The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rappocciolo, G.
Right arrow Articles by Rinaldo, C. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rappocciolo, G.
Right arrow Articles by Rinaldo, C. R., Jr
The Journal of Immunology, 2006, 176: 1741-1749.
Copyright © 2006 by The American Association of Immunologists

DC-SIGN Is a Receptor for Human Herpesvirus 8 on Dendritic Cells and Macrophages1

Giovanna Rappocciolo2,*, Frank J. Jenkins*,{dagger}, Heather R. Hensler*, Paolo Piazza*, Mariel Jais*, Luann Borowski*, Simon C. Watkins{ddagger} and Charles R. Rinaldo, Jr*,{dagger}

* Department of Infectious Diseases and Microbiology, Graduate School of Public Health, and {dagger} Department of Pathology and {ddagger} Department of Cell Biology and Physiology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human herpesvirus 8 (HHV-8) causes Kaposi’s sarcoma and pleural effusion lymphoma. In this study, we show that dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN; CD209) is a receptor for HHV-8 infection of myeloid DCs and macrophages. DC-SIGN was required for virus attachment to these cells and DC-SIGN-expressing cell lines. HHV-8 binding and infection were blocked by anti-DC-SIGN mAb and soluble DC-SIGN, and mannan, a natural ligand for DC-SIGN. Infection of DCs and macrophages with HHV-8 led to production of viral proteins, with little production of viral DNA, similar to HHV-8 infection of vascular endothelial cells. Infection of DCs resulted in down-regulation of DC-SIGN, a decrease in endocytic activity, and an inhibition of Ag stimulation of CD8+ T cells. We propose that DC-SIGN serves as a portal for immune dysfunction and oncogenesis caused by HHV-8 infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human herpesvirus 8 (HHV-8),3 also termed Kaposi’s sarcoma (KS)-associated herpesvirus, is a {gamma} herpesvirus that is the etiologic agent of KS and rare malignancies, including primary effusion lymphomas and certain forms of multicentric Castleman’s disease (1). HHV-8 has been detected in KS spindle cells, which are of mixed vascular and lymphatic endothelial cell and macrophage origin (2), and monocytes that are found in proximity to KS lesions (3). Virus persists in a latent form in these cell types as well as in B lymphocytes.

Herpesvirus entry into susceptible cells requires at least two separate binding events (4). In HHV-8 infection, viral glycoproteins K8.1 and gB bind to cell surface heparan sulfate, and infection of endothelial cells can be blocked by soluble heparin (5, 6, 7). This binding likely enhances the efficiency of viral infection by concentrating the virus on the cell surface and may serve to position the virus for binding to a second receptor(s) involved in entry of virus into the cell. The integrin {alpha}3beta1 (CD49c/29) has been shown to serve as a receptor for HHV-8 infection of vascular endothelial cells and human foreskin fibroblasts (8). The identification of receptor(s) on other cell types has not been determined.

Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN; CD209) is a type II C-type lectin that is expressed on myeloid DCs in the dermis, mucosa, lymph nodes, lung and thymus (9, 10), and IL-4-treated, monocyte-derived DCs (11). It is also expressed on macrophages of the lung alveolae (9), placenta (12), and inflammatory lesions (13), and IL-13-activated, monocyte-derived macrophages (9, 13, 14, 15). DC-SIGN and other C-type lectins act as pathogen recognition receptors that alert macrophages and DCs to take up and process pathogens for Ag presentation to T cells (15, 16). Certain viruses, parasites, yeast, and bacteria can subvert this immune function by using DC-SIGN as a receptor for infection of myeloid lineage DCs (16, 17).

Although DCs have occasionally been found in KS lesions (18), there have been conflicting reports on the ability of DCs to be infected with HHV-8 (19, 20, 21). Given the tropism of HHV-8 for monocytes and infection of DCs by several other viruses (22), we hypothesized that HHV-8 could infect DCs, and that the virus used DC-SIGN as a receptor. In this study, we show that myeloid DCs and macrophages can be infected with HHV-8 in vitro using DC-SIGN as a receptor. Infection of DCs resulted in a loss of Ag processing and presentation functions. This finding is of importance to the understanding of the immunopathology and oncogenesis of HHV-8 infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Donors

HHV-8-seronegative blood donors were used in this study as determined by a serum immunofluorescence assay (23). Informed consent was obtained according to University of Pittsburgh guidelines.

Cell lines

BCBL-1 cells (HHV-8-positive/EBV-negative human B cells) were obtained through the National Institutes of Health AIDS Research and Reference Reagent Program (Division of AIDS, National Institutes of Allergy and Infectious Diseases) and used as the source of virus. EBV-transformed, Raji B-LCL and B-LCL-DC-SIGN (formerly known as B-THP-1 and B-THP-1-DC-SIGN cells, respectively; Ref.24) were a gift from Dr. D. Littman (New York University, New York, NY) and Dr. V. KewalRamani (National Cancer Institute, Bethesda, MD). K562 cells (American Type Culture Collection), an erythroleukemia cell line that does not constitutively express DC-SIGN, were stably transfected with plasmid pcDNA-DC-SIGN (National Institutes of Health AIDS Research and Reference Reagent Program) constructed with a G418-resistance cassette for selection using LipofectAMINE 2000 (Invitrogen Life Technologies) as per the manufacturer’s instructions; controls were K562 cells stably transfected with pcDNA3.1-V5/His (empty vector) and untreated K562 cells. The stable transfectant cell lines were selected and maintained in medium containing G418 (Invitrogen Life Technologies). Cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated FCS and L-glutamine and were negative for mycoplasma (Mycoplasma PCR ELISA; Roche).

Monocyte-derived DCs and macrophages

Enriched CD14+ monocytes were obtained from PBMCs and used for generation of immature DCs as previously described (25). For some experiments, DCs were matured with trimeric CD40L at 1 µg/ml (Amgen). Macrophages were generated from CD14+ monocytes by culture in RPMI 1640 medium with 5% heat inactivated, pooled human AB+ serum, and 1000 U/ml recombinant human GM-CSF for 7 days, and treated overnight with 20 ng/ml human IL-13 (PeproTech).

Endothelial cells

Primary human adult dermal microvascular endothelial cells (HMVEC-d; catalog CC2543; Cambrex) were cultured according to the supplier’s instructions. Cells were removed from the culture wells for surface marker staining and subculture by treatment with 0.1 mM EDTA in PBS with 0.25% BSA.

Virus purification and infection of cells

Concentrated supernatants and cell-free lysates from 2 x 109 12-O-tetradecanoylphorbol-13-acetate (TPA; Sigma-Aldrich)-induced BCBL-1 cells were used as source of HHV-8 and purified by a method modified from Cerimele et al. (26). Briefly, the HHV-8 lytic cycle was induced in BCBL-1 cells by treatment with 20 ng/ml TPA for 4 days. The cells were then centrifuged, and the supernatant was removed and saved. The cell pellet was lysed by three freeze-thaw cycles, centrifuged, and pooled with the original supernatant. These virus preparations were then put through a 0.45-µm filter to eliminate cell debris and concentrated overnight at 4°C with 7% polyethylene glycol 6000 (EMB Biosciences). The precipitate was centrifuged at 15,000 x g for 1 h at 4°C, resuspended in a small amount of PBS with Ca2+ and Mg2+ and 0.1% BSA, and microcentrifuged at 13,000 rpm (Biofuge Fresco). The resulting supernatant was then centrifuged through a 25% w/v sucrose cushion at 40,000 rpm at 4°C for 1 h in a Beckman SW41 rotor. The pellet was soaked overnight at 4°C in 1 ml of PBS containing 0.1% BSA and then layered over a 5–45% sucrose gradient in PBS, and centrifuged at 40,000 rpm at 4°C for 1 h in a Beckman SW41 rotor. The visible band at the center of the gradient was collected, dialyzed against PBS at 4°C overnight, and then frozen at 70°C. In some experiments, pooled pellets recovered from the 25% sucrose cushion were layered over a discontinuous Nycodenz (Sigma-Aldrich) gradient and centrifuged at 24,000 rpm at 10°C in a Beckman SW28 rotor (27). Virus was collected at the gradient interface, and dialyzed overnight against sterile PBS and stored as described above.

An average of 1 x 107 copies of viral DNA were used to infect 1 x 106 cells for 2 h at 37°C, washed and incubated for up to 5 days in AIM-V medium at 37°C in 5% CO2. For blocking studies, cells were pretreated with 20 µg/ml anti-human-DC-SIGN mAb (clone 120507; R&D Systems, or clone DCN46; BD Biosciences), anti-CD11a mAb (BD Biosciences), mouse IgG (Sigma-Aldrich), or 100 µg/ml mannan (Sigma-Aldrich), for 1 h at 4°C before exposure to HHV-8.

Quantitative PCR assay for HHV-8

HHV-8 DNA levels were measured by a quantitative, real-time TaqMan PCR assay with a primer set specific for the HHV-8 alkaline exonuclease gene (ORF37) previously shown to be highly specific for HHV-8 DNA (28). Standardization of HHV-8 DNA quantitation was done using known quantities of HHV-8 DNA (Applied Biosystems). To determine viral DNA levels in infected cells, cellular DNA was extracted using the QIAamp Blood DNA Miniprep kit (Qiagen). To determine viral DNA levels from encapsidated virus in cell culture supernatants, the experimental samples were pelleted by ultracentrifugation and treated with DNase I (Invitrogen Life Technologies) followed by phenol chloroform extraction. To verify that this assay detected lytic virus replication, we analyzed DNA samples from BCBL-1 cells containing the ORF-50 gene under the control of a tetracycline-responsive promoter (gift from J. Jung, New England Regional Primate Research Center, Worcester, MA) (29). We induced viral replication by the addition of 20 ng/ml doxycycline and collected samples at 24, 48, and 72 h for isolation of DNA and analysis of HHV-8 viral load by our real-time TaqMan PCR assay. Our results demonstrated a significant increase in viral DNA in the induced cells relative to the uninduced cells (data not shown).

Immunofluorescence assays

Immunofluorescence was done on HHV-8-infected or mock-infected cells using anti-HHV-8 ORF 73 rat mAb and mouse mAbs directed against ORF-K8.1A/B and ORF 59 (Applied Biosystems). Cells were counted and spotted on poly-L-lysine-coated slides, fixed in 4% paraformaldehyde for 20 min, and permeabilized with buffer (0.5% BSA, 0.1% saponin, 0.1% NaN3) for 20 min at room temperature. Cells were incubated with the primary Ab for 30 min at 4°C, washed extensively in buffer, and then stained with FITC-conjugated goat anti-mouse or anti-rat IgG (Sigma-Aldrich). Controls were omission of the primary Ab and staining of uninfected cells. In some experiments, cells were also stained by direct immunofluorescence with FITC-conjugated, anti-DC-SIGN mAb (clone 120507; R&D Systems), or with K8.1A/B or ORF 59 mAbs directly conjugated with PE or Texas Red using a Zenon labeling kit (Molecular Probes). To avoid nonspecific binding of IgG, a blocking step was added using SuperBlock Blocking Buffer (Pierce).

For confocal microscopy, DCs were cultured as per our standard procedures on glass coverslips, fixed with 2% paraformaldehyde, washed in PBS, blocked with BSA/normal goat serum for 40 min, washed in PBS, stained with anti-K8.1A/B mAb for 1 h and with goat anti-mouse CY3-conjugated F(ab')1 (Jackson Immuno) for 1 h. After washing, the cells were treated with anti-ORF73 mAb and anti-DC-SIGN-FITC mAb (clone DCN 46; BD Biosciences) for 1 h, washed, and stained with CY5 conjugated goat anti-rat serum (Jackson Immuno) for 1 h. Images were taken with an FluoView BX61 confocal microscope (Olympus America).

Blocking of HHV-8 binding

For receptor-blocking studies, cells were incubated with purified, [3H]thymidine-labeled HHV-8 at 4°C by the method of Akula et al. (5, 8). In some experiments, cells were incubated with anti-DC-SIGN mAb (20 µg/ml; clone 120507) or mannan (100 µg/ml) for 1 h at 4°C before adding the radiolabeled virus. Purified [3H]thymidine-labeled HHV-8 were also incubated with 10 µg/ml soluble DC-SIGN (a gift from S. Butera, Centers for Disease Control and Prevention, Atlanta, GA) at 37°C for 1 h, added to the cell pellets, and measured for radioactivity as described (5).

Flow cytometric analysis of cell phenotypes

Expression of cell surface molecules was examined by flow cytometry (Beckman Coulter XL) using FITC-conjugated mAb specific for MHC class I (HLA-ABC) and MHC class II (HLA-DR), CD80, CD86, CD83, and DC-SIGN for 30 min at 4°C, and then fixed with 1% paraformaldehyde. Cells were also treated with unconjugated anti-{alpha}3beta1 integrin mAb (VLA-3; Chemicon International) and FITC-conjugated goat anti-mouse IgG antisera for 30 min each at 4°C. Cells were stained with isotype Ab or goat-anti-mouse IgG FITC as controls.

Flow cytometric analysis of endocytosis

DCs were infected with HHV-8 for 24 or 48 h, or were mock-infected, and assessed for endocytic capacity of FITC-dextran (Mr, 40,000) (1 mg/ml; Sigma-Aldrich) at 37°C or 4°C for 30 min, washed four times with cold PBS containing 1% FCS and 0.01% NaN3, and analyzed (Beckman Coulter XL); cells incubated at 4°C were the control.

ELISPOT assay

Single-cell IFN-{gamma} production by CD8+ T cells was measured by an ELISPOT assay using peptides representing known HLA A*0201 influenza A virus M158–66 and EBV BMLF1 280–288 epitopes (25). CD8+ T cells were obtained from CD14+ cell-depleted PBMC by positive, immunomagnetic bead selection (Miltenyi Biotec), which does not interfere with Ag activation of the CD8+ T cells (data not shown).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
HHV-8 infects DCs

We examined whether DCs that are of myeloid origin and express DC-SIGN could be infected with HHV-8. Representative data from eight independent experiments using a range of virus input multiplicities show that viral DNA copies per cell remained low in infected DCs during 72 h of infection, and there was no detectable virus found in the supernatants of infected cells (Fig. 1A). Although viral DNA replication was very low in the infected DCs, viral proteins coded by ORF-K8.1 and ORF-59 could be detected up to 48 h postinfection, while ORF 73 LNA-1 expression became apparent at 48 and 72 h of infection (Fig. 1B and data not shown). Expression of both K 8.1 and ORF 59 diminished greatly after 48 h (Fig. 1B) and was not detected by 72 h postinfection (data not shown). The peak level of cells expressing these proteins reached 50–75% by 24–48 h (Fig. 1B; Table I); LNA-1 was expressed in 72% (range 60–100%) of DCs at 48 h (Fig. 1B) and 72 h (data not shown).


Figure 1
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 1. HHV-8 infects DCs. A, Quantitative DNA results from real-time PCR assay on total DNA that was extracted from infected (inf) DCs (red dotted line) and the corresponding DNase-resistant, concentrated cell culture supernatants (sup, pink dotted line). Uninfected cells and supernatants were negative for HHV-8 DNA (red solid line and pink solid line, respectively). B, Immunofluorescence on DCs that were infected with HHV-8 and stained with either mAb anti-K8.1A/B (cytoplasmic red fluorescence), anti-ORF 59 (cytoplasmic and perinuclear red fluorescence), or anti-LNA-1 (ORF 73, nuclear green fluorescence). Uninfected DCs were negative for each of the three HHV-8 proteins (Fig. 1B, top row). Cells were counterstained with DAPI (blue) (x600). Data are from one experiment representative of eight independent experiments.

 

View this table:
[in this window]
[in a new window]
  		Table I. HHV-8 infection of primary cells and cell lines expressing DC-SIGN

 
Thus, there is gene expression of K8.1 and ORF59 proteins during the first 24–48 h of infection of DCs, which is replaced by LNA-1 gene expression by 72 h of infection. Moreover, although complete viral replication was lacking, there was a small but significant amount of cell death associated with infection, as the percentage of viable DCs in the HHV-8-infected cultures at 72 h was 83 (±3.1)%, compared with 92 (±4.7)% in uninfected DCs (n = 8; p = 0.05; paired t test). Finally, studies done in parallel confirmed that HHV-8 infection of the DCs was similar to that of vascular endothelial cells (30), which represent major targets of HHV-8 infection in vivo. Thus, the data show a lack of a significant increase in viral DNA, but production of viral proteins, over several days of HHV-8 infection of HMVEC-d (Fig. 2).


Figure 2
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 2. HHV-8 infection of endothelial cells. A, Quantitative DNA results from real-time PCR assay on total DNA that was extracted from infected (inf) HMVEC-d (blue dotted line) and the corresponding DNase-resistant concentrated cell culture supernatants (sup, light blue dotted line). Uninfected cells and supernatants were negative for HHV-8 DNA (blue solid line and light blue solid line, respectively). Data are from one experiment representative of three independent experiments. B, Immunofluorescence results on HMVEC-d that were infected with HHV-8 and stained with either anti-K8.1A/B mAb (cytoplasmic red fluorescence) or anti-LNA-1 (ORF73) mAb (nuclear green fluorescence) at 24 and 72 h, respectively. Uninfected HMVEC-d were used as controls. Cells were counterstained with DAPI (blue) (x600). Data are from one experiment representative of six independent experiments.

 
Anti-DC-SIGN mAb and mannan inhibit HHV-8 infection of DCs

We next determined that HHV-8 infection of DCs could be inhibited by blocking with anti-DC-SIGN mAb (clone 120507) as indicated by a 92% decrease in K8.1A/B expression at 24 h (Fig. 3A; Table I) and 48 h (data not shown). Treatment of the DCs with anti-CD11a mAb had no effect on HHV-8 infection. These results were confirmed using a different anti-DC-SIGN mAb (clone DCN46) (data not shown). DCs infected with HHV-8 for 24 h were also positive for the ORF 59 viral protein (Table I) and were the same cells expressing DC-SIGN (Fig. 3B). Treatment of the DCs with anti-DC-SIGN mAb before HHV-8 infection blocked 91% of ORF 59 expression (Fig. 3B; Table I) and also inhibited the binding of the fluorochrome dye-labeled anti-DC-SIGN mAb to DC-SIGN (Fig. 3B). Similar levels of inhibition of HHV-8 infection were noted after pretreatment of the DCs with mannan, a natural ligand for DC-SIGN (data not shown).


Figure 3
View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 3. Infection of DCs with HHV-8 is blocked by anti DC-SIGN mAb. A, Immunofluorescence results on DCs that were left untreated or treated with either anti-DC-SIGN mAb (clone 120507) or anti-CD11a mAb, infected with HHV-8, incubated, and stained with anti-K8.1A/B mAb (red) at 24 h. Uninfected DCs were used as controls. B, Immunofluorescence results on DCs that were either treated with anti-DC-SIGN mAb (clone 120507) or left untreated, infected, and stained at 24 h with anti-DC-SIGN mAb (green) and anti-ORF 59 mAb (red). The overlay of combined colors for anti-DC-SIGN and ORF 59 is shown. Cells were counterstained with DAPI (blue) (x600). Data are from one experiment representative of eight independent experiments.

 
DC-SIGN-transfected cells are susceptible to HHV8 infection

K562 cells and B-LCL do not constitutively express DC-SIGN and were not susceptible to HHV-8 infection (Fig. 4; Table I). In contrast, K562 cells and B-LCLs stably transfected with a plasmid that overexpresses DC-SIGN (K562 DC-SIGN and B-LCL-DC-SIGN) were susceptible to HHV-8 infection as evidenced by expression of HHV-8 K8.1 and ORF 59 proteins at 24 h (Fig. 4; Table I). In additional experiments, we confirmed that HHV-8 infected these cells via DC-SIGN by blocking infection with anti-DC-SIGN mAb (Table I).


Figure 4
View larger version (53K):
[in this window]
[in a new window]
  		FIGURE 4. DC-SIGN expression renders resistant cells susceptible to HHV-8 infection. A, Immunofluorescence results on K562 and K562-DC-SIGN cells that were infected with HHV-8 and stained with anti-K8.1A/B mAb at 24 h (red). B, Immunofluorescence results on B-LCL and B-LCL DC-SIGN that were infected with HHV-8 and stained after 24 h with anti-K8.1A/B mAb (red). Cells were counterstained with DAPI (x600). Data are from one experiment representative of five independent experiments.

 
HHV-8 binds to DC-SIGN and binding is inhibited by anti-DC-SIGN mAb, soluble DC-SIGN and mannan

To prove that DC-SIGN was actually a surface receptor for binding of HHV-8, we assessed binding of radioactively labeled virus to DCs and B-LCL-DC-SIGN at 4°C to allow binding but prevent internalization of virus (8). Binding of radiolabeled HHV-8 was inhibited by >60% with either anti-DC-SIGN mAb or mannan (Fig. 5A). Furthermore, pretreatment of radiolabeled HHV-8 with soluble DC-SIGN greatly inhibited the binding of the virus to these cells (Fig. 5B), confirming that HHV-8 binds DC-SIGN. Finally, anti-DC-SIGN mAb-mediated inhibition of virus binding to DCs was dose-dependent (Fig. 5C).


Figure 5
View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 5. HHV-8 binds to DC-SIGN. A, Inhibition of binding of radioactively labeled HHV-8 by treatment of target cells with anti-DC-SIGN mAb. DCs or B-LCL-DC-SIGN were pretreated with anti-DC-SIGN mAb (clone 120507) or mannan, or left untreated. Each bar represents the mean percent of binding inhibition (± SE) (compared with untreated cells) from two duplicate determinations. B, Inhibition of binding of radioactively labeled HHV-8 by treatment of virus with soluble DC-SIGN. Results are the mean (± SE) percentage of inhibition of binding of soluble DC-SIGN-treated HHV-8 compared with binding of radiolabeled untreated virus to each cell type from two determinations. C, Dose response of inhibition of virus binding to DCs by treatment with anti-DC-SIGN mAb. Each bar represents the mean of duplicate reactions (± SE) from duplicate determinations. Data are from one experiment representative of three independent experiments.

 
HHV-8 infects macrophages through DC-SIGN

Monocytes and macrophages are known to be infected with HHV-8 in vivo (18) but are refractory to infection in vitro unless activated (31). DC-SIGN is up-regulated by treatment of monocyte-derived macrophages with IL-13 in vitro (9, 15) and in inflammatory, activated macrophages in vivo (13, 32). We, therefore, determined whether DC-SIGN was a receptor on these cells for HHV-8. Non-IL-13-stimulated, monocyte-derived macrophages expressed low levels of DC-SIGN at 24 h and supported limited HHV-8 infection, i.e., K8.1 and ORF 59 expression, median, 8% (Table I). In contrast, IL-13-treated macrophages expressed high levels of DC-SIGN by 24 h (Fig. 6A). Although there was little increase in viral DNA in these cultures (data not shown), the majority of IL-13-treated macrophages expressed ORF 59 (median, 88%) (Fig. 6B; Table I) and K8.1 (median, 63%) (Fig. 6C; Table I) by 24 h. DC-SIGN was coexpressed in HHV-8 infected macrophages as shown in Fig. 6B. Finally, HHV-8 infection of the non-IL-13-treated macrophages was completely blocked by pretreatment of the cells with anti-DC-SIGN mAb (Table I) or mannan (data not shown). More importantly, this effect was also noted in IL-13-treated macrophages, where anti-DC-SIGN mAb decreased expression of ORF 59 by 100% (Table I) and K8.1 by 84% (Fig. 6C; Table I).


Figure 6
View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 6. HHV-8 infection of IL-13-activated macrophages is related to DC-SIGN expression. A, Flow cytometric analysis showing expression of DC-SIGN on HHV-8-infected (empty histogram, broken line) or uninfected (empty histogram, solid line) IL-13-activated macrophages. Full histogram, isotype controls. B, Immunofluorescence results on IL-13-activated macrophages that were infected with HHV-8 for 24 h and stained for ORF 59 (red) and DC-SIGN (green). The overlay of combined colors for anti-DC-SIGN and ORF59 is shown. C, Immunofluorescence results on IL-13-activated macrophages that were pretreated with anti-DC-SIGN mAb (clone 120507) or mouse IgG, infected with HHV-8 for 24 h and stained for anti-K8.1 mAb (red). Cells were counterstained with DAPI (x600). Data are from one experiment representative of four independent experiments.

 
{alpha}3beta1 integrin is not essential for HHV-8 infection of DC-SIGN-expressing cells

The {alpha}3beta1 integrin has been identified as a receptor for HHV-8 in human foreskin fibroblasts and endothelial cells (8). We found that, in contrast to DC-SIGN, this integrin was not expressed on the DC-SIGN transfected B-LCLs and K562 cells that were susceptible to HHV-8 infection (Table I). Moreover, {alpha}3beta1 integrin was expressed on non-IL-13-activated macrophages, only a few of which expressed DC-SIGN and supported HHV-8 infection.

HHV-8 infection results in a decrease in DC-SIGN expression

Surface expression of DC-SIGN is known to decrease due to internalization after binding to HIV1 or its natural ligand ICAM-3 (33). In our studies, DC-SIGN expression was down-regulated on DCs after 24–72 h of infection with HHV-8 (p < 0.001 determined by two-tailed paired Student’s t test) (Fig. 7A). The loss of DC-SIGN expression on the surface of infected DCs was much greater than that induced by maturation of DCs by treatment with CD40L (e.g., mean fluorescence intensity (MFI) for DC-SIGN on HHV-8-infected DCs at 72 h postinfection (MFI = 10), compared with uninfected DCs (MFI = 245), and CD40L-stimulated DCs (MFI = 205) (data not shown). We also observed a strong down-regulation of expression of DC-SIGN on the surface of both activated macrophages (Fig. 6A) and B-LCL-DC-SIGN (data not shown) after infection with HHV-8. There were insignificant increases in expression of DC maturation markers HLA-DR and CD83 by 24 h (Fig. 7B). Expression of CD83 declined to background levels by 72 h. HHV-8 infected DCs also had lower expression of HLA ABC at 48 and 72 h.


Figure 7
View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 7. Effect of HHV-8 infection of DCs on expression of DC-SIGN and costimulatory molecules. A, DC-SIGN expression on uninfected or HHV-8 infected DCs. Data are mean MFI (±SE) from seven independent experiments. B, Expression of HLA-ABC, HLA-DR, CD83, and DC-SIGN on infected DCs. Blue histogram, HHV-8-infected DCs; yellow histogram, uninfected DCs; empty histogram, fine line, and broken line = isotype controls for the infected and uninfected DCs, respectively. Data are from one experiment representative of 10 independent experiments. C, Confocal microscopy of HHV-8-infected DCs stained with anti-DC-SIGN (green) and anti-ORF 59 (red) mAbs at 24 h (left panel) and 48 h (center panel) after infection. Uninfected DCs served as controls (right panel). Data are from one experiment representative of two independent experiments.

 
Further analysis of HHV-8-infected DCs stained for DC-SIGN and ORF 59 and examined by confocal microscopy at 24 and 48 h showed that DC-SIGN was strongly down-regulated and localized in the cytoplasm of infected DCs (Fig. 7C). This suggests that DC-SIGN molecules have been internalized as a result of viral infection, or alternatively could have changed their structure, and therefore could not be revealed with the mAb used in these assays. However, there appeared to be no new molecules synthesized and/or brought to the cell surface during the time span of these experiments (Fig. 7C).

HHV-8 infection of DCs impairs their function

HHV-8 is known to code for several proteins with immunomodulatory properties that are considered important in its pathogenesis (34). We therefore determined whether HHV-8 infection induced impairment of Ag-processing and -presentation functions of DCs (35). We found a strong inhibition of endocytosis in HHV-8-infected DCs when compared with uninfected, immature DCs or CD40L-matured DCs (Fig. 8A). Because Ag uptake is pivotal to subsequent Ag presentation, we examined whether HHV-8-infected DCs could stimulate virus Ag-specific memory CD8+ T lymphocytes. HHV-8-infected DCs were poor APCs when compared with uninfected DCs, as shown by decreased production of IFN-{gamma} by autologous CD8+ T cells in response to immunodominant influenza A virus and EBV peptides (Fig. 8B). Additionally, as mature DCs are more efficient at presenting Ag to T cells, we determined whether HHV-8 infected mature DCs were able to present Ag more efficiently than infected, immature DCs. Notably, maturation of DCs by treatment with CD40L before infection was not sufficient to overcome the inhibitory effect of HHV-8 infection on DC Ag-presenting function (Fig. 8B).


Figure 8
View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 8. Effect of HHV-8 infection of DCs on endocytosis and Ag-specific activation of CD8+ T cells. A, Endocytosis of FITC-dextran by uninfected DCs (left panel, open histogram) or HHV-8 infected DCs (right panel, open histogram). CD40L-matured DCs (middle panel, open histogram) and cells incubated at 4°C (shaded histograms) served as controls. Data are from one experiment representative of five independent experiments. B, The ability of HHV-8-infected or uninfected, immature (iDC), and CD40L-matured DCs (mDC) to stimulate Ag-specific IFN-{gamma} production in autologous CD8+ T cells. Each bar is the mean ± SE of triplicate wells. Data are from one experiment representative of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
DC-SIGN is a type II C-type lectin that serves as a recognition receptor on DCs for several viruses and other, diverse pathogens (16). Binding to DC-SIGN and other C-type lectins leads to Ag processing and presentation to T cells for activation of adaptive immune responses. However, some pathogens can subvert this process and use DC-SIGN to establish infection in the host. In this study, we show that DC-SIGN is a receptor for HHV-8 on myeloid DCs and macrophages. Evidence that DC-SIGN is a receptor for HHV-8 is based on established criteria (4), and includes: 1) DC-SIGN was present on DCs and macrophages that are susceptible to infection by the virus, 2) expression of DC-SIGN rendered normally resistant cell lines susceptible to HHV-8 infection, and 3) HHV-8 infection was blocked by mAbs directed against DC-SIGN, and by soluble DC-SIGN and a natural ligand of DC-SIGN (mannan).

Immature, monocyte-derived DCs supported viral K8.1 and ORF 59 gene expression, which was blocked by pretreatment of the cells with anti-DC-SIGN mAb or mannan, or by pretreatment of the virus with soluble DC-SIGN. Expression of K8.1 and ORF 59 subsequently declined in the DCs, with persistence of latent (LNA-1) viral gene expression. Analysis of HHV-8 DNA demonstrated very low levels of viral DNA in infected DCs with no evidence of virus production. However, the viral lytic genes K8.1 and ORF 59 and the latency gene LNA-1 were expressed in the infected cells. The expression of some but not all viral genes with highly restricted DNA replication has also been noted for HHV-8 infection of human fibroblasts and endothelial cells (30). Indeed, microarray analysis has shown that HHV-8 infection of fibroblasts and endothelial cells does not result in a productive, replicative cycle (30). Instead, there is expression of a subset of lytic cycle gene transcripts (including ORF 59 and K8.1) which quickly subsides, followed by persistent expression of latency gene transcripts (ORF 73). Moreover, only latent, and not productive lytic, HHV-8 infection has been noted in various types of infected cells (8, 36, 37, 38). Infection of DCs by HHV-8 appears similar to infection of these other cell types in vitro in that there is a burst of gene transcription resulting in a nonproductive, latent viral infection without significant viral DNA replication. This limited lytic gene expression with the persistence of latent genes in vitro appears to be unique to this {gamma} herpesvirus (30), and requires further study.

Our study is the first to demonstrate HHV-8 infection of myeloid lineage DCs. This is supported by a recent report that HHV-8 can infect CD34+ stem cell precursors of DCs in vitro (39). We propose that DCs that express DC-SIGN in vivo, i.e., mature myeloid DCs and immature plasmacytoid DCs (9, 10, 40), should be susceptible to infection by HHV-8. Interestingly, DCs have occasionally been found in KS lesions (18). Previously, however, there have been conflicting reports on the presence of HHV-8 in DCs (19, 20, 21). Notably, these studies only examined DCs for natural, in vivo infection with HHV-8 in persons who were either not assessed, or were negative, for HHV-8 Abs. Further studies are ongoing to examine DCs for infection with HHV-8 in persons who are HHV-8 seropositive or have KS.

The results show that macrophages were also susceptible to HHV-8 infection via DC-SIGN. Low numbers of unstimulated macrophages expressed DC-SIGN in association with their in vitro transformation from monocytes. These unstimulated macrophages were susceptible to a low level of infection with HHV-8, which was blocked by anti-DC-SIGN mAb or mannan. Activation of macrophages with IL-13 greatly enhanced expression of DC-SIGN, confirming previous reports (9, 15), and rendered them highly permissive for HHV-8 infection. Moreover, HHV-8 infection was blocked by pretreatment of the activated macrophages with anti-DC-SIGN mAb or mannan. Previously, it has been reported that monocyte-derived macrophages from some normal donors that are stimulated in vitro with allogeneic PBMCs are susceptible to HHV-8 infection (31). Moreover, treatment of blood monocytes from KS patients in vitro with proinflammatory cytokines results in persistence of HHV-8 (41). IL-13 is produced by Th2 cells, as well as eosinophils, mast cells, and basophils. Interestingly, HHV-8 encodes a viral macrophage inhibitory protein II that is associated with infiltration of Th2 cells and eosinophils in KS lesions (42). Production of IL-13 by these infiltrating cells could potentially enhance HHV-8 infection of monocytes and macrophages proximal to the KS lesion by up-regulating expression of DC-SIGN.

Integrins, particularly the {alpha}3beta1 dimer, have been shown to serve as receptors for HHV-8 infection of endothelial cells and fibroblasts (8). We have found that expression of DC-SIGN on cells normally resistant to HHV-8 infection (B-LCLs and K562 cells) rendered them susceptible to HHV-8 infection, although neither of these cells expressed {alpha}3beta1 on their surface. Nevertheless, herpesviruses can have more than one receptor in the same cell (4), as has recently been reported for epidermal growth factor receptor and {alpha}Vbeta3 integrin for infection of fibroblasts by human CMV (43). Thus, {alpha}3beta1 or other integrins could serve together with DC-SIGN as receptors for HHV-8 in certain types of cells. Moreover, heparan sulfate is also known to bind HHV-8 on endothelial cells and fibroblasts (5, 6, 7). Indeed, the residual level of binding and infection of HHV-8 in cells pretreated with anti-DC-SIGN mAb in our studies, which is similar to that reported for other microbial agents that use DC-SIGN as a receptor (44, 45), suggests that there are additional receptors for HHV-8 on these cells. Studies designed to distinguish the roles of DC-SIGN and other receptors in HHV-8 infection are in progress.

Binding of HHV-8 to DC-SIGN induced down-regulation of this molecule on DCs, which appears to be sequestered in the cytoplasm of the cells. Uptake of DC-SIGN into lysosomal compartments is known to occur after binding of other pathogens to the molecule (46), and could be involved in processing of HHV-8 Ags. We have also shown that HHV-8 infection of DCs induced profound perturbations in two of their most important functions, i.e., a decrease in endocytosis, or Ag uptake, and Ag presentation to CD8+ T cells. The latter effect could be due in part to a decrease in expression of MHC class I molecules that is a well-documented feature of HHV-8 infection (47, 48). Indeed, we noted a decrease in MHC class I expression on HHV-8-infected DCs. These modulatory effects on the DCs were due to HHV-8 infection, as we used purified HHV-8 in these studies to rule out possible effects on the DCs of other factors in crude virus preparations. Taken together, these results indicate that HHV-8 infection of DCs leads to defects in Ag processing and presentation that could be important in immune evasion of the virus. This DC dysfunction could also relate to our previous findings of a relatively nonrobust CD8+ T cell response to HHV-8 after primary HHV-8 infection (49).

These results suggest that a major block in cells resistant to HHV-8 infection is the lack of expression of a proper receptor such as DC-SIGN. Indeed, we have found that activated B lymphocytes express DC-SIGN, and microvascular endothelial cells express a C-type lectin homolog of DC-SIGN, DC-SIGNR (DC-SIGN related; CD209R), and that these serve as receptors for HHV-8 on these cells (our unpublished observations). Finally, it should be noted that the cellular host range of HHV-8 infection can be expanded artificially to include cells normally resistant to HHV-8 infection by treatment of these cells with Polybrene (50), which enhances receptor-independent infection (51).

We conclude that expression of the type II C-type lectin, DC-SIGN, is necessary for receptor-mediated infection of myeloid DCs and macrophages. DC-SIGN could serve as a portal for HHV-8 infection of cells that are known to express DC-SIGN in vivo, i.e., tissue-resident DCs and alveolar macrophages (10, 52), macrophages activated by proinflammatory cytokines (9) and activated B lymphocytes (our unpublished results). This novel finding on the role of DC-SIGN in the infectious process of HHV-8 could have important implications for designing effective strategies for inhibiting HHV-8 infection and associated cancers.


   Acknowledgments
 
We gratefully acknowledge helpful discussions with P. Spear, P. Gupta, D. Rowe, P. Moore, Y. Chang and O. Flore, and the assistance of M. Tomaszewski, A. Pollard, and S. Alber. We thank Amgen for the gift of CD40L (MTA n.200312724).


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the National Institutes of Health Grants R01 CA82053 and U01 AI35041. Back

2 Address correspondence and reprint requests to Dr. Giovanna Rappocciolo, Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, 604 Parran Hall, 130 DeSoto Street, Pittsburgh, PA 15261. E-mail address: giovanna{at}pitt.edu Back

3 Abbreviations used in this paper: HHV-8, human herpesvirus 8; KS, Kaposi’s sarcoma; DC-SIGN, dendritic cell-specific ICAM-3 grabbing nonintegrin; HMVEC-d, human adult dermal microvascular endothelial cell; MFI, mean fluorescence intensity. Back

Received for publication September 7, 2005. Accepted for publication November 17, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Boshoff, C., R. Weiss. 2002. AIDS-related malignancies. Nat. Rev. Cancer 2: 373-382. [Medline]
  2. Bubman, D., E. Cesarman. 2003. Pathogenesis of Kaposi’s sarcoma. Hematol. Oncol. Clin. North Am. 17: 717-745. [Medline]
  3. Blasig, C., C. Zietz, B. Haar, F. Neipel, S. Esser, N. H. Brockmeyer, E. Tschachler, S. Colombini, B. Ensoli, M. Sturzl. 1997. Monocytes in Kaposi’s sarcoma lesions are productively infected by human herpesvirus 8. J. Virol. 71: 7963-7968. [Abstract]
  4. Spear, P. G., R. Longnecker. 2003. Herpesvirus entry: an update. J. Virol. 77: 10179-10185. [Free Full Text]
  5. Akula, S. M., N. P. Pramod, F. Z. Wang, B. Chandran. 2001. Human herpesvirus 8 envelope-associated glycoprotein B interacts with heparan sulfate-like moieties. Virology 284: 235-249. [Medline]
  6. Birkmann, A., K. Mahr, A. Ensser, S. Yaguboglu, F. Titgemeyer, B. Fleckenstein, F. Neipel. 2001. Cell surface heparan sulfate is a receptor for human herpesvirus 8 and interacts with envelope glycoprotein K8.1. J. Virol. 75: 11583-11593. [Abstract/Free Full Text]
  7. Wang, F. Z., S. M. Akula, N. P. Pramod, L. Zeng, B. Chandran. 2001. Human herpesvirus 8 envelope glycoprotein K8.1A interaction with the target cells involves heparan sulfate. J. Virol. 75: 7517-7527. [Abstract/Free Full Text]
  8. Akula, S. M., N. P. Pramod, F. Z. Wang, B. Chandran. 2002. Integrin {alpha}3beta1 (CD 49c/29) is a cellular receptor for Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) entry into the target cells. Cell 108: 407-419. [Medline]
  9. Soilleux, E. J., L. S. Morris, G. Leslie, J. Chehimi, Q. Luo, E. Levroney, J. Trowsdale, L. J. Montaner, R. W. Doms, D. Weissman, et al 2002. Constitutive and induced expression of DC-SIGN on dendritic cell and macrophage subpopulations in situ and in vitro. J. Leukocyte Biol. 71: 445-457. [Abstract/Free Full Text]
  10. Turville, S. G., P. U. Cameron, A. Handley, G. Lin, S. Pohlmann, R. W. Doms, A. L. Cunningham. 2002. Diversity of receptors binding HIV on dendritic cell subsets. Nat. Immunol. 3: 975-983. [Medline]
  11. Relloso, M., A. Puig-Kroger, O. M. Pello, J. L. Rodriguez-Fernandez, G. de la Rosa, N. Longo, J. Navarro, M. A. Munoz-Fernandez, P. Sanchez-Mateos, A. L. Corbi. 2002. DC-SIGN (CD209) expression is IL-4 dependent and is negatively regulated by IFN, TGF-beta, and anti-inflammatory agents. J. Immunol. 168: 2634-2643. [Abstract/Free Full Text]
  12. Soilleux, E. J., L. S. Morris, B. Lee, S. Pohlmann, J. Trowsdale, R. W. Doms, N. Coleman. 2001. Placental expression of DC-SIGN may mediate intrauterine vertical transmission of HIV. J. Pathol. 195: 586-592. [Medline]
  13. Soilleux, E. J., L. S. Morris, J. Trowsdale, N. Coleman, J. J. Boyle. 2002. Human atherosclerotic plaques express DC-SIGN, a novel protein found on dendritic cells and macrophages. J. Pathol. 198: 511-516. [Medline]
  14. Soilleux, E. J., L. S. Morris, S. Rushbrook, B. Lee, N. Coleman. 2002. Expression of human immunodeficiency virus (HIV)-binding lectin DC-SIGNR: consequences for HIV infection and immunity. Hum. Pathol. 33: 652-659. [Medline]
  15. Chehimi, J., Q. Luo, L. Azzoni, L. Shawver, N. Ngoubilly, R. June, G. Jerandi, M. Farabaugh, L. J. Montaner. 2003. HIV-1 transmission and cytokine-induced expression of DC-SIGN in human monocyte-derived macrophages. J. Leukocyte Biol. 74: 757-763. [Abstract/Free Full Text]
  16. Geijtenbeek, T. B., Y. van Kooyk. 2003. Pathogens target DC-SIGN to influence their fate DC-SIGN functions as a pathogen receptor with broad specificity. APMIS 111: 698-714. [Medline]
  17. Cambi, A., C. G. Figdor. 2003. Dual function of C-type lectin-like receptors in the immune system. Curr. Opin. Cell Biol. 15: 539-546. [Medline]
  18. Ensoli, B., C. Sgadari, G. Barillari, M. C. Sirianni, M. Sturzl, P. Monini. 2001. Biology of Kaposi’s sarcoma. Eur. J. Cancer 37: 1251-1269. [Medline]
  19. Rettig, M. B., H. J. Ma, R. A. Vescio, M. Pold, G. Schiller, D. Belson, A. Savage, C. Nishikubo, C. Wu, J. Fraser, et al 1997. Kaposi’s sarcoma-associated herpesvirus infection of bone marrow dendritic cells from multiple myeloma patients. Science 276: 1851-1854. [Abstract/Free Full Text]
  20. Olsen, S. J., K. Tarte, W. Sherman, E. E. Hale, M. T. Weisse, A. Orazi, B. Klein, Y. Chang. 1998. Evidence against KSHV infection in the pathogenesis of multiple myeloma. Virus Res. 57: 197-202. [Medline]
  21. Chauhan, D., A. Bharti, N. Raje, E. Gustafson, G. S. Pinkus, J. L. Pinkus, G. Teoh, T. Hideshima, S. P. Treon, J. D. Fingeroth, K. C. Anderson. 1999. Detection of Kaposi’s sarcoma herpesvirus DNA sequences in multiple myeloma bone marrow stromal cells. Blood 93: 1482-1486. [Abstract/Free Full Text]
  22. Rinaldo, C. R., Jr, P. Piazza. 2004. Virus infection of dendritic cells: portal for host invasion and host defense. Trends Microbiol. 12: 337-345. [Medline]
  23. Jenkins, F. J., L. J. Hoffman, A. Liegey-Dougall. 2002. Reactivation of and primary infection with human herpesvirus 8 among solid-organ transplant recipients. J. Infect. Dis. 185: 1238-1243. [Medline]
  24. Wu, L., T. D. Martin, M. Carrington, V. N. KewalRamani. 2004. Raji B cells, misidentified as THP-1 cells, stimulate DC-SIGN-mediated HIV transmission. Virology 318: 1 7-23. [Medline]
  25. Wang, Q. J., X. L. Huang, G. Rappocciolo, F. J. Jenkins, W. H. Hildebrand, Z. Fan, E. K. Thomas, C. R. Rinaldo, Jr. 2002. Identification of an HLA A*0201-restricted CD8+ T-cell epitope for the glycoprotein B homolog of human herpesvirus 8. Blood 99: 3360-3366. [Abstract/Free Full Text]
  26. Cerimele, F., F. Curreli, S. Ely, A. E. Friedman-Kien, E. Cesarman, O. Flore. 2001. Kaposi’s sarcoma-associated herpesvirus can productively infect primary human keratinocytes and alter their growth properties. J. Virol. 75: 2435-2443. [Abstract/Free Full Text]
  27. Akula, S. M., F.-Z. Wang, J. Vieira, B. Chandran. 2001. Human herpesvirus 8 interaction with target cells involves heparan sulfate. Virology 282: 245-255. [Medline]
  28. Stamey, F. R., M. M. Patel, B. P. Holloway, P. E. Pellett. 2001. Quantitative, fluorogenic probe PCR assay for detection of human herpesvirus 8 DNA in clinical specimens. J. Clin. Microbiol. 39: 3537-3540. [Abstract/Free Full Text]
  29. Nakamura, H., M. Lu, Y. Gwack, J. Souvlis, S. L. Zeichner, J. U. Jung. 2003. Global changes in Kaposi’s sarcoma-associated virus gene expression patterns following expression of a tetracycline-inducible Rta transactivator. J. Virol. 77: 4205-4220. [Abstract/Free Full Text]
  30. Krishnan, H. H., P. P. Naranatt, M. S. Smith, L. Zeng, C. Bloomer, B. Chandran. 2004. Concurrent expression of latent and a limited number of lytic genes with immune modulation and antiapoptotic function by Kaposi’s sarcoma-associated herpesvirus early during infection of primary endothelial and fibroblast cells and subsequent decline of lytic gene expression. J. Virol. 78: 3601-3620. [Abstract/Free Full Text]
  31. Blackbourn, D. J., E. Lennette, B. Klencke, A. Moses, B. Chandran, M. Weinstein, R. G. Glogau, M. H. Witte, D. L. Way, T. Kutzkey, et al 2000. The restricted cellular host range of human herpesvirus 8. AIDS 14: 1123-1133. [Medline]
  32. van Lent, P. L., C. G. Figdor, P. Barrera, K. van Ginkel, A. Sloetjes, W. B. van den Berg, R. Torensma. 2003. Expression of the dendritic cell-associated C-type lectin DCSIGN by inflammatory matrix metalloproteinase-producing macrophages in rheumatoid arthritis synovium and interaction with intercellular adhesion molecule 3-positive T cells. Arthritis Rheum. 48: 360-369. [Medline]
  33. Engering, A., T. B. Geijtenbeek, S. J. van Vliet, M. Wijers, E. van Liempt, N. Demaurex, A. Lanzavecchia, J. Fransen, C. G. Figdor, V. Piguet, Y. van Kooyk. 2002. The dendritic cell-specific adhesion receptor DC-SIGN internalizes antigen for presentation to T cells. J. Immunol. 168: 2118-2126. [Abstract/Free Full Text]
  34. Means, R. E., J. K. Choi, H. Nakamura, Y. H. Chung, S. Ishido, J. U. Jung. 2002. Immune evasion strategies of Kaposi’s sarcoma-associated herpesvirus. Curr. Top Microbiol. Immunol. 269: 187-201. [Medline]
  35. Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. Liu, B. Pulendran, K. Palucka. 2000. Immunobiology of dendritic cells. Annu. Rev. Immunol. 18: 767-811. [Medline]
  36. Naranatt, P. P., S. M. Akula, C. A. Zien, H. H. Krishnan, B. Chandran. 2003. Kaposi’s sarcoma-associated herpesvirus induces the phosphatidylinositol 3-kinase-PKC-{zeta}-MEK-ERK signaling pathway in target cells early during infection: implications for infectivity. J. Virol. 77: 1524-1539.
  37. Renne, R., D. Blackbourn, D. Whitby, J. Levy, D. Ganem. 1998. Limited transmission of Kaposi’s sarcoma-associated herpesvirus in cultured cells. J. Virol. 72: 5182-5188. [Abstract/Free Full Text]
  38. Vieira, J., P. O’Hearn, L. Kimball, B. Chandran, L. Corey. 2001. Activation of Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8) lytic replication by human cytomegalovirus. J. Virol. 75: 1378-1386. [Abstract/Free Full Text]
  39. Larcher, C., V. A. Nguyen, C. Furhapter, S. Ebner, E. Solder, H. Stossel, N. Romani, N. Sepp. 2005. Human herpesvirus-8 infection of umbilical cord-blood-derived CD34+ stem cells enhances the immunostimulatory function of their dendritic cell progeny. Exp. Dermatol. 14: 41-49. [Medline]
  40. Geijtenbeek, T. B., R. Torensma, S. J. van Vliet, G. C. van Duijnhoven, G. J. Adema, Y. van Kooyk, C. G. Figdor. 2000. Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses. Cell 100: 575-585. [Medline]
  41. Monini, P., S. Colombini, M. Sturzl, D. Goletti, A. Cafaro, C. Sgadari, S. Butto, M. Franco, P. Leone, S. Fais, et al 1999. Reactivation and persistence of human herpesvirus-8 infection in B cells and monocytes by Th-1 cytokines increased in Kaposi’s sarcoma. Blood 93: 4044-4058. [Abstract/Free Full Text]
  42. Weber, K. S., H. J. Grone, M. Rocken, C. Klier, S. Gu, R. Wank, A. E. Proudfoot, P. J. Nelson, C. Weber. 2001. Selective recruitment of Th2-type cells and evasion from a cytotoxic immune response mediated by viral macrophage inhibitory protein-II. Eur. J. Immunol. 31: 2458-2466. [Medline]
  43. Wang, X., D. Y. Huang, S. M. Huong, E. S. Huang. 2005. Integrin {alpha}vbeta3 is a coreceptor for human cytomegalovirus. Nat. Med. 11: 515-521. [Medline]
  44. Halary, F., A. Amara, H. Lortat-Jacob, M. Messerle, T. Delaunay, C. Houles, F. Fieschi, F. Arenzana-Seisdedos, J. F. Moreau, J. Dechanet-Merville. 2002. Human cytomegalovirus binding to DC-SIGN is required for dendritic cell infection and target cell trans-infection. Immunity 17: 653-664. [Medline]
  45. Tassaneetrithep, B., T. H. Burgess, A. Granelli-Piperno, C. Trumpfheller, J. Finke, W. Sun, M. A. Eller, K. Pattanapanyasat, S. Sarasombath, D. L. Birx, et al 2003. DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells. J. Exp. Med. 197: 823-829. [Abstract/Free Full Text]
  46. van Kooyk, Y., T. B. Geijtenbeek. 2003. DC-SIGN: escape mechanism for pathogens. Nat. Rev. Immunol. 3: 697-709. [Medline]
  47. Coscoy, L., D. Ganem. 2000. Kaposi’s sarcoma-associated herpesvirus encodes two proteins that block cell surface display of MHC class I chains by enhancing their endocytosis. Proc. Natl. Acad. Sci. USA 97: 8051-8056. [Abstract/Free Full Text]
  48. Ishido, S., C. Wang, B. S. Lee, G. B. Cohen, J. U. Jung. 2000. Downregulation of major histocompatibility complex class I molecules by Kaposi’s sarcoma-associated herpesvirus K3 and K5 proteins. J. Virol. 74: 5300-5309. [Abstract/Free Full Text]
  49. Wang, Q. J., F. J. Jenkins, L. P. Jacobson, L. A. Kingsley, R. D. Day, Z. W. Zhang, Y. X. Meng, P. E. Pellett, K. G. Kousoulas, A. Baghian, C. R. Rinaldo, Jr. 2001. Primary human herpesvirus 8 infection generates a broadly specific CD8+ T-cell response to viral lytic cycle proteins. Blood 97: 2366-2373. [Abstract/Free Full Text]
  50. Bechtel, J. T., Y. Liang, J. Hvidding, D. Ganem. 2003. Host range of Kaposi’s sarcoma-associated herpesvirus in cultured cells. J. Virol. 77: 6474-6481. [Abstract/Free Full Text]
  51. Davis, H. E., J. R. Morgan, M. L. Yarmush. 2002. Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption on target cell membranes. Biophys. Chem. 97: 159-172. [Medline]
  52. Soilleux, E. J.. 2003. DC-SIGN (dendritic cell-specific ICAM-grabbing nonintegrin) and DC-SIGN-related (DC-SIGNR): friend or foe?. Clin. Sci. 104: 437-446. [Medline]



This article has been cited by other articles:


Home page
 J. Virol.Home page
J. West and B. Damania
Upregulation of the TLR3 Pathway by Kaposi's Sarcoma-Associated Herpesvirus during Primary Infection
J. Virol., June 1, 2008; 82(11): 5440 - 5449.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y.-Y. Liu, W.-F. Cai, H.-Z. Yang, B. Cui, Z.-R. Chen, H.-Z. Liu, J. Yan, W. Jin, H.-M. Yan, B.-M. Xin, et al.
Bacillus Calmette-Guerin and TLR4 Agonist Prevent Cardiovascular Hypertrophy and Fibrosis by Regulating Immune Microenvironment
J. Immunol., June 1, 2008; 180(11): 7349 - 7357.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
G. Rappocciolo, H. R. Hensler, M. Jais, T. A. Reinhart, A. Pegu, F. J. Jenkins, and C. R. Rinaldo
Human Herpesvirus 8 Infects and Replicates in Primary Cultures of Activated B Lymphocytes through DC-SIGN
J. Virol., May 15, 2008; 82(10): 4793 - 4806.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
H. J. Garrigues, Y. E. Rubinchikova, C. M. DiPersio, and T. M. Rose
Integrin {alpha}V 3 Binds to the RGD Motif of Glycoprotein B of Kaposi's Sarcoma-Associated Herpesvirus and Functions as an RGD-Dependent Entry Receptor
J. Virol., February 1, 2008; 82(3): 1570 - 1580.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
H. Raghu, N. Sharma-Walia, M. V. Veettil, S. Sadagopan, A. Caballero, R. Sivakumar, L. Varga, V. Bottero, and B. Chandran
Lipid Rafts of Primary Endothelial Cells Are Essential for Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8-Induced Phosphatidylinositol 3-Kinase and RhoA-GTPases Critical for Microtubule Dynamics and Nuclear Delivery of Viral DNA but Dispensable for Binding and Entry
J. Virol., August 1, 2007; 81(15): 7941 - 7959.
[Abstract] [Full Text] [PDF]

Home page
 J. Gen. Virol.Home page
R. Hochreiter, C. Ptaschinski, S. L. Kunkel, and R. Rochford
Murine gammaherpesvirus-68 productively infects immature dendritic cells and blocks maturation
J. Gen. Virol., July 1, 2007; 88(7): 1896 - 1905.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
L. Mark, O. B. Spiller, M. Okroj, S. Chanas, J. A. Aitken, S. W. Wong, B. Damania, A. M. Blom, and D. J. Blackbourn
Molecular Characterization of the Rhesus Rhadinovirus (RRV) ORF4 Gene and the RRV Complement Control Protein It Encodes
J. Virol., April 15, 2007; 81(8): 4166 - 4176.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
J.-H. Wang, A. M. Janas, W. J. Olson, V. N. KewalRamani, and L. Wu
CD4 Coexpression Regulates DC-SIGN-Mediated Transmission of Human Immunodeficiency Virus Type 1
J. Virol., March 1, 2007; 81(5): 2497 - 2507.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
W. Wu, J. Vieira, N. Fiore, P. Banerjee, M. Sieburg, R. Rochford, W. Harrington Jr, and G. Feuer
KSHV/HHV-8 infection of human hematopoietic progenitor (CD34+) cells: persistence of infection during hematopoiesis in vitro and in vivo
Blood, July 1, 2006; 108(1): 141 - 151.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available