HIV is a fast-replicating virus in vivo and in cell cultures. We have established simple experimental systems to evaluate the role of cell contacts during viral spread. We first report that continuous and gentle rocking of Jurkat cells or primary CD4+
lymphocytes has profoundly deleterious effects on the ability of HIV to propagate. An altered viral replication in shaken lymphocytes was observed with a panel of laboratory-adapted and primary HIV strains with X4 or R5 tropism. Rocking did not significantly affect cell viability and metabolism, nor did it affect the surface expression of various receptors. Once productively infected, shaken cells released normal amounts of virions, indicating that the late steps of viral replication are not affected in mobile cells. We thus hypothesized that virus transfer to recipient cells was altered in shaken lymphocytes due to reduced contact times between cells. To directly demonstrate this, we have designed a flow cytometry-based assay to measure the relative contributions of free virus and cell-associated virus in the propagation of infectivity. With this assay, we confirm previous reports demonstrating that the infectivity of HIV during cell-to-cell transmission is much greater than that with free virus (11
). Up to 50% of targets became productively infected within 24 h of coculturing in a process requiring a direct contact between donor cells and recipients. This assay is quantitative, the efficiency of viral transfer being dependent on the percentage of productively infected donors as well as on the donor-to-target-cell ratio. A time course analysis demonstrated a two-phase kinetics of Gag transfer and expression in targets. The first phase is rapid (2 to 4 h) and allows the detection of Gag antigens in 5 to 10% of target cells. This time frame is too short to correspond to newly synthesized viral proteins in targets and likely represents the transmission of incoming virions or viral material. This first phase was insensitive to the reverse transcriptase inhibitor NVP. The second phase is slower (12 to 24 h), in large part sensitive to NVP, and requires adequate receptors and fusogenic viral envelope glycoprotein interactions. It thus corresponds to the de novo synthesis of virus in targets.
It has been reported that a high level of coreceptor-independent HIV transfer is induced by contacts between primary CD4+
T cells (7
) associated with a nonspecific endocytosis of virions in intracellular vesicles in targets. These coreceptor-independent events were barely detected in our assay but may correspond to the 5 to 10% of target cells carrying Gag antigens at early time points, after contact with cells producing viruses with either functional or defective envelope glycoproteins. The sensitivity of our assay is probably too low to detect a large number of these nonspecific events (29
). The flow cytometry assay thus represents a convenient tool to follow the transfer and productive infection of targets through cell-to-cell interactions.
Small numbers of syncytia between donor and target Jurkat cells were observed by visual examination of the cocultures. Moreover, by using differential fluorescent labeling of donors and targets, we estimated that fewer than 10% of Jurkat cells in cocultures were engaged in fusion or clustering events upon analysis by flow cytometry. Syncytia were even rarer with primary lymphocytes, at least during the 24-h time frame of the survey. The kinetics of HIV Gag transfer are indeed more rapid than those of cell-cell fusion (24
). With the exception of their detection in some regions of lymphoid tissue and the nervous system, syncytia are barely detectable in HIV-infected individuals. Our flow cytometry assay of Gag detection thus allows the analysis of cell-to-cell transfer without extensive syncytium formation, a situation reminiscent of the infection in vivo.
Several lines of evidence strongly suggest that Gag detection in CFSE+ targets reflects the productive transfer of infection rather than an increased clustering of cells into doublets or more. First, the number of cell-cell conjugates, as measured by double-fluorescent labeling, was not proportional to Gag expression in CFSE+ cells. Second, the nonfusogenic HIV F522Y mutant, which retains its ability to bind to its receptor, did not induce detectable Gag transfer to CFSE+ cells. Third, Gag detection was significantly decreased by NVP and was thus associated with a reverse transcription step.
This efficient viral spread through direct cell contacts is likely mediated by the induction of virological synapses (22
). Virological synapses are defined by a macromolecular and structural organization of the junction zone between infected cells and recipients. These synapses involve cellular and retroviral proteins, as well as components of rafts and cytoskeleton (5
). However, it is not formally demonstrated that HIV Gag movement across the synaptic junction corresponds to productive infection (24
). The combined use of our flow cytometry assay, which provides a functional assessment of productive viral transfer, and of the analysis of synapse formation by fluorescent microscopy will help to decipher the functional role of virological synapses. For instance, it will be worthwhile to examine the effects of chemicals (i.e., raft and cytoskeleton disorganizers, etc.), negative transdominant proteins, and small interfering RNA against cellular proteins of interest, from both functional and structural points of view.
The influence of viral proteins can also be easily studied with our functional assay of productive cell-to-cell viral transfer. We have shown here that a fusogenic envelope is required in this test. It will be of interest to analyze other Env mutants, for instance those previously described to alter the polarized budding of HIV in lymphocytes (14
). The Vpu protein is known to facilitate virion release from the cell. Interestingly, a vpu
-defective virus has been previously identified in an in vitro assay selecting rapidly spreading viral strains (20
). Accordingly, we observed that a Δvpu
HIV strain is transmitted in our assay at an efficiency similar or even better than that of its WT counterpart (not shown). Another protein of interest is Nef, which is known to increase the infectivity of free virions, whereas WT and Δnef
viruses replicate similarly in most cell culture systems (1
). This apparent discrepancy may be due to the cell-to-cell propagation of infection in cultures, which may not require Nef. Current experiments in our laboratory are aimed at examining this point.
In conclusion, we report here that HIV replication is dramatically impaired in shaken cells. We show that shaking prevents efficient interactions between cells and hence viral transfer. We also demonstrate that cell-to-cell transmission is the predominant mode of viral propagation in cultures. These findings help to explain why, in infected individuals, HIV replicates mostly in lymphoid tissues, where high lymphocyte concentrations and slow movements will facilitate cellular cross talk and viral transmission. In contrast, circulating, highly motile lymphocytes will probably transmit HIV infection much less efficiently. Finally, from a practical point of view, the flow cytometry assay of productive HIV transfer may prove useful in studying the effects of antiviral drugs or neutralizing antibodies in a physiologically relevant situation of viral propagation.