We found that HIV-1 replication under conditions where CD4+
-T-cell life span is limited to 3 days is dependent on the cell-associated transfer of virus. In particular, dendritic cell-mediated transfer of HIV-1 to T cells was absolutely essential for wild type virus replication over multiple passages. We demonstrate here that the establishment of productive infection in CD4+
T cells mediated by the cell-associated form of HIV-1 was much more rapid compared to that of cell-free infection. Furthermore, the efficiency of virus transfer was dependent on establishment of close contacts between the virus-bound cell and the target T cell. The inability of cell-free virus to sustain replication in T cells under rapid-turnover conditions probably relates to the relative inefficiency of the initial adsorption of free virus particles to the T-cell surface, which is diffusion limited (8
). We speculate that the adhesion molecule mediated juxtaposition of the two cell membranes in a cell-associated virus infection would in essence increase the net effective concentration of the virus receptor in the vicinity of the bound virus and hence enhance transfer of attached virus to target cells.
Furthermore, the intrinsic affinity between the T-cell surface CD4 and virion-associated native gp120 is low (38
), thus implying that the virus relies on the presence of a large number of attachment factors to bring it into close contact with the cell membrane. Our results demonstrate that DC bind HIV-1 particles with a higher affinity than that exhibited by CD4+
T cells. More importantly, we demonstrate that the kinetics of binding of free virus particles by DC or THP1-DC-SIGN cells and subsequent transfer to CD4+
T cells was faster in its establishment of productive infection than that mediated by cell-free virus stocks of HIVLai
T cells. Finally, the viral burst size was significantly larger in the DC- and THP1-DC-SIGN-T-cell cocultures than in T-cell cultures infected by the cell-free virus stocks within 48 h postinfection. Finally, this ability of DC to mediate rapid trans
infection of activated T cells is unique, since monocyte-derived macrophages, B cells, and T cells pulsed with virus in a similar manner, fail to promote trans
infection to similar levels with rapid kinetics upon coculture with T cells (Fig. and unpublished observations) (37
Based on these results, we propose the following model that could potentially account for the high level of virus replication in the peripheral lymphoid organs in the context of a robust virus-specific CTL response. During HIV-1 infection in vivo, large numbers of HIV-1 particles are produced daily (~1010
), which can be taken up by these immature DC, and transmitted to T cells in the peripheral lymphoid organs via formation of supramolecular activation clusters between the virus-bound DC and the T cell (5
). These DC-T-cell conjugates hence, are the “factories” that drive virus production in vivo. The control of HIV spread in vivo is presumably due to a strong CTL response induced by the lentivirus infection in the peripheral lymphoid organs (7
). Under the restrictive conditions of host immune response, virus has a short window of opportunity to establish a productive infection. In fact, some estimates predict the virus generation time in vivo to be in the order of 2.6 days or less (44
). Direct infection of T cells by cell-free virus particles is rate limited due to the low affinity of the oligomeric virion gp120 for CD4, the sole virus-attachment factor on CD4+
T cells (55
). Furthermore, the cell-free virus half-life in plasma is less than 2 h (44
), thus further limiting the opportunity for HIV to directly infect CD4+
T cells. Presence of large numbers of virus attachment factors on DC (including DC-SIGN, which binds HIV-1 gp120 with a higher affinity than CD4) could allow for the efficient uptake of virus particles. Once they encounter foreign antigen, immunologically favored cellular interactions of DC with CD4+
T cells in the paracortical regions of the lymphoid organs could increase the efficiency with which virus is transmitted to these CD4+
T cells. Hence, we propose that in addition to the postulated role of DC in facilitating initial spread after mucosal exposure of virus (15
), DC could be involved in the continued presentation of HIV-1 to activated CD4+
T cells in the lymphatic tissues, thus sustaining a high level of virus replication under rapid-turnover conditions in vivo.
Finally, we demonstrate here that initial HIV-1 attachment to DC can also occur in a DC-SIGN-independent fashion since virus binding to DC and subsequent trans
infection of T cells was not inhibited in the presence of saturating amounts of mannan, DC-SIGN neutralizing antibodies, or CD4 neutralizing antibodies (Fig. ). These results are consistent with the recently published observations which demonstrate a lack of DC-SIGN expression on rhesus macaque monocyte-derived DC (59
) and certain human DC subsets, namely, Langerhans cells (16
) and plasmacytoid DC (43
). More importantly these DC subsets were competent for virus binding and subsequent transfer of virus to T cells (43
). It is known that peripheral blood monocyte-derived DC express at least three different C-type lectin receptors, including DC-SIGN (54
). Since we observed no significant inhibition of virus attachment and subsequent transmission to T cells in the presence of mannan and CD4 neutralizing antibodies (Fig. ), we conclude that in addition to DC-SIGN, CD4, and C-type lectin (mannose) receptors, there are yet-unknown mechanisms of DC-specific virus attachment. A possible implication of this work is that methods designed to block initial attachment of HIV-1 to DC in the genital mucosa (presumably the initial cell type that encounters virus in the periphery) should include strategies beyond those that target DC-SIGN and mannose specific C-type lectin receptors.
Hence, this study has several implications for in vivo HIV-1 pathogenesis, especially in regards to the apparent ability of the virus to establish and maintain high levels of virus replication under stressful conditions of the host immune response. We predict that in the setting of an HIV-1-infected lymph node, DC-T-cell interactions would provide a favorable milieu for the multiple rounds of virus replication that contribute to the HIV-1-induced pathology and immunodeficiency. If sustaining a vigorous level of HIV-1 replication is a kinetic race fought with virus-specific CTL, then antivirals designed to prevent HIV-1 interactions with DC that do not obviate their important role in antigen presentation may tip the balance of this battle in favor of the human immune system. Finally, our in vitro culture system recapitulates ongoing virus replication in the lymphatic tissues and thus provides a unique model system to study HIV-1 replication. Studies of viral fitness in such an in vitro environment would provide novel insights into the contributions of virus accessory genes to HIV-1 replication.