) is considered the etiologic agent of AIDS (1, 3, 25). Although the biology of HIV has been intensively explored, the relationship between HIV-1 infection and AIDS is not clear. Animal models for AIDS are thus essential for understanding the pathogenesis of HIV-1-induced immunodeficiency and for defining effective antiviral therapies and vaccines (10). A small and well-characterized laboratory animal, such as the mouse, would be especially useful (27). However, we are not aware of any report of HIV infection of murine cells in vitro or in vivo. The following two major types of in vivo murine models for HIV have been developed so far: (i) transgenic mice carrying either intact (16) or partial (17, 32) HIV-1 genomes, and (ii) immunodeficient mice engrafted with human fetal lymphoid tissue (SCID-hu mice) (22, 24) or adult human peripheral blood leukocytes (PBL-SCID mice) (23). An additional and simple approach for the development of a persistent HIV infection in a small-animal model would be the injection of HIV or HIV-infected human cells directly into immunocompetent animals. In fact, it has been reported that rabbits can be infected with HIV under special experimental conditions (8, 14). We suspected that a similar phenomenon could also occur in mice if suitable numbers of virus producer cells were injected into an immunocompetent mouse host. The results reported in this article indicate that this experimental procedure can be very effective in establishing a persistent infection of normal mice with HIV.
*
MATERIALS AND METHODS Cells and culture conditions. The human histiocytic lymphoma cell line U937 (30) (kindly provided by A. Siccardi, Milan, Italy) was cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum. U937 cells were infected with HIV-1 (HTLV III-B strain) as described elsewhere (19). Fifty days after HIV infection, chronically virus-infected U937 cells were cloned. A virus producer cloned U937 cell line (clone 10) was selected and used for the in vivo experiments described below. These cloned U937 cells produced large amounts of HIV in vivo when transplanted subcutaneously in nude mice treated with antibodies to mouse alpha/beta interferon (26). Mice. Six-week-old male Swiss mice (Charles River S.p.A., Milan, Italy) were kept under special biosafety level 3 conditions during the course of the experiments. Mice were injected intraperitoneally (i.p.) with 0.5 ml of 4% thioglycolate broth 4 days before the injection of HIVinfected cells. In 3 sets of experiments, a total of 70 mice were inoculated i.p. with HIV-infected U937 cells (2 x 106 cells per mouse) and 30 control mice were injected with the same number of uninfected U937 cells. Before injection, HIV-infected cells were treated for 24 h with human tumor necrosis factor alpha (100 U/ml), which results in a marked increase in the production of infectious HIV particles (19). Serologic testing of anti-HIV antibodies. Aliquots of orbital venous blood were collected from individual tagged mice at different times after injection of virus-infected cells. Serum samples were tested for HIV-1-specific antibodies with the commercial HIV-1 enzyme-linked immunosorbent assay
(ELISA) microplates (Ortho Diagnostics Systems) following a modified procedure. Briefly, 200 pl of serum, diluted 1:100 in phosphate-buffered saline (PBS), was added to the appropriate wells. After a 1-h incubation at 37°C, wells were extensively washed and 200 ,ul of a 1:1,000 dilution of
Corresponding author. 1649
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peroxidase-conjugated anti-mouse immunoglobulin G (IgG) (Cappel Laboratories) was added. After 60 min at 37°C, microplates were washed five times with PBS and 200 VI of peroxidase substrate solution was added to each well. After 30 min at room temperature, the reaction was stopped by the addition of 50 j,l of 4 N H2SO4 and the A492 was read by means of a flow multiscan reader. Western blot (immunoblot) analyses were carried out with nitrocellulose strips containing HIV-1 proteins (Du Pont, Wilmington, Del.) and biotin-labelled goat anti-mouse IgG (Sigma, St. Louis, Mo.). Western blot strips were probed with a 1:50 dilution of mouse serum. Before Western blot analysis, mouse sera were adsorbed against uninfected U937 cells (108 viable cells incubated with 0.4 ml of serum for 1 h at 37°C). Gene amplification by PCR. DNA from cultured cells was extracted by standard procedures (29). Mouse peripheral blood mononuclear cells (PBMC), peritoneal cells, and cocultivated cells were lysed as described previously (13), and aliquots of the lysate were added directly to the polymerase chain reaction (PCR). PCR was carried out in a reaction mixture of 25 VI containing 0.5 ,uM (each) primers, 200 ,uM (each) deoxynucleoside triphosphates, and 1.5 mM MgCl2. After 30 cycles of amplification (95°C for 90 s, 50°C for 90 s, and 72°C for 2 min) followed by a final extension at 72°C for 10 min, PCR products were resolved on 1.75% SeaKem (FMC) agarose gels, transferred to a Hybond (Amersham) membrane, and hybridized with 32P-labelled detector oligonucleotides. PCR experiments were performed with envl 401 and 404 and gag 881 and 882 primer pairs. The corresponding probes were envl 405 (6654 5' GCGGGAGAAT GATAATG GAGAAAGGAG, HIV HXB2) and gag 883 (28). Amplification and detection of the HLA-DQ a gene fragment were performed with the GH26-GH27 primer pair and the corresponding probe, RH54 (5). All PCR experiments included a set of reaction mixtures containing GAPDH primer pairs (12) as a control for amplification (data not shown). Detection of p24 antigenemia. Serum aliquots were harvested from tagged mice and tested for the presence of detectable p24 levels by means of an antigen-capture ELISA (Du Pont). RT assays. The assay to measure the human reverse transcriptase (RT) activity in culture supernatants was performed as described in detail elsewhere (19). Before the RT activity was tested, 1 ml of cell supernatant was centrifuged for 15 min at 95,000 rpm in a TLA-100 rotor on a Beckman TL-100 bench-top ultracentrifuge. The pellet was resuspended in 70 VI of TNE (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA [pH 8], 0.1% Triton X-100), and 30 ,ul was added to the reaction mixture (19). For evaluating the possible presence of murine RT activity, the same samples were tested with a different reaction mixture containing MnCl2 instead of MgCI2, as described in detail elsewhere (6). Culture of mouse PBMC and cocultivation techniques. At different days after the i.p. injection of HIV-infected U937 cells, blood samples from individual tagged mice were collected and PBMC were isolated by Ficoll-Hypaque (density, 1.09 g/cm3). The PBMC were treated for 48 h with phytohemagglutinin (20 ,ug/ml) and then for 24 h with human recombinant interleukin-2 (50 U/ml). Next, the PBMC were cocultivated with human uninfected cells (i.e., U937 cells or CEM cells, a T-lymphoma cell line) at a donor cell/target cell ratio of approximately 1:1. After a cocultivation period of 8 to 10 days, the cocultivated human target cells were tested for HIV infection. Aliquots of cell supernatant from coculti-
J. VIROL.
vated target cells were tested for the presence of detectable p24 levels by means of an antigen-capture ELISA (Du Pont). DNAs were extracted from cocultivated target cells and tested for the presence of HIV sequences by PCR techniques using gag and env primers for HIV (28). Nucleotide sequence accession number. The nucleotide sequence accession number for envl 405 is GenBank K03455. RESULTS Long-lasting antibody response to HIV-1 in mice injected with HIV-infected U937 cells. IgG antibodies to HIV were detected by IgG ELISA in all the mice inoculated with HIV-infected U937 cells. Figure 1 shows the kinetics of the antibody response to HIV in four representative mice injected with HIV-infected U937 cells. The titers of anti-HIV antibodies in the sera reached the maximum level 60 to 100 days after the injection of HIV-infected cells. Subsequently, there were fluctuations in the antibody titers in these mice, but all mice continued to have high antibody titers 200 to 500 days after the initial inoculation of HIV-infected U937 cells (Fig. 1A through D). The Western blot analyses of these sera revealed the presence of detectable antibodies to gpl20 and p24 HIV proteins even 500 days after the original injection with HIV-infected U937 cells (Fig. 2, lane 2). Detection of HIV-specific gag and env sequences in the DNAs of mouse cells from seropositive Swiss mice. By PCR techniques, we analyzed blood samples from seropositive mice for the presence of HIV-specific sequences in the DNAs extracted 350 to 500 days after the injection of HIV-infected U937 cells. As shown in Fig. 3A (lane 3) and B (lane 4), DNAs extracted from the blood of long-termseropositive mice exhibited detectable levels of HIV-specific gag and env sequences. In order to determine the approximate number of HIV genomic copies present in the DNA from these blood samples, we performed PCR assays in which decreasing amounts of DNA from 8E5/LAV cells (which contain a single integrated copy of proviral DNA per genome [9]) were mixed with a constant amount of spleen DNA from normal Swiss mice. Comparison of the signals in the same PCR assays showed that the DNA sample from one seropositive mouse (D5) contained approximately 1 HIV genomic copy per 100 to 1,000 cell genomes (data not shown). HIV-specific sequences were also found in DNA samples from peritoneal cells of seropositive mice (Fig. 3A, lane 5). To rule out the possibility that these HIV-positive signals were due to some contamination with surviving human U937 cells, we performed PCR analysis of DNA using primers and the probe for the HLA-DQ a gene. As shown in Fig. 4, human sequences were not detected in the HIV-positivemouse DNA. Virus expression in mice injected with HIV-infected cells. The data reported above indicated that the HIV genomic sequences were present in some mouse PBMC and peritoneal cells from seropositive Swiss mice for a long time after a single i.p. injection of HIV-infected U937 cells. It was of interest, therefore, to investigate whether HIV could be expressed in and/or isolated from cells of seropositive mice by different techniques. Detectable levels of p24 antigenemia (40 to 60 pg/ml) were found in the serum samples of four of eight seropositive mice more than 500 days after the original injection of HIV-infected U937 cells. Moreover, when PBMC were harvested from three PCR-positive mice and injected intravenously into four recipient untreated mice, an
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0 100 200 300 400 500 200 300 400 5oo DAYS DAYS FIG. 1. Kinetics of the antibody response to HIV in four representative Swiss mice injected i.p. with cloned HIV-infected U937 cells. Serum samples from individual tagged mice were taken at different times after cell injection. At each time, serum samples from control mice were also tested for anti-HIV antibodies. Serum samples were tested for HIV-1-specific antibodies with HIV-1 ELISA microplates as described in Materials and Methods. 0, control mice; 0, mice injected with HIV-infected U937 cells. O.D., optical density.
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increasing antibody response to HIV was found in the serum samples of the mice. In contrast, antibodies were not detected in the sera of recipient mice injected with PBMC from control mice (Fig. 5). These results suggested that HIV proteins were expressed in vivo in some PBMC from seropositive Swiss mice. In other experiments, PBMC, peritoneal cells, spleen cells, and cells from mesenteric lymph nodes were cultured in vitro for a few days to determine the p24 levels in the cell supernatants. Low levels of extracellular and/or intracellular p24 (i.e., 10 to 20 pg/ml) were found 5 to 15 days after in vitro cultivation. Supernatants from spleen cells and cells from lymph nodes were also ultracentrifuged and tested for the presence of human and murine RT activities. Significant levels of human RT activity were found in these samples, whereas no enzymatic activity was revealed with the typical reaction buffer for murine RT (data not shown). In some experiments, PBMC, peritoneal cells, or spleen cells from seropositive mice were cocultivated with uninfected human U937 or human CEM cells. The cocultivated target cells were then tested for the presence of HIV-specific sequences by PCR analysis of the extracted DNAs. Figure 3C shows one example of the PCR analysis of CEM cells after cocultivation with spleen cells from a seropositive mouse compared with that of CEM cells prior to cocultivation. The DNA of cocultivated CEM cells exhibited env and gag HIV-specific sequences (Fig. 3C, lane 4). Table 1 summarizes representative data for individual mice in which the evidence of HIV infection was obtained by different techniques. A total of 32 mice were randomly sacrificed 350 to 500 days after the original injection. Eighteen of these mice were found to be infected with HIV as evaluated by the capability of their PBMC or spleen cells to
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FIG. 2. Western blot analyses of sera from Swiss mice injected with HIV-infected U937 cells. Western blot analyses were carried out with nitrocellulose strips containing HIV-1 proteins as described in Materials and Methods. Strips were probed with serum from a control mouse (lane 1); pooled serum from four mice harvested 512 days after the original injection with HIV-infected U937 cells (lane 2), and control human serum, positive for antibodies to HIV (lane 3).
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3 1 2 FIG. 4. PCR evaluation of the absence of human DNA sequences in the HIV-positive mouse DNA. Blood DNA from a representative HIV-PCR-positive mouse (D5) was tested by PCR for the presence of HLA-DQ a sequences by using the specific primer pair and probe as described in Materials and Methods. DNA is from HIV-1-infected U937 cells (clone 10) (lane 1); a blood sample from the D5 HIV-PCR-positive mouse (lane 2) (see also Fig. 2B, lane 4); splenocytes from a control Swiss mouse (lane 3). The inset (upper right corner) shows the GAPDH gene amplification from the DNA of the same samples.
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FIG. 3. Detection by PCR analysis of HIV sequences in mouse cells from Swiss mice injected with HIV-infected U937 cells. Integrated HIV-1 sequences were detected in different mouse cell types by PCR analysis of DNA using primer pairs specific for HIV-1 env and gag sequences (left and right sets of lanes in each panel, respectively) as described in Materials and Methods. (A) DNA from HIV-1-infected U937 cells (clone 10) (lane 1), a blood sample from a control mouse (lane 2), a blood sample from a seropositive mouse (C9) (lane 3), peritoneal cells from a control Swiss mouse (lane 4), and peritoneal cells from a seropositive mouse (V7) (lane 5); (B) DNA from HIV-1-infected U937 cells (lane 1), a blood sample from a normal Swiss mouse (lane 2), splenocytes from a control Swiss mouse (lane 3), and a blood sample from a seropositive mouse (D5) (lane 4); (C) DNA from HIV-1-infected U937 cells (lane 1), splenocytes from a control Swiss mouse (lane 2), splenocytes from a seropositive mouse (C9) (lane 3), CEM cells cocultivated with splenocytes from the C9 mouse (lane 4), and CEM cells before cocultivation (lane 5).
spleen cells, even beyond 500 days after the original injection. These HIV-positive signals did not reflect contamination with in vivo surviving U937 cells, as PCR analysis did not reveal any presence of human-specific sequences. (ii) Mouse cells were capable of expressing virus proteins (i.e., p24 and RT) and infecting human target cells, indicating the competence of the mouse cells for infectious-virus production. It has generally been assumed that mouse cells cannot be infected with HIV because they lack some properties essential for virus replication. Mouse cells do not bind HIV, 0.6
0.4o
o infect human target cells. Together these results indicate that cells from seropositive mice injected with HIV-infected U937 cells were persistently infected with HIV.
DISCUSSION The data reported in this article represent, to the best of our knowledge, the first experimental evidence to indicate that mouse cells can be infected in vivo with HIV. (Filice and coworkers [7] reported a long-lasting antibody response to HIV in mice injected i.p. with HIV-infected human H9 cells, but there was no experimental evidence to show that mouse cells had been infected with HIV-1.) In this study, the following two major findings clearly demonstrate that mouse cells had been persistently infected with HIV. (i) HIV-1-specific sequences were detected by PCR in the DNAs from mouse cells, such as PBMC and
0.3 0.2
0.1
0~~~~~~~ 10
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days after Injection of PBMC FIG. 5. Antibody response to HIV in mice injected with PBMC from seropositive Swiss mice. Three HIV-PCR-positive and three control mice were sacrificed 455 days after the original injection of U937 cells, and their PBMC were pooled and injected intravenously into recipient 6-week-old male Swiss mice (2 x 106 cells per mouse; four mice per group). At different days after the PBMC injection, serum samples from individual mice were tested for the presence of antibodies to HIV as described in Materials and Methods. Bar, standard error (shown when it exceeds the diameter of the symbol). 0, PBMC from control Swiss mice; *, PBMC from seropositive Swiss mice. O.D., optical density.
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TABLE 1. Evidence of persistent HIV infection of mouse cells from mice injected with HIV-1-infected U937 cells Mouse
C9 C9 D5 C4 IVi
II16 V8 IV2 C7
Day'
352 380 352 354 354 380 420 420 531
HIV DNA in mouse PBMCb
HIV isolation by in vitro cocultivationc
env
gag
Donor cells
Target cells
Testd
+ ND + ND ND + + + ND
+ ND + ND ND + + + ND
PBMC Spleen cells PBMC PC PBMC PBMC PBMC PBMC PC
U937 CEM CEM
p24 PCR p24 PCR p24 p24 p24 p24; PCR p24
U937 U937
U937 CEM CEM U937
a Day after i.p. injection of mice with HIV-infected U937 cells. b Blood samples from tagged mice were collected, and PBMC were isolated by Ficoll-Hypaque. DNA aliquots were processed by PCR analysis for the detection of HIV sequences with specific primers for HIV env andgag as described in Materials and Methods. +, presence of specific HIV sequences; ND, not determined. c PBMC, peritoneal cells (PC), or spleen cells from the indicated tagged mice were cocultivated with human uninfected target cells at a donor cell/target cell ratio of approximately 1:1. Before cocultivation, PBMC and spleen cells were treated for 48 h with phytohemagglutinin (20 1Lg/ml) and, subsequently, for 24 h with interleukin-2 (50 U/ml). After a cocultivation period of 8 to 10 days, the cocultivated human target cells were tested for HIV infection by different experimental approaches. d Evidence of HIV infection of target cells, determined as follows. Aliquots of cell supematant from cocultivated target cells were tested for the presence of detectable p24 levels as described in Materials and Methods. DNAs were extracted from cocultivated target cells and tested for the presence of HIV sequences by PCR techniques with gag and env primers for HIV.
apparently because of the species-specific properties of the HIV receptor (i.e., human CD4) (15). In addition, the existence of other cell components restricting infection has been postulated because expression of human CD4 in transfected murine cells permits HIV-1 binding but not viral infection (21). Likewise, murine cells transfected with HIV-1 provirus produce virus particles much less efficiently than transfected human cells (18), probably because of a decreased transactivation of the HIV-1 long terminal repeat by the tat protein (11). Lastly, a human cell factor essential for HIV-1 rev action has recently been described (31). In spite of all these intrinsic limitations, our data clearly indicate that at least some of these restrictive steps for HIV replication in mouse cells can be overcome in vivo. In this regard, it is of interest to mention, however, that mouse cells from infected mice produced low amounts of infectious virus, suggesting that some restrictions for virus replication may still be present in this system. Although the mechanisms involved in the HIV infection of mice reported herein are still unclear, several experimental aspects may have been essential: (i) the injection of mice with a cloned line of HIV-infected cells capable of producing large amounts of HIV (26), (ii) the use of immunocompetent mice, and (iii) the i.p. injection of HIV-infected cells into thioglycolate-treated mice. The possible importance of this last approach has also been emphasized by others (8). It is possible that mouse peritoneal macrophages are initially infected with HIV in vivo, perhaps through such mechanisms as Fc-receptor-mediated endocytosis, cell contact with HIV-infected U937 cells, and cell fusion processes. The finding that HIV-infected PBMC are present in these mice even 500 days after the initial i.p. injection of HIVinfected human cells indicates that some crucial selection steps did occur in vivo, resulting in an altered HIV tropism for mouse cells. It has been suggested that expanded HIV-1 tropism can occur by phenotypic mixing with murine endogenous retroviruses (20). Although we cannot rule out the possibility that phenotypic mixing occurred in some of these mice, we were unable to find any evidence that the HIVpositive human U937 cells, obtained after cocultivation with
mouse PBMC from seropositive mice, were also infected with some putative mouse retroviruses (data not shown). Other mechanisms might be suggested. The HTLV III-B strain originally used for the in vitro infection of U937 cells has been shown to be a mixture of several viruses (25). A great deal of variability in HIV cellular tropism exists among different HIV strains (2, 4), and a number of selection steps are likely to occur in vivo in an immunocompetent host. In this regard, it is of interest that, even with a molecularly cloned HIV strain in human cells, marked alterations in the host range of HIV were observed after passage through various cell types (2). Our data could be interpreted to suggest, therefore, that the selection of HIV variants with additional tropism for mouse cells can occur in vivo in an immunocompetent mouse host. Work to determine the possible spectrum of pathology induced by HIV infection in mice and to characterize the HIV recovered from infected mice is in progress. ACKNOWLEDGMENTS We thank G. B. Rossi (Rome, Italy) and I. Gresser (Villejuif, France) for helpful suggestions. This work was supported in part by a grant from Ministero della Sanita/ISS (IV Progetto di Ricerche sull'AIDS 1990). REFERENCES 1. Barre-Sinoussi, F., J. C. Chermann, F. Rey, M. T. Nugeyre, S. Chamaret, J. Gruest, C. Dauget, C. Axler-Blin, F. Vezinet-Brun, C. Rouzioux, W. Rozenbaum, and L. Montaigner. 1983. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220: 868-870. 2. Cheng-Mayer, C., D. Seto, and J. A. Levy. 1991. Altered host range of HIV-1 after passage through various human cell types. Virology 181:288-294. 3. Coffin, J., A. Haase, J. A. Levy, L. Montagnier, S. Oroszlan, N. Teich, H. Temin, K. Toyoshima, H. Varmus, P. Vogt, and R. Weiss. 1986. Human immunodeficiency viruses. Science 232: 697. 4. Collman, R., N. F. Hassan, R. Walker, B. Godfrey, J. Cutilli, J. C. Hastings, H. Friedman, S. D. Douglas, and N. Nathanson.
1654
5.
6.
7.
8. 9.
10.
11.
12. 13.
LOCARDI ET AL.
1989. Infection of monocyte-derived macrophages with human immunodeficiency virus type 1 (HIV-1). J. Exp. Med. 170:11491163. Erlich, H. A., and T. L. Bugawan. 1989. HLA class II gene polymorphism: DNA typing, evolution, and relation to disease susceptibility, p. 202-204. In H. A. Erlich (ed.), PCR technology: principles and applications for DNA amplification. Stockton Press, New York. Ferrantini, M., F. Belardelli, and C. Locardi. 1988. Role of endogenous alpha/beta interferon in the selection of virus nonproducer Friend leukemia cells after serial intraperitoneal passages in syngenic mice. J. Virol. 62:600-605. Filice, G., P. M. Cereda, P. Orsolini, L. Soldini, E. Romero, and E. G. Rondanelli. 1989. Detection of IgG anti-HIV antibodies in mice experimentally infected with HIV-1. Microbiologica 12: 75-80. Filice, G., P. M. Cereda, and 0. E. Varnier. 1988. Infection of rabbit with human immunodeficiency virus. Nature (London) 335:366-368. Folks, T. M., D. Powell, M. Lightfoote, S. Koenig, A. S. Fauci, S. Benn, A. Rabson, D. Daugherty, H. E. Gendelman, M. D. Hoggan, S. Venkatesan, and M. A. Martin. 1986. Biological and biochemical characterization of a cloned leu-3- cell surviving infection with the acquired immune deficiency syndrome retrovirus. J. Exp. Med. 164:280-290. Gardner, M. B., and P. A. Luciw. 1989. Animal models of AIDS. FASEB J. 3:2593-2606. Hart, C. E., C. Ou, J. C. Galphin, J. Moore, L. T. Bacheler, J. J. Wasmuth, S. R. Petteway, and G. Schochetman. 1989. Human chromosome 12 is required for elevated HIV-1 expression in human-hamster hybrid cells. Science 246:488-491. Jacobsen, H., J. Mestan, S. Mittnacht, and C. W. Dieffenbach. 1989. Beta interferon subtype 1 induction by tumor necrosis factor. Mol. Cell. Biol. 9:3037-3042. Kawasaki, E. S. 1989. Sample preparation from blood, cells, and other fluids, p. 147-148. In M. A. Innins, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.), PCR protocols: a guide to methods and applications. Academic Press, Inc., New York.
14. Kulaga, H., T. Folks, R. Rutledge, M. E. Truckenmiller, E. Gugel, T. J. Kindt. 1989. Infection of rabbits with human immunodeficiency virus 1. J. Exp. Med. 169:321-326. 15. Landau, N. R., M. Warton, and D. R. Littman. 1988. The envelope glycoprotein of the human immunodeficiency virus binds to the immunoglobulin-like domain of CD4. Nature (London) 334:159-162. 16. Leonard, J. M., J. W. Abramczuk, D. S. Pezen, R. Ruthledge, J. H. Belcher, F. Hakim, G. Shearer, L. Lamperth, W. Travis, T. Fredrickson, A. Notkins, and M. A. Martin. 1988. Development of disease and virus recovery in transgenic mice containing HIV proviral DNA. Science 242:1665-1670. 17. Leonard, J. M., J. S. Khillan, H. E. Gendelman, A. Adachi, S. Lorenzo, H. Westphal, M. A. Martin, and M. S. Meltzer. 1989. The human immunodeficiency virus long terminal repeat is preferentially expressed in Langerhans' cells in transgenic mice. AIDS Res. Hum. Retroviruses 5:421-430. 18. Levy, J. A., C. Cheng-Mayer, D. Dina, and P. A. Luciw. 1986. AIDS retrovirus (ARV-2) clone replicates in transfected human and animal fibroblasts. Science 232:998-1001.
J. VIROL. 19. Locardi, C., C. Petrini, G. Boccoli, U. Testa, C. Dieffenbach, S. Butto, and F. Belardelli. 1990. Increased human immunodeficiency virus (HIV) expression in chronically infected U937 cells upon in vitro differentiation by hydroxyvitamin D3: roles of interferon and tumor necrosis factor in regulation of HIV production. J. Virol. 64:5874-5882. 20. Lusso, P., F. Di Marzo Veronese, B. Ensoli, G. Franchini, C. Jemma, E. S. De Rocco, V. S. Kalyanaraman, and R. C. Gallo. 1990. Expanded HIV-1 cellular tropism by phenotypic mixing with murine endogenous retroviruses. Science 24:848-852. 21. Maddon, P. J., A. G. Dalgleish, J. S. McDougal, P. R. Clapham, R. A. Weiss, and R. Axel. 1986. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell 47:333-348. 22. McCune, J. M., R. Namikawa, H. Kaneshima, L. D. Shultz, M. Lieberman, and I. L. Weissman. 1988. The SCID-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function. Science 241:1632-1639. 23. Mosier, D. E., R. J. Gulizia, S. M. Baird, D. B. Wilson, D. H. Spector, and S. A. Spector. 1991. Human immunodeficiency virus infection of human-PBL-SCID mice. Science 251:791-794. 24. Namikawa, R., H. Kaneshima, M. Lieberman, I. L. Weissman, and J. M. McCune. 1988. Infection of the SCID-hu mouse by HIV-1. Science 242:1684-1686. 25. Popovic, M., M. G. Sarngadharan, E. Read, and R. C. Gallo. 1984. Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 224:497-500. 26. Puddu, P., C. Locardi, P. Sestili, F. Varano, C. Petrini, A. Modesti, L. Masuelli, I. Gresser, and F. Belardelli. 1991. Human immunodeficiency virus (HIV)-infected tumor xenografts as an in vivo model for antiviral therapy: role of alpha/beta interferon in restriction of tumor growth in nude mice injected with HIV-infected U937 tumor cells. J. Virol. 65:2245-2253. 27. Ruprecht, R. M., L. D. Bernard, T. C. Chou, M. A. Gama Sosa, F. Fazely, J. Koch, P. L. Sharma, and S. Mullaney. 1990. Murine models for evaluating antiretroviral therapy. Cancer Res. 50: 5618-5627. 28. Simmonds, P., P. Balfe, J. F. Peutherer, C. A. Ludlam, J. 0. Bishop, and A. J. L. Brown. 1990. Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. J. Virol. 64:864-872. 29. Strauss, W. M. 1987. Preparation of genomic DNA from mammalian tissue, p. 2.2.1-2.2.3. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecular biology. John Wiley & Sons, Inc., New York. 30. Sunstrom, C., and K. Nilsson. 1976. Establishment and characterization of a human histiocytic lymphoma cell line (U937). Int. J. Cancer 17:565-577. 31. Trono, D., and D. Baltimore. 1990. A human cell factor is essential for HIV-1 Rev action. EMBO J. 9:4155-4160. 32. Vogel, J., S. H. Hinrichs, R. K. Reynolds, P. A. Luciw, and G. Jay. 1988. The HIV tat gene induces dermal lesions resembling Kaposi's sarcoma in transgenic mice. Nature (London) 335:606611.
Vol. 66, No. 3
0022-538X/92/031649-06$02.00/0 Copyright © 1992, American Society for Microbiology
Persistent Infection of Normal Mice with Human Immunodeficiency Virus CHIARA LOCARDI, PATRIZIA PUDDU, MARIA FERRANTINI, ELEONORA PARLANTI, PAOLA SESTILI, FRANCO VARANO, AND FILIPPO BELARDELLI* Laboratory of Virology, Istituto Superiore di Sanitez, Viale Regina Elena 299, 00161 Rome, Italy Received 11 July 1991/Accepted 22 November 1991
In this article, we report the establishment of persistent HIV type 1 infection of normal Swiss mice after a single intraperitoneal injection with high-producing HIV-infected U937 cells. Anti-HIV antibodies were found more than 500 days after the original injection, and p24 antigenemia was detected in approximately 50%37s of the mice. By polymerase chain reaction (PCR) techniques, HIV-specific gag and env sequences were detected in DNA samples from peripheral blood mononuclear cells (PBMC) and peritoneal cells of seropositive mice 300 to 500 days after inoculation with HIV-infected cells. These DNA samples did not contain human DNA sequences, as determined by PCR analysis using primers and the probe for the HLA-DQ at gene. Low levels of p24 and detectable human reverse transcriptase activity were found in cultures of PBMC and peritoneal macrophages. Cocultivation of PBMC, peritoneal cells, and spleen cells with human uninfected U937 or CEM (a T lymphoma cell line) cells resulted in HIV infection of the target cells, as determined by PCR analysis and/or p24 assays. The intravenous injection of untreated Swiss mice with the PBMC from PCR-positive mice resulted in the development of an increasing antibody response to HIV in the recipient animals. Together these results indicate that cells from seropositive Swiss mice were persistently infected with HIV and were capable of producing infectious virus. The development of persistent HIV infection in an immunocompetent mouse may represent the starting point for further studies aimed at defining the host mechanisms involved in the restriction of virus replication, defining the pathogenesis of HIV infection, and testing antiviral compounds and vaccines.
Human immunodeficiency virus type 1 (HIV-1) is considered the etiologic agent of AIDS (1, 3, 25). Although the biology of HIV has been intensively explored, the relationship between HIV-1 infection and AIDS is not clear. Animal models for AIDS are thus essential for understanding the pathogenesis of HIV-1-induced immunodeficiency and for defining effective antiviral therapies and vaccines (10). A small and well-characterized laboratory animal, such as the mouse, would be especially useful (27). However, we are not aware of any report of HIV infection of murine cells in vitro or in vivo. The following two major types of in vivo murine models for HIV have been developed so far: (i) transgenic mice carrying either intact (16) or partial (17, 32) HIV-1 genomes, and (ii) immunodeficient mice engrafted with human fetal lymphoid tissue (SCID-hu mice) (22, 24) or adult human peripheral blood leukocytes (PBL-SCID mice) (23). An additional and simple approach for the development of a persistent HIV infection in a small-animal model would be the injection of HIV or HIV-infected human cells directly into immunocompetent animals. In fact, it has been reported that rabbits can be infected with HIV under special experimental conditions (8, 14). We suspected that a similar phenomenon could also occur in mice if suitable numbers of virus producer cells were injected into an immunocompetent mouse host. The results reported in this article indicate that this experimental procedure can be very effective in establishing a persistent infection of normal mice with HIV.
*
MATERIALS AND METHODS Cells and culture conditions. The human histiocytic lymphoma cell line U937 (30) (kindly provided by A. Siccardi, Milan, Italy) was cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum. U937 cells were infected with HIV-1 (HTLV III-B strain) as described elsewhere (19). Fifty days after HIV infection, chronically virus-infected U937 cells were cloned. A virus producer cloned U937 cell line (clone 10) was selected and used for the in vivo experiments described below. These cloned U937 cells produced large amounts of HIV in vivo when transplanted subcutaneously in nude mice treated with antibodies to mouse alpha/beta interferon (26). Mice. Six-week-old male Swiss mice (Charles River S.p.A., Milan, Italy) were kept under special biosafety level 3 conditions during the course of the experiments. Mice were injected intraperitoneally (i.p.) with 0.5 ml of 4% thioglycolate broth 4 days before the injection of HIVinfected cells. In 3 sets of experiments, a total of 70 mice were inoculated i.p. with HIV-infected U937 cells (2 x 106 cells per mouse) and 30 control mice were injected with the same number of uninfected U937 cells. Before injection, HIV-infected cells were treated for 24 h with human tumor necrosis factor alpha (100 U/ml), which results in a marked increase in the production of infectious HIV particles (19). Serologic testing of anti-HIV antibodies. Aliquots of orbital venous blood were collected from individual tagged mice at different times after injection of virus-infected cells. Serum samples were tested for HIV-1-specific antibodies with the commercial HIV-1 enzyme-linked immunosorbent assay
(ELISA) microplates (Ortho Diagnostics Systems) following a modified procedure. Briefly, 200 pl of serum, diluted 1:100 in phosphate-buffered saline (PBS), was added to the appropriate wells. After a 1-h incubation at 37°C, wells were extensively washed and 200 ,ul of a 1:1,000 dilution of
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peroxidase-conjugated anti-mouse immunoglobulin G (IgG) (Cappel Laboratories) was added. After 60 min at 37°C, microplates were washed five times with PBS and 200 VI of peroxidase substrate solution was added to each well. After 30 min at room temperature, the reaction was stopped by the addition of 50 j,l of 4 N H2SO4 and the A492 was read by means of a flow multiscan reader. Western blot (immunoblot) analyses were carried out with nitrocellulose strips containing HIV-1 proteins (Du Pont, Wilmington, Del.) and biotin-labelled goat anti-mouse IgG (Sigma, St. Louis, Mo.). Western blot strips were probed with a 1:50 dilution of mouse serum. Before Western blot analysis, mouse sera were adsorbed against uninfected U937 cells (108 viable cells incubated with 0.4 ml of serum for 1 h at 37°C). Gene amplification by PCR. DNA from cultured cells was extracted by standard procedures (29). Mouse peripheral blood mononuclear cells (PBMC), peritoneal cells, and cocultivated cells were lysed as described previously (13), and aliquots of the lysate were added directly to the polymerase chain reaction (PCR). PCR was carried out in a reaction mixture of 25 VI containing 0.5 ,uM (each) primers, 200 ,uM (each) deoxynucleoside triphosphates, and 1.5 mM MgCl2. After 30 cycles of amplification (95°C for 90 s, 50°C for 90 s, and 72°C for 2 min) followed by a final extension at 72°C for 10 min, PCR products were resolved on 1.75% SeaKem (FMC) agarose gels, transferred to a Hybond (Amersham) membrane, and hybridized with 32P-labelled detector oligonucleotides. PCR experiments were performed with envl 401 and 404 and gag 881 and 882 primer pairs. The corresponding probes were envl 405 (6654 5' GCGGGAGAAT GATAATG GAGAAAGGAG, HIV HXB2) and gag 883 (28). Amplification and detection of the HLA-DQ a gene fragment were performed with the GH26-GH27 primer pair and the corresponding probe, RH54 (5). All PCR experiments included a set of reaction mixtures containing GAPDH primer pairs (12) as a control for amplification (data not shown). Detection of p24 antigenemia. Serum aliquots were harvested from tagged mice and tested for the presence of detectable p24 levels by means of an antigen-capture ELISA (Du Pont). RT assays. The assay to measure the human reverse transcriptase (RT) activity in culture supernatants was performed as described in detail elsewhere (19). Before the RT activity was tested, 1 ml of cell supernatant was centrifuged for 15 min at 95,000 rpm in a TLA-100 rotor on a Beckman TL-100 bench-top ultracentrifuge. The pellet was resuspended in 70 VI of TNE (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA [pH 8], 0.1% Triton X-100), and 30 ,ul was added to the reaction mixture (19). For evaluating the possible presence of murine RT activity, the same samples were tested with a different reaction mixture containing MnCl2 instead of MgCI2, as described in detail elsewhere (6). Culture of mouse PBMC and cocultivation techniques. At different days after the i.p. injection of HIV-infected U937 cells, blood samples from individual tagged mice were collected and PBMC were isolated by Ficoll-Hypaque (density, 1.09 g/cm3). The PBMC were treated for 48 h with phytohemagglutinin (20 ,ug/ml) and then for 24 h with human recombinant interleukin-2 (50 U/ml). Next, the PBMC were cocultivated with human uninfected cells (i.e., U937 cells or CEM cells, a T-lymphoma cell line) at a donor cell/target cell ratio of approximately 1:1. After a cocultivation period of 8 to 10 days, the cocultivated human target cells were tested for HIV infection. Aliquots of cell supernatant from coculti-
J. VIROL.
vated target cells were tested for the presence of detectable p24 levels by means of an antigen-capture ELISA (Du Pont). DNAs were extracted from cocultivated target cells and tested for the presence of HIV sequences by PCR techniques using gag and env primers for HIV (28). Nucleotide sequence accession number. The nucleotide sequence accession number for envl 405 is GenBank K03455. RESULTS Long-lasting antibody response to HIV-1 in mice injected with HIV-infected U937 cells. IgG antibodies to HIV were detected by IgG ELISA in all the mice inoculated with HIV-infected U937 cells. Figure 1 shows the kinetics of the antibody response to HIV in four representative mice injected with HIV-infected U937 cells. The titers of anti-HIV antibodies in the sera reached the maximum level 60 to 100 days after the injection of HIV-infected cells. Subsequently, there were fluctuations in the antibody titers in these mice, but all mice continued to have high antibody titers 200 to 500 days after the initial inoculation of HIV-infected U937 cells (Fig. 1A through D). The Western blot analyses of these sera revealed the presence of detectable antibodies to gpl20 and p24 HIV proteins even 500 days after the original injection with HIV-infected U937 cells (Fig. 2, lane 2). Detection of HIV-specific gag and env sequences in the DNAs of mouse cells from seropositive Swiss mice. By PCR techniques, we analyzed blood samples from seropositive mice for the presence of HIV-specific sequences in the DNAs extracted 350 to 500 days after the injection of HIV-infected U937 cells. As shown in Fig. 3A (lane 3) and B (lane 4), DNAs extracted from the blood of long-termseropositive mice exhibited detectable levels of HIV-specific gag and env sequences. In order to determine the approximate number of HIV genomic copies present in the DNA from these blood samples, we performed PCR assays in which decreasing amounts of DNA from 8E5/LAV cells (which contain a single integrated copy of proviral DNA per genome [9]) were mixed with a constant amount of spleen DNA from normal Swiss mice. Comparison of the signals in the same PCR assays showed that the DNA sample from one seropositive mouse (D5) contained approximately 1 HIV genomic copy per 100 to 1,000 cell genomes (data not shown). HIV-specific sequences were also found in DNA samples from peritoneal cells of seropositive mice (Fig. 3A, lane 5). To rule out the possibility that these HIV-positive signals were due to some contamination with surviving human U937 cells, we performed PCR analysis of DNA using primers and the probe for the HLA-DQ a gene. As shown in Fig. 4, human sequences were not detected in the HIV-positivemouse DNA. Virus expression in mice injected with HIV-infected cells. The data reported above indicated that the HIV genomic sequences were present in some mouse PBMC and peritoneal cells from seropositive Swiss mice for a long time after a single i.p. injection of HIV-infected U937 cells. It was of interest, therefore, to investigate whether HIV could be expressed in and/or isolated from cells of seropositive mice by different techniques. Detectable levels of p24 antigenemia (40 to 60 pg/ml) were found in the serum samples of four of eight seropositive mice more than 500 days after the original injection of HIV-infected U937 cells. Moreover, when PBMC were harvested from three PCR-positive mice and injected intravenously into four recipient untreated mice, an
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0 100 200 300 400 500 200 300 400 5oo DAYS DAYS FIG. 1. Kinetics of the antibody response to HIV in four representative Swiss mice injected i.p. with cloned HIV-infected U937 cells. Serum samples from individual tagged mice were taken at different times after cell injection. At each time, serum samples from control mice were also tested for anti-HIV antibodies. Serum samples were tested for HIV-1-specific antibodies with HIV-1 ELISA microplates as described in Materials and Methods. 0, control mice; 0, mice injected with HIV-infected U937 cells. O.D., optical density.
0
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increasing antibody response to HIV was found in the serum samples of the mice. In contrast, antibodies were not detected in the sera of recipient mice injected with PBMC from control mice (Fig. 5). These results suggested that HIV proteins were expressed in vivo in some PBMC from seropositive Swiss mice. In other experiments, PBMC, peritoneal cells, spleen cells, and cells from mesenteric lymph nodes were cultured in vitro for a few days to determine the p24 levels in the cell supernatants. Low levels of extracellular and/or intracellular p24 (i.e., 10 to 20 pg/ml) were found 5 to 15 days after in vitro cultivation. Supernatants from spleen cells and cells from lymph nodes were also ultracentrifuged and tested for the presence of human and murine RT activities. Significant levels of human RT activity were found in these samples, whereas no enzymatic activity was revealed with the typical reaction buffer for murine RT (data not shown). In some experiments, PBMC, peritoneal cells, or spleen cells from seropositive mice were cocultivated with uninfected human U937 or human CEM cells. The cocultivated target cells were then tested for the presence of HIV-specific sequences by PCR analysis of the extracted DNAs. Figure 3C shows one example of the PCR analysis of CEM cells after cocultivation with spleen cells from a seropositive mouse compared with that of CEM cells prior to cocultivation. The DNA of cocultivated CEM cells exhibited env and gag HIV-specific sequences (Fig. 3C, lane 4). Table 1 summarizes representative data for individual mice in which the evidence of HIV infection was obtained by different techniques. A total of 32 mice were randomly sacrificed 350 to 500 days after the original injection. Eighteen of these mice were found to be infected with HIV as evaluated by the capability of their PBMC or spleen cells to
1
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FIG. 2. Western blot analyses of sera from Swiss mice injected with HIV-infected U937 cells. Western blot analyses were carried out with nitrocellulose strips containing HIV-1 proteins as described in Materials and Methods. Strips were probed with serum from a control mouse (lane 1); pooled serum from four mice harvested 512 days after the original injection with HIV-infected U937 cells (lane 2), and control human serum, positive for antibodies to HIV (lane 3).
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B *
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3 1 2 FIG. 4. PCR evaluation of the absence of human DNA sequences in the HIV-positive mouse DNA. Blood DNA from a representative HIV-PCR-positive mouse (D5) was tested by PCR for the presence of HLA-DQ a sequences by using the specific primer pair and probe as described in Materials and Methods. DNA is from HIV-1-infected U937 cells (clone 10) (lane 1); a blood sample from the D5 HIV-PCR-positive mouse (lane 2) (see also Fig. 2B, lane 4); splenocytes from a control Swiss mouse (lane 3). The inset (upper right corner) shows the GAPDH gene amplification from the DNA of the same samples.
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FIG. 3. Detection by PCR analysis of HIV sequences in mouse cells from Swiss mice injected with HIV-infected U937 cells. Integrated HIV-1 sequences were detected in different mouse cell types by PCR analysis of DNA using primer pairs specific for HIV-1 env and gag sequences (left and right sets of lanes in each panel, respectively) as described in Materials and Methods. (A) DNA from HIV-1-infected U937 cells (clone 10) (lane 1), a blood sample from a control mouse (lane 2), a blood sample from a seropositive mouse (C9) (lane 3), peritoneal cells from a control Swiss mouse (lane 4), and peritoneal cells from a seropositive mouse (V7) (lane 5); (B) DNA from HIV-1-infected U937 cells (lane 1), a blood sample from a normal Swiss mouse (lane 2), splenocytes from a control Swiss mouse (lane 3), and a blood sample from a seropositive mouse (D5) (lane 4); (C) DNA from HIV-1-infected U937 cells (lane 1), splenocytes from a control Swiss mouse (lane 2), splenocytes from a seropositive mouse (C9) (lane 3), CEM cells cocultivated with splenocytes from the C9 mouse (lane 4), and CEM cells before cocultivation (lane 5).
spleen cells, even beyond 500 days after the original injection. These HIV-positive signals did not reflect contamination with in vivo surviving U937 cells, as PCR analysis did not reveal any presence of human-specific sequences. (ii) Mouse cells were capable of expressing virus proteins (i.e., p24 and RT) and infecting human target cells, indicating the competence of the mouse cells for infectious-virus production. It has generally been assumed that mouse cells cannot be infected with HIV because they lack some properties essential for virus replication. Mouse cells do not bind HIV, 0.6
0.4o
o infect human target cells. Together these results indicate that cells from seropositive mice injected with HIV-infected U937 cells were persistently infected with HIV.
DISCUSSION The data reported in this article represent, to the best of our knowledge, the first experimental evidence to indicate that mouse cells can be infected in vivo with HIV. (Filice and coworkers [7] reported a long-lasting antibody response to HIV in mice injected i.p. with HIV-infected human H9 cells, but there was no experimental evidence to show that mouse cells had been infected with HIV-1.) In this study, the following two major findings clearly demonstrate that mouse cells had been persistently infected with HIV. (i) HIV-1-specific sequences were detected by PCR in the DNAs from mouse cells, such as PBMC and
0.3 0.2
0.1
0~~~~~~~ 10
20
30
40
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days after Injection of PBMC FIG. 5. Antibody response to HIV in mice injected with PBMC from seropositive Swiss mice. Three HIV-PCR-positive and three control mice were sacrificed 455 days after the original injection of U937 cells, and their PBMC were pooled and injected intravenously into recipient 6-week-old male Swiss mice (2 x 106 cells per mouse; four mice per group). At different days after the PBMC injection, serum samples from individual mice were tested for the presence of antibodies to HIV as described in Materials and Methods. Bar, standard error (shown when it exceeds the diameter of the symbol). 0, PBMC from control Swiss mice; *, PBMC from seropositive Swiss mice. O.D., optical density.
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TABLE 1. Evidence of persistent HIV infection of mouse cells from mice injected with HIV-1-infected U937 cells Mouse
C9 C9 D5 C4 IVi
II16 V8 IV2 C7
Day'
352 380 352 354 354 380 420 420 531
HIV DNA in mouse PBMCb
HIV isolation by in vitro cocultivationc
env
gag
Donor cells
Target cells
Testd
+ ND + ND ND + + + ND
+ ND + ND ND + + + ND
PBMC Spleen cells PBMC PC PBMC PBMC PBMC PBMC PC
U937 CEM CEM
p24 PCR p24 PCR p24 p24 p24 p24; PCR p24
U937 U937
U937 CEM CEM U937
a Day after i.p. injection of mice with HIV-infected U937 cells. b Blood samples from tagged mice were collected, and PBMC were isolated by Ficoll-Hypaque. DNA aliquots were processed by PCR analysis for the detection of HIV sequences with specific primers for HIV env andgag as described in Materials and Methods. +, presence of specific HIV sequences; ND, not determined. c PBMC, peritoneal cells (PC), or spleen cells from the indicated tagged mice were cocultivated with human uninfected target cells at a donor cell/target cell ratio of approximately 1:1. Before cocultivation, PBMC and spleen cells were treated for 48 h with phytohemagglutinin (20 1Lg/ml) and, subsequently, for 24 h with interleukin-2 (50 U/ml). After a cocultivation period of 8 to 10 days, the cocultivated human target cells were tested for HIV infection by different experimental approaches. d Evidence of HIV infection of target cells, determined as follows. Aliquots of cell supematant from cocultivated target cells were tested for the presence of detectable p24 levels as described in Materials and Methods. DNAs were extracted from cocultivated target cells and tested for the presence of HIV sequences by PCR techniques with gag and env primers for HIV.
apparently because of the species-specific properties of the HIV receptor (i.e., human CD4) (15). In addition, the existence of other cell components restricting infection has been postulated because expression of human CD4 in transfected murine cells permits HIV-1 binding but not viral infection (21). Likewise, murine cells transfected with HIV-1 provirus produce virus particles much less efficiently than transfected human cells (18), probably because of a decreased transactivation of the HIV-1 long terminal repeat by the tat protein (11). Lastly, a human cell factor essential for HIV-1 rev action has recently been described (31). In spite of all these intrinsic limitations, our data clearly indicate that at least some of these restrictive steps for HIV replication in mouse cells can be overcome in vivo. In this regard, it is of interest to mention, however, that mouse cells from infected mice produced low amounts of infectious virus, suggesting that some restrictions for virus replication may still be present in this system. Although the mechanisms involved in the HIV infection of mice reported herein are still unclear, several experimental aspects may have been essential: (i) the injection of mice with a cloned line of HIV-infected cells capable of producing large amounts of HIV (26), (ii) the use of immunocompetent mice, and (iii) the i.p. injection of HIV-infected cells into thioglycolate-treated mice. The possible importance of this last approach has also been emphasized by others (8). It is possible that mouse peritoneal macrophages are initially infected with HIV in vivo, perhaps through such mechanisms as Fc-receptor-mediated endocytosis, cell contact with HIV-infected U937 cells, and cell fusion processes. The finding that HIV-infected PBMC are present in these mice even 500 days after the initial i.p. injection of HIVinfected human cells indicates that some crucial selection steps did occur in vivo, resulting in an altered HIV tropism for mouse cells. It has been suggested that expanded HIV-1 tropism can occur by phenotypic mixing with murine endogenous retroviruses (20). Although we cannot rule out the possibility that phenotypic mixing occurred in some of these mice, we were unable to find any evidence that the HIVpositive human U937 cells, obtained after cocultivation with
mouse PBMC from seropositive mice, were also infected with some putative mouse retroviruses (data not shown). Other mechanisms might be suggested. The HTLV III-B strain originally used for the in vitro infection of U937 cells has been shown to be a mixture of several viruses (25). A great deal of variability in HIV cellular tropism exists among different HIV strains (2, 4), and a number of selection steps are likely to occur in vivo in an immunocompetent host. In this regard, it is of interest that, even with a molecularly cloned HIV strain in human cells, marked alterations in the host range of HIV were observed after passage through various cell types (2). Our data could be interpreted to suggest, therefore, that the selection of HIV variants with additional tropism for mouse cells can occur in vivo in an immunocompetent mouse host. Work to determine the possible spectrum of pathology induced by HIV infection in mice and to characterize the HIV recovered from infected mice is in progress. ACKNOWLEDGMENTS We thank G. B. Rossi (Rome, Italy) and I. Gresser (Villejuif, France) for helpful suggestions. This work was supported in part by a grant from Ministero della Sanita/ISS (IV Progetto di Ricerche sull'AIDS 1990). REFERENCES 1. Barre-Sinoussi, F., J. C. Chermann, F. Rey, M. T. Nugeyre, S. Chamaret, J. Gruest, C. Dauget, C. Axler-Blin, F. Vezinet-Brun, C. Rouzioux, W. Rozenbaum, and L. Montaigner. 1983. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220: 868-870. 2. Cheng-Mayer, C., D. Seto, and J. A. Levy. 1991. Altered host range of HIV-1 after passage through various human cell types. Virology 181:288-294. 3. Coffin, J., A. Haase, J. A. Levy, L. Montagnier, S. Oroszlan, N. Teich, H. Temin, K. Toyoshima, H. Varmus, P. Vogt, and R. Weiss. 1986. Human immunodeficiency viruses. Science 232: 697. 4. Collman, R., N. F. Hassan, R. Walker, B. Godfrey, J. Cutilli, J. C. Hastings, H. Friedman, S. D. Douglas, and N. Nathanson.
1654
5.
6.
7.
8. 9.
10.
11.
12. 13.
LOCARDI ET AL.
1989. Infection of monocyte-derived macrophages with human immunodeficiency virus type 1 (HIV-1). J. Exp. Med. 170:11491163. Erlich, H. A., and T. L. Bugawan. 1989. HLA class II gene polymorphism: DNA typing, evolution, and relation to disease susceptibility, p. 202-204. In H. A. Erlich (ed.), PCR technology: principles and applications for DNA amplification. Stockton Press, New York. Ferrantini, M., F. Belardelli, and C. Locardi. 1988. Role of endogenous alpha/beta interferon in the selection of virus nonproducer Friend leukemia cells after serial intraperitoneal passages in syngenic mice. J. Virol. 62:600-605. Filice, G., P. M. Cereda, P. Orsolini, L. Soldini, E. Romero, and E. G. Rondanelli. 1989. Detection of IgG anti-HIV antibodies in mice experimentally infected with HIV-1. Microbiologica 12: 75-80. Filice, G., P. M. Cereda, and 0. E. Varnier. 1988. Infection of rabbit with human immunodeficiency virus. Nature (London) 335:366-368. Folks, T. M., D. Powell, M. Lightfoote, S. Koenig, A. S. Fauci, S. Benn, A. Rabson, D. Daugherty, H. E. Gendelman, M. D. Hoggan, S. Venkatesan, and M. A. Martin. 1986. Biological and biochemical characterization of a cloned leu-3- cell surviving infection with the acquired immune deficiency syndrome retrovirus. J. Exp. Med. 164:280-290. Gardner, M. B., and P. A. Luciw. 1989. Animal models of AIDS. FASEB J. 3:2593-2606. Hart, C. E., C. Ou, J. C. Galphin, J. Moore, L. T. Bacheler, J. J. Wasmuth, S. R. Petteway, and G. Schochetman. 1989. Human chromosome 12 is required for elevated HIV-1 expression in human-hamster hybrid cells. Science 246:488-491. Jacobsen, H., J. Mestan, S. Mittnacht, and C. W. Dieffenbach. 1989. Beta interferon subtype 1 induction by tumor necrosis factor. Mol. Cell. Biol. 9:3037-3042. Kawasaki, E. S. 1989. Sample preparation from blood, cells, and other fluids, p. 147-148. In M. A. Innins, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.), PCR protocols: a guide to methods and applications. Academic Press, Inc., New York.
14. Kulaga, H., T. Folks, R. Rutledge, M. E. Truckenmiller, E. Gugel, T. J. Kindt. 1989. Infection of rabbits with human immunodeficiency virus 1. J. Exp. Med. 169:321-326. 15. Landau, N. R., M. Warton, and D. R. Littman. 1988. The envelope glycoprotein of the human immunodeficiency virus binds to the immunoglobulin-like domain of CD4. Nature (London) 334:159-162. 16. Leonard, J. M., J. W. Abramczuk, D. S. Pezen, R. Ruthledge, J. H. Belcher, F. Hakim, G. Shearer, L. Lamperth, W. Travis, T. Fredrickson, A. Notkins, and M. A. Martin. 1988. Development of disease and virus recovery in transgenic mice containing HIV proviral DNA. Science 242:1665-1670. 17. Leonard, J. M., J. S. Khillan, H. E. Gendelman, A. Adachi, S. Lorenzo, H. Westphal, M. A. Martin, and M. S. Meltzer. 1989. The human immunodeficiency virus long terminal repeat is preferentially expressed in Langerhans' cells in transgenic mice. AIDS Res. Hum. Retroviruses 5:421-430. 18. Levy, J. A., C. Cheng-Mayer, D. Dina, and P. A. Luciw. 1986. AIDS retrovirus (ARV-2) clone replicates in transfected human and animal fibroblasts. Science 232:998-1001.
J. VIROL. 19. Locardi, C., C. Petrini, G. Boccoli, U. Testa, C. Dieffenbach, S. Butto, and F. Belardelli. 1990. Increased human immunodeficiency virus (HIV) expression in chronically infected U937 cells upon in vitro differentiation by hydroxyvitamin D3: roles of interferon and tumor necrosis factor in regulation of HIV production. J. Virol. 64:5874-5882. 20. Lusso, P., F. Di Marzo Veronese, B. Ensoli, G. Franchini, C. Jemma, E. S. De Rocco, V. S. Kalyanaraman, and R. C. Gallo. 1990. Expanded HIV-1 cellular tropism by phenotypic mixing with murine endogenous retroviruses. Science 24:848-852. 21. Maddon, P. J., A. G. Dalgleish, J. S. McDougal, P. R. Clapham, R. A. Weiss, and R. Axel. 1986. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell 47:333-348. 22. McCune, J. M., R. Namikawa, H. Kaneshima, L. D. Shultz, M. Lieberman, and I. L. Weissman. 1988. The SCID-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function. Science 241:1632-1639. 23. Mosier, D. E., R. J. Gulizia, S. M. Baird, D. B. Wilson, D. H. Spector, and S. A. Spector. 1991. Human immunodeficiency virus infection of human-PBL-SCID mice. Science 251:791-794. 24. Namikawa, R., H. Kaneshima, M. Lieberman, I. L. Weissman, and J. M. McCune. 1988. Infection of the SCID-hu mouse by HIV-1. Science 242:1684-1686. 25. Popovic, M., M. G. Sarngadharan, E. Read, and R. C. Gallo. 1984. Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 224:497-500. 26. Puddu, P., C. Locardi, P. Sestili, F. Varano, C. Petrini, A. Modesti, L. Masuelli, I. Gresser, and F. Belardelli. 1991. Human immunodeficiency virus (HIV)-infected tumor xenografts as an in vivo model for antiviral therapy: role of alpha/beta interferon in restriction of tumor growth in nude mice injected with HIV-infected U937 tumor cells. J. Virol. 65:2245-2253. 27. Ruprecht, R. M., L. D. Bernard, T. C. Chou, M. A. Gama Sosa, F. Fazely, J. Koch, P. L. Sharma, and S. Mullaney. 1990. Murine models for evaluating antiretroviral therapy. Cancer Res. 50: 5618-5627. 28. Simmonds, P., P. Balfe, J. F. Peutherer, C. A. Ludlam, J. 0. Bishop, and A. J. L. Brown. 1990. Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. J. Virol. 64:864-872. 29. Strauss, W. M. 1987. Preparation of genomic DNA from mammalian tissue, p. 2.2.1-2.2.3. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecular biology. John Wiley & Sons, Inc., New York. 30. Sunstrom, C., and K. Nilsson. 1976. Establishment and characterization of a human histiocytic lymphoma cell line (U937). Int. J. Cancer 17:565-577. 31. Trono, D., and D. Baltimore. 1990. A human cell factor is essential for HIV-1 Rev action. EMBO J. 9:4155-4160. 32. Vogel, J., S. H. Hinrichs, R. K. Reynolds, P. A. Luciw, and G. Jay. 1988. The HIV tat gene induces dermal lesions resembling Kaposi's sarcoma in transgenic mice. Nature (London) 335:606611.
