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Original Article |
ganem{at}socrates.ucsf.edu
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Key Words: Kaposi's sarcoma–associated herpesvirus • KSHV • SCID-hu • transmission • real-time PCR
Kaposi's sarcoma–associated herpesvirus (KSHV), also called human herpesvirus (HHV)-8, is a novel member of the lymphotropic human herpesvirus family. KSHV sequences were identified initially in Kaposi's sarcoma (KS) lesions of AIDS patients 1. The virus has also been detected in a variety of AIDS-associated lymphoproliferative disorders, namely body cavity or primary effusion lymphoma (PEL; reference 2) and multicentric Castleman's disease 3. KS itself is a hyperproliferation of poorly differentiated endothelial cells and is associated with extreme neovascularization. To date, a wealth of epidemiological evidence points to a central role for KSHV in the development of KS (for review see references 4567). For instance, KSHV DNA 8 and seroreactivity 9 directed against latent or lytic antigens can be detected in virtually all cases of KS. Seropositivity for KSHV precedes disease and is a major independent risk factor for KS in AIDS patients 1011121314. Viral DNA can also be isolated from KS lesions of HIV-1–seronegative patients 8, suggesting that KSHV is the principal viral factor involved in all epidemiological forms of this disease.
Like all herpesviruses, KSHV has two modes of infection: lytic replication, which generates infectious progeny and destroys the host cell, and latent replication, in which the viral genome persists in its host cell but with dramatically restricted gene expression and without cell destruction 15. Transcripts of latent viral genes (e.g., K12/kaposin, orf73/LANA, orf72/v-cyclin) are found in most KS tumor cells 16171819, and a subset of these transcripts (e.g., kaposin and v-cyclin) may confer a cell-autonomous proliferative advantage to the cells expressing them 2021. By contrast, <3% of infected cells in KS lesions or in KSHV+ lymphomas display evidence of lytic KSHV gene expression 162223. Certain lytic genes (K1, vGCR, vIRF) also transform cultures of NIH3T3 cells 242526; however, the relevance of these observations to tumorigenesis in vivo remains to be established.
A major impediment to the study of KSHV biology has been the absence of a suitable animal or culture model for de novo infection. PEL cell lines harbor latent KSHV genomes and can be induced to produce virions by treatment with phorbol-12-myristate-13-acetate (TPA) or sodium butyrate 22. However, most established cell lines or primary explants support only limited replication or maintenance of KSHV 2728. Unlike its cousin, Epstein-Barr virus 29, KSHV could not be propagated in SCID mice reconstituted with human peripheral blood cells 30, and transmission of KSHV to other conventional laboratory animals has not been reported. This suggests that the cell type most susceptible to KSHV replication and latency might be highly specialized or not readily propagated ex vivo. To alleviate the problem of inadequate in vitro systems to study natural infection, we decided to test whether KSHV could infect cells in human Thy/Liv organs grafted onto SCID mice.
The SCID-hu Thy/Liv mouse model uses C.B-17 scid/scid mice as recipients of human fetal liver and fetal thymus implants. Upon coimplantation of both tissues under the murine kidney capsule, human hematopoietic and lymphoid precursor cells reconstitute an organ that faithfully reproduces human multilineage hematopoiesis, including thymopoiesis 3132. T lymphocytes in various stages of development comprise the bulk (>90%) of cells in the implant, but cells of all hematopoietic lineages (including monocytes and B cells) and stromal endothelial cells are present. After direct injection of virus into the implant, replication of a number of human viruses is observed in the lymphoid (e.g., HIV-1 33, HTLV-1 [34], HHV-6 35, and varicella-zoster virus 36) and stromal (e.g., HIV-1 37, CMV 38, and measles virus 39) compartments of the graft. Depending on the biology of the particular virus, the resulting infection may be noncytopathic, (e.g., CMV 38) or may induce severe target cell depletion (e.g., HIV-1 40 and HHV-6 35).
This paper shows that KSHV derived from cultured PEL cells can initiate a productive infection in SCID-hu Thy/Liv mice. Accumulation of viral DNA depends on intact virions and can be inhibited by ganciclovir. Latent and lytic transcripts can be detected principally in CD19+ lymphocytes, but the morphology of the implant remains intact and no cytopathic effects are seen during acute infection. SCID-hu Thy/Liv mice represent the first animal model in which KSHV replication and persistence can be observed.
KSHV Infection of SCID-hu Thy/Liv Mice.
PCR and Reverse Transcriptase–PCR.
Materials and Methods
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Abstract
Materials and Methods
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Virus Growth.
BCBL-1 cells at a concentration of 5 x 105 cells/ml were induced with 20 ng/ml TPA (Calbiochem) for 12 h, reseeded in fresh medium (RPMI 1640, 10% FCS, penicillin and streptomycin, L-glutamine, sodium bicarbonate, and β-ME; all from GIBCO BRL), and cultured for an additional 4 d in 5% CO2 at 37°C. This induction protocol results in 
106 DNA genome equivalents per milliliter of culture 22. Debris was removed by centrifugation for 10 min at 5,000 rpm in a GS4 rotor, and virus was pelleted by centrifugation for 2 h at 14,000 rpm in a SS-38 rotor. Virus from 180 ml supernatant was pelleted, and the pellet was resuspended in 1,000 µl PBS, of which 50 µl was injected per implant. HIV-1 NL4-3 was prepared as previously described 41.
SCID-hu Thy/Liv mice were generated by coimplantation of fragments of human second trimester fetal liver and thymus under the kidney capsule of male homozygous C.B-17 scid/scid (SCID) mice 31. Implants were infected 4–7 mo after implantation by direct inoculation as previously described 42. In brief, mice were anesthetized, the left kidney was surgically exposed, and the implant was inoculated with 50 µl of virus stock. The abdominal wall incision was sutured, and the skin was closed with staples. Where indicated, ganciclovir sodium salt (Cytovene; Hoffman-La Roche) was administered intraperitoneally twice daily at 50 mg/kg/d from the day of virus inoculation. At indicated times after inoculation, mice were killed and their implants surgically removed and processed for PCR. For flow cytometry, cell sorting, and quantitative PCR, single-cell suspensions were obtained by grinding the implants between glass plates. The cells were counted in a Coulter counter and divided in aliquots for the different assays.
Qualitative reverse transcriptase (RT)-PCR and subsequent Southern hybridization for lytic messages was performed as described by Renne et al. 27. Qualitative RT-PCR was performed using the same procedure with plasmid pDD4 17 as hybridization probe. A description of the primers used in these experiments is shown in Table . In brief, RNA was isolated using RNAzol (Tel-Test, Inc.) according to the supplier's protocol. 500 ng total RNA was reverse transcribed using 200 U of Mo-MuLV RT (GIBCO BRL) in a total volume of 20 µl containing 125 µM dATP, dGTP, and dTTP, 20 U RNasin (Promega Corp.), and 120 pmol random hexanucleotide primers (Boehringer Mannheim). After incubation at 42°C for 35 min, and the reaction was stopped by heating to 95°C for 5 min, and then 80 µl of a PCR mix containing 10x PCR Mg-Buffer (Perkin-Elmer Corp.), 100 pmol of each primer, and 5 U of Taq polymerase (Perkin-Elmer Corp.) was added and amplified over 30 cycles (30 s at 94°C, 1 min at 58°C, and 1 min 30 s at 72°C).
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Cell Sorting and Flow Cytometry.
Single-cell suspensions were incubated with biotinylated mAbs directed against human CD4, CD8, and CD3–biotin (all from Becton Dickinson) for 0.5 h in PBS and 2% FBS on ice and depleted using Dynabeads (Dynal Inc.) according to recommendations from the manufacturer. Pre- and postdepletion cell populations were stained with streptavidin–FITC and PE-conjugated mAbs directed against human CD19 (Becton Dickinson) and separated on a FACSVantageTM (Becton Dickinson) cell sorter to isolate CD19+ B cells, CD4+, CD8+, and CD3+ T cells, and cells negative for all four markers. Sort-purified cells were frozen as pellets and stored at –80°C before processing.
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107 genome equivalents) directly into each implant, according to established procedures 32. As an average implant contains 108 human cells, the maximal multiplicity of infection is an estimated 0.1 genome equivalents per cell. However, it is likely that this substantially overestimates the true multiplicity of infection, because it is unlikely that all of the input virions are infectious (in many viruses, the particle/pfu ratio can be as high as 102–104). Each cohort included animals injected with UV-inactivated KSHV to serve as negative control; UV irradiation should completely abolish infectivity by cross-linking the double-stranded DNA genome 454647. At various times after inoculation, the mice were killed. To have all cell types represented, the implants were removed in toto, and 2.5 µg of total implant DNA (corresponding to 4.5 x 105 cells) was assayed for the presence of viral DNA by quantitative real-time DNA PCR using primers lat-273F, lat-335R, and lat-294T (Table ). Fig. 1 A shows a standard curve for the real-time quantitative DNA PCR to demonstrate the linear range of the assay, and Fig. 1 B shows a time course of infection. KSHV-specific DNA was low or undetectable in most implants 7 d after inoculation, peaked with a mean signal of 100 times background at day 14, and then receded to lower levels at days 21 and 28. This increase in KSHV genome copy number between days 7 and 14 after inoculation and subsequent reduction provides evidence for initial viral replication in the implant, as would be expected in a de novo infection. Exposure of KSHV virions to UV irradiation blocked infectivity.
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50 copies of episomal KSHV DNA per cell, we estimate that this level of viral DNA would correspond to infection of 1 cell in 1,000 in the implant (see Fig. 7). As CD19+ B cells represent between 0.2 and 2% of the total number of cells in the implant 31, the actual density of infection in this permissive subpopulation is likely to be higher. No difference in the levels of KSHV-specific DNA or transcripts was found between implants derived from different fetal sources (n = 4), indicating that susceptibility to KSHV infection is independent of the donor background.
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We also developed similar qualitative techniques to detect evidence for latent transcription (Fig. 2), using primers specific for either the latent nonspliced orf73 message or spliced orf72 message (primers #7209, #7307, and #7324; Table ). As for orf29, correct amplification products using orf72-specific primers can only derive from properly processed mRNA. Amplification products with orf73 primers could derive from either DNA or RNA; however, we estimate that >90% of the signal is derived from amplification of mRNA. Fig. 2 shows the location of the primers and the result of a control amplification from BCBL-1 RNA. Lanes 7–9 show evidence of latent message as judged by the presence of amplification products of the expected size (Fig. 2 B). Using primers specific for spliced orf72 message, no signal was generated in the absence of RT (compare lanes 7 and 8 to 4 and 5). Using primers for unspliced orf73 message, a faint band was detected in the absence of RT, presumably resulting from residual viral DNA (lane 6), but was strongly augmented in the presence of RT (lane 9).
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0.005 by X2). Human GAPDH (glyceraldehyde-3-phosphate-dehydrogenase)-specific message could be amplified in each case. This demonstrates that infection was dependent on intact, functional virions. Up to 50% of implants harbored levels of lytic transcripts above the cutoff detectable in this assay (P 0.1 by X2), suggesting that lytic KSHV replication was supported in these instances (summarized in Table ). To demonstrate that these results were not specific to the BCBL-1 isolate of KSHV, the experiments were repeated with KSHV purified from the equivalent number of induced BC-3 cells (data not shown). As in previous experiments, KSHV latency–specific messages could be amplified from all implants injected with BC-3–derived KSHV but not from those injected with UV-inactivated virus. These results establish that both PEL-derived isolates are functional in this assay.
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Ganciclovir Interferes with Establishment and Maintenance of Infection.
As ganciclovir inhibits KSHV replication in vitro 4849, we investigated whether the drug would also inhibit KSHV infection in the SCID-hu Thy/Liv model. Six infected animals were treated with twice-daily intraperitoneal injection of 50 mg/kg/d ganciclovir beginning at the time of KSHV inoculation. To concentrate our analysis specifically on productively infected cells, we quantitatively analyzed spliced transcript levels rather than viral DNA.
ABI Taqman® technology was used for real-time quantitative RT-PCR analysis 43. RNA was prepared and amplified in duplicate using primers specific for either spliced lytic (primers tac29-5F, tac29-68R, and tac29-22T) or spliced latent messages (primer lat-273F or TAQ-F1 with lat-335R and lat-294T; Table and Fig. 4). Primers for amplification of the lytic orf29 message were located on opposite sites of intron #1, and the fluorescently labeled oligonucleotide probe spanned the splice junction. Hence, only correctly spliced late messages for orf29 were detected (Fig. 4 B). For the amplification of latent messages, primers specific for the spliced orf72 message were used (Fig. 4 A). As these primers differed in sequence from those employed for qualitative RT-PCR, RNA from each implant was analyzed twice for each transcript (qualitatively and quantitatively) using two different primer pairs. Implants that yielded significant signal using real-time quantitative RT-PCR always scored positive in the qualitative PCR, and samples that did not result in an amplified product using the qualitative RT-PCR and radioactive hybridization never showed a signal using real-time quantitative PCR and fluorescent detection. Only a single amplification product was produced in the quantitative RT-PCR reaction (data not shown). The amount of product was quantified at each cycle using a third specific, fluorescently labeled oligonucleotide present during the reaction. Hence, the number of cycles (ct) needed for the fluorescence intensity to reach a threshold value is a direct measure of the amount of amplified product. Based on control reactions using RNA from latently infected BCBL-1 cells, sensitivity limits were established to be 1–10 cells per reaction for the spliced latent orf72 assay and 100–1,000 cells per reaction for the lytic orf29 assays (Fig. 4c and Fig. d), consistent with the observation that <3% of the population undergoes spontaneous lytic reactivation at any given time and the levels of orf29 mRNA in this population are much lower. The assay was linearly dependent on input RNA over three orders of magnitude.
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99% CD3+ (data not shown).
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Coinfection of SCID-hu Thy/Liv Mice with HIV-1 and KSHV.
To test whether an interaction between KSHV and HIV-1 might occur in coinfected implants, cohorts of SCID-hu Thy/Liv mice were injected with a mixture of HIV-1 and KSHV or with each virus alone. The pathogenic, syncytium-inducing HIV-1 isolate NL4-3 was used at an input inoculum of 103 tissue culture (TC)ID50. This strain induces massive T cell depletion starting at day 14 after inoculation, with <85% of implant thymocytes being destroyed by day 28–35 42. Accordingly, we chose to examine the coinfected implants on day 14, when HIV-1 infection is well established but before the onset of T cell death complicates the analysis of effects on viral load. KSHV viral load and HIV-1 p24 and RNA levels were quantitated at this time point (Fig. 7). Relative to animals infected with KSHV alone, no difference was noted in the levels of KSHV DNA in animals coinfected with HIV-1 and KSHV. The mean KSHV-specific DNA PCR signal in these two cohorts was 10–100 times the signal obtained from animals infected with UV-inactivated virus or HIV-1 alone, suggesting that KSHV replicated as in previous experiments and that its level of replication was unaffected by HIV-1. Correspondingly, the level of HIV-1 p24 in the implant was reduced only minimally in animals coinfected with KSHV as compared with animals infected with HIV-1 alone. We conclude that KSHV did not significantly influence HIV-1 replication, at least in the short period of acute coinfection.
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Flow cytometric analysis demonstrates that KSHV replicates primarily in CD19+ B cell populations of the implant, which is consistent with the tropism described for KSHV in humans 47515253. As KSHV does not replicate in SCID mice 30 that have received human peripheral blood leukocytes (a model that does not support hematopoiesis; reference 29), our observations suggest that immature or developing cell populations may serve as the predominant target of KSHV infection. Alternatively, it is possible that transmission to human blood cells requires a lymphoid microenvironment not reproduced by the implant. Dissecting the tissue tropism of KSHV in detail and mapping KSHV gene expression in different host cells will be one of the major uses of the SCID-hu Thy/Liv system.
The presence of lytic replication during the early phase of infection suggests another potential application of this model: to assess the impact of antivirals and biological response modifiers on KSHV replication in vivo, similar to its established use in the development of anti-HIV drugs. The susceptibility of KSHV infection to ganciclovir in vivo faithfully mirrors prior in vitro findings and affirms that this model, once optimized, may become a useful part of preclinical drug development.
Coinfection with HIV-1 did not enhance or interfere with KSHV replication, and the presence of KSHV did not influence the replication kinetics or cytopathogenicity induced by HIV-1. KSHV encodes viral chemokines that block interactions between HIV-1 and CCR5/CCR3 in vitro 545556, raising the speculation that this mechanism might also be operational in vivo (for review see reference 57). The absence of a significant effect of KSHV on HIV-1 replication argues against this, although the small number of KSHV-infected cells in the implant does not allow us to entirely exclude the possibility of interactions in vivo. HIV-1 itself is known to induce a number of cytokines (IFN-
, TNF-
, IL-1, and IL-6), and these as well as HIV-1 Tat itself have been proposed to be involved in the development of KS (for review see reference 58). Although analogous changes in cytokine expression may occur in HIV-1–infected SCID-hu Thy/Liv implants compared with infected PBMCs, no effect of HIV-1 on KSHV replication was observed, suggesting that HIV-1–induced cofactors did not have an impact on KSHV replication in this context. Ongoing experiments are now testing the possibility that interactions between HIV-1 and KSHV might be observed after coinfection with different isolates of HIV-1 and/or after longer periods of time.
KSHV infection of the Thy/Liv implant did not produce detectable histological phenotypes reminiscent of known KSHV-related human diseases. Certainly the model was not expected to reproduce KS, a neoplasm involving poorly understood cells of endothelial lineage 5. Although some CD34+ cells are doubtless present in the implant, it is unlikely that the full range of endothelial precursors, stromal components, and growth factors needed to support KS development would be present in this system. Rather, the model was designed to examine infection of the lymphoid compartment. The model successfully produces transient lytic infection and more sustained viral persistence and appears to mirror the correct tissue tropism of infection. However, it did not give evidence for lymphoid depletion or lymphoproliferation. Although the small number of B cells present in the implant severely constrained our ability to score for B cell depletion, the proportion of CD19+ cells did not appear to diminish over time. Similarly, we did not observe expansion of the B cell population and did not detect features reminiscent of Castleman's disease or PEL. However, it is important to remember that both of these phenotypes are exceedingly rare manifestations of human KSHV infection. Judging from the fact that 5–7% of healthy subjects are KSHV seropositive, it is likely that most primary infections with this agent are subclinical or asymptomatic. Thus, it is not clear that the relatively bland histologic picture generated here is not representative of many human primary infections. We are continuing to study the model to see if disease appears at late times after inoculation or if the system can be experimentally modified to allow induction of cell injury or proliferation. In the meantime, in addition to its potential uses in drug screening, the model offers an excellent opportunity to better define in detail which subsets of CD19+ cells are most permissive for KSHV and whether and how efficiently T cells and macrophages can be targets of infection.
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Kinetic PCR analysis was supported by Robert M. Grant and Jerry Kropp of the Gladstone/UCSF Laboratory of Clinical Virology (#P30 MH59037; University of California at San Francisco/Macy's Center for Creative Therapies). This work was supported by the Howard Hughes Medical Institute and Public Health Service grants to D. Ganem and National Institutes of Health grants AI-65309 and AI-40312 to J.M. McCune. J.M. McCune is an Elisabeth Glaser Scientist supported by the Elizabeth Glaser Pediatric AIDS Foundation. R. Renne is a Fellow of the Leukemia Society.
Submitted: 19 May 1999
Revised: 21 August 1999
Accepted: 28 September 1999
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) shows the copy number (logarithmic scale) per 2.5 µg of total implant DNA. The horizontal axis shows times after inoculation. The column labeled UV presents the signal obtained from implants that received UV-inactivated virus. The median of each group is indicated by the dashed line.
) probes compared with c-myc (
). The number of latent BCBL-1 cells per reaction is shown on the vertical axis, and the corresponding ct value as determined by real-time quantitative RT-PCR is shown on the horizontal axis.
