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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 18, 2004 as doi:10.1096/fj.04-1755fje. |
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Research Center in Infectious Diseases, CHUL Research Center, and Faculty of Medicine, Laval University, Quebec, Canada
1Correspondence: Laboratory of Human Immuno-Retrovirology Research Center in Infectious Diseases, RC709 CHUL Research Center, 2705 Laurier Blvd., Quebec, QC G1V 4G2, Canada. E-mail: michel.j.tremblay{at}crchul.ulaval.ca
SPECIFIC AIMS
Some specific virus-cell interactions have been demonstrated to promote efficient transmission of HIV-1 from cell types naturally nonpermissive for virus infection to susceptible cells. The most convincing example is the association between viral envelope glycoprotein gp120 and dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3) -grabbing nonintegrin (DC-SIGN), which results in capture and transmission of HIV-1 to CD4+ T cells. Experiments with established human lymphoid cell lines, primary peripheral blood mononuclear cells, and ex vivo lymphoid tissue culture system have indicated that several steps in the HIV-1 life cycle are modulated by virion-associated proteins of host origin. In this study, the contribution of some specific virus-associated host proteins in HIV-1 dissemination to susceptible target cells is investigated.
PRINCIPAL FINDINGS
1. ICAM-1-bearing viruses are more efficiently transmitted by monocytoid cells than ICAM-3-bearing virions or HIV-1 lacking both host-derived proteins
To examine whether the nature of virally embedded host constituents can influence HIV-1 transmission, we developed a virus transmission assay relying on LuSIV indicator cells. In that the Mono Mac 6 monocytoid cell line is not susceptible to productive HIV-1 infection due to an almost undetectable level of CD4 (unpublished observations), our initial experiments were carried out using such cells to capture tested virus preparations to LuSIV cells. Flow cytometric analyses indicated that Mono Mac 6 expresses a significant amount of surface LFA-1 (a natural counter-receptor for ICAM-1 and ICAM-3) but are negative for DC-SIGN. Mono Mac 6 cells were found to act as weak transmitters of NL4-3 lacking (NL4-3) and bearing ICAM-3 (NL4-3/ICAM-3). An efficient time-dependent spreading of HIV-1 was obtained when using HIV-1 particles bearing host ICAM-1 (NL4-3/ICAM-1) along with Mono Mac 6.
2. A more efficient transmission of HIV-1 is seen when LFA-1-expressing cells are used in combination with ICAM-1-bearing virions
Previous observations have revealed that DC-SIGN interactions with virus-associated Env glycoproteins (gp120) promote efficient infection in trans of susceptible targets. To compare capacity of DC-SIGN and LFA-1 to mediate HIV-1 transmission, we used HEK293T that transiently express surface DC-SIGN or LFA-1. Significant levels of surface DC-SIGN and LFA-1 were obtained when cells were transfected with similar amounts of the appropriate expression vector (5 µg) (DC-SIGN expression vector: % of positive cells of 96.9 and mean fluorescence intensity of 18.8; LFA-1 expression vector: % of positive cells of 94.4 and mean fluorescence intensity of 12.6) (Fig. 1
A). HIV-1 transmission was weakly increased by presence of DC-SIGN for viruses devoid of foreign ICAM-1 (Fig. 1B
). However, virus transmission was significantly more efficient when LFA-1-expressing HEK293T cells were used along with NL4-3/ICAM-1 viruses. To confirm that ICAM-1/LFA-1 interactions are more important than gp120/DC-SIGN interactions in HIV-1 transmission, our next series of investigations was performed by transfecting HEK293T cells with a constant amount of DC-SIGN expression vector (5 µg) and increasing amounts of LFA-1 expression plasmid (0.01, 0.1, and 1 µg). This experimental strategy resulted in an increase in HIV-1 transmission depending of the amount of LFA-1 expression vector introduced into HEK293T cells (Fig. 1C
). Pretreatment of LFA-1+ HEK293T cells with MEM30 (an anti-LFA-1 CD11a chain antibody that blocks binding to ICAM-1) prevented HIV-1 transmission (Fig. 1D
), indicating that ICAM-1/LFA-1 interactions are indeed critical for HIV-1 capture and transmission to target cells. Because of the weak capacity of DC-SIGN-expressing HEK293T cells to achieve transmission of tested virus preparations and since ICAM-3 is the natural ligand of DC-SIGN, we tested whether acquisition of foreign ICAM-3 by virions would render such DC-SIGN-positive cells more efficient transmitters of HIV-1. Consistent with our previous data, exposure of ICAM-1-bearing viruses to LFA-1-expressing cells led to capture and transmission of HIV-1 to LuSIV cells (5-fold increase). HIV-1 transmission by DC-SIGN-expressing HEK293T cells was inefficient despite insertion of host ICAM-3 within mature HIV-1 particles.
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3. ICAM-1-bearing virions trapped on the surface of LFA-1-expressing cells are efficiently transmitted to histocultures of human lymphoid tissue
Key pathogenesis events and most viral replication take place in lymphoid tissues. Thus, the importance of ICAM-1/LFA-1 interactions in HIV-1 transmission was tested using an established HIV-1 model system based on human tonsillar tissue blocks cultured ex vivo. Transfer of isogenic NL4-3 and NL4-3/ICAM-1 virus particles to histocultures of human lymphoid tissue was unaffected by surface expression of DC-SIGN on parental HEK293T cells. In contrast to data with HEK293T/DC-SIGN cells, we found that transmission of HIV-1 to human lymphoid tissue cultured ex vivo was enhanced when using HEK293T/LFA-1 cells in combination with ICAM-1-bearing virions (4-fold increase). This experiment was repeated with two other donors, with reproducible results.
4. ICAM-1-bearing virions and viruses lacking ICAM-1 are similarly transmitted by primary human cells of macrophage/dendritic lineage
We next determined the role played by virus-anchored host ICAM-1 in transmission and propagation of HIV-1 using monocyte-derived macrophages (MDM) and dendritic cells (DC) as transmitter cells. Virus transmission was comparable when MDM were pulsed with isogenic X4-tropic virions either bearing or lacking host ICAM-1 before incubation with unseparated PBMCs. A similar observation was made using R5-tropic virus stocks differing only by absence or presence of host-derived ICAM-1. Experiments performed with immature and mature DC revealed that X4- and R5-using virions either lacking or bearing host-derived ICAM-1 were equally transmitted to autologous CD4+ T lymphocytes.
CONCLUSIONS AND SIGNIFICANCE
Our data demonstrate that interactions between virion-anchored host ICAM-1 and cell surface LFA-1 take over gp120/DC-SIGN and ICAM-3/DC-SIGN interactions during HIV-1 dissemination, at least when transmitter cells consisted of CD4-negative epithelial cells (HEK293T) or monocytoid cells that express little CD4 (Mono Mac 6). The importance of the interaction of ICAM-1 with LFA-1 in HIV-1 transfer could not be illustrated when using transmitter cells such as MDM and DC (immature and mature).
DC-SIGN binds efficiently to viral envelope glycoproteins from R5- and X4-tropic HIV-1 isolates and acts as a major ICAM-3 receptor on DC; it plays a key role in establishing the initial interaction between DC and other cell types, such as T cells. It was unexpected to discover that the association between ICAM-1 and LFA-1 is much more efficient than the combined action of gp120/DC-SIGN and ICAM-3/DC-SIGN interactions when DC-SIGN-expressing HEK293T cells were used to accomplish transfer of HIV-1. Several findings may contribute to solving this mystery. For example, contact between ICAM-3 and DC-SIGN mediates a transient, loose adhesion between DC and T cells, suggesting that the strength of this interaction is not of a high order of magnitude. Differences in experimental methodologies may be responsible to some extent for our data. Most published HIV-1 capture and transmission assays have relied on pulsing of transmitters cells with infectious virus for a longer period (>2 h, sometimes 48 h) than the 45 min incubation used in our work with HEK293T-DC-SIGN transfectants. It can be proposed that attachment of HIV-1 mediated by gp120/DC-SIGN and ICAM-3/DC-SIGN interactions require more time to resume than does virus attachment resulting from ICAM-1/LFA-1 interactions. This is supported by affinity and kinetic analyses revealing that ICAM-1 interacts with high affinity and a slow dissociation rate constant with LFA-1.
Experiments with MDM and DC demonstrated that interactions between virus-anchored ICAM-1 and its potential natural ligands (integrins LFA-1 and Mac-1) are not playing a dominant role in HIV-1 uptake and eventual transfer from such transmitter cells, perhaps related to the fact that the attachment process of HIV-1 can occur via CD4-independent mechanisms involving ligation of highly glycosylated groups or basic residues found on gp120 with polyanionic sulfated chains or lectin-like domains on specific cell surface receptors other than DC-SIGN. Gp120, for example, has been shown to associate with the glycolipid galactocerebroside and its sulfated derivative (sulfatide), glycosaminoglycan heparan sulfate, placental membrane binding protein (now called DC-SIGN), and macrophage mannose receptor (MMR). A recent study shows that MMR plays a cardinal role in binding and transmission of HIV-1 to T cells by primary human macrophages. Mannose receptor is primarily responsible for HIV-1 binding on dermal DC. Heparan sulfate proteoglycans such as syndecans are the main class of attachment receptors for HIV-1 on different cell types such as macrophages and endothelial cells and are thought to play a cardinal role in HIV-1 transmission. Our data demonstrate that results from HIV-1 transmission experiments carried out in transfected cells or established cell lines cannot be extrapolated to primary human cells such as MDM and DC due to differences in cell surface constituents that can interact with HIV-1. It can be proposed that cellular type might also influence the rapidity with which HIV-1 is endocytosed upon intimate contact and the fate of HIV-1 once found within endolysosomal apparatus. This is illustrated by rapid internalization of DC-SIGN and MMR upon interactions between HIV-1 and DC. In contrast, HEK293T cells display very low efficiency of HIV-1 endocytosis. Thus, the ability of DC-SIGN to transmit HIV-1 to susceptible cells most likely is related to its capacity to be endocytosed.
This study provides new insight into the mechanism of HIV-1 transmission and indicates that, under some circumstances, virus dissemination is not only influenced by interactions between virus-encoded constituents (gp120) and appropriate ligands (DC-SIGN) but also by interactions between host molecules incorporated within HIV-1 and their natural counter-receptors (Fig. 2
). This work illustrates that HIV-1 capture and transfer to susceptible target cells involves many interactions between the virion and cell surface. We present evidence that this phenomenon is influenced by the identity of virus transmitter cells.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1755fje; doi: 10.1096/fj.04-1755fje
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