It has been proposed that changes in cell surface concentrations of coreceptors may control infections by human immunodeficiency virus type 1 (HIV-1), but the mechanisms of coreceptor function and the concentration dependencies of their activities are unknown. To study these issues and to generate stable clones of adherent cells able to efficiently titer diverse isolates of HIV-1, we generated two panels of HeLa-CD4/CCR5 cells in which individual clones express either large or small quantities of CD4 and distinct amounts of CCR5. The panels were made by transducing parental HeLa-CD4 cells with the retroviral vector SFF-CCR5. Derivative clones expressed a wide range of CCR5 quantities which were between 7.0 × 102 and 1.3 × 105 molecules/cell as measured by binding antibodies specific for CCR5 and the chemokine [125I]MIP1β. CCR5 was mobile in the membranes, as indicated by antibody-induced patching. In cells with a large amount of CD4, an unexpectedly low trace of CCR5 (between 7 × 102 and 2.0 × 103molecules/cell) was sufficient for maximal susceptibility to all tested HIV-1, including primary patient macrophagetropic and T-cell-tropic isolates. Indeed, the titers as indicated by immunoperoxidase staining of infected foci were as high as the tissue culture infectious doses measured in human peripheral blood mononuclear cells. In contrast, cells with a small amount of CD4 required a much larger quantity of CCR5 for maximal infection by macrophagetropic HIV-1 (ca. 1.0 × 104 to 2.0 × 104 molecules/cell). Cells that expressed low and high amounts of CD4 were infected with equal efficiencies when CCR5 concentrations were above threshold levels for maximal infection. Our results suggest that the concentrations of CD4 and CCR5 required for efficient infections by macrophagetropic HIV-1 are interdependent and that the requirements for each are increased when the other component is present in a limiting amount. We conclude that CD4 and CCR5 directly or indirectly interact in a concentration-dependent manner within a pathway that is essential for infection by macrophagetropic HIV-1. In addition, our results suggest that multivalent virus-receptor bonds and diffusion in the membrane contribute to HIV-1 infections.

In addition to the primary cell surface receptor CD4, CCR5 or another coreceptor is necessary for infections by human immunodeficiency virus type 1 (HIV-1), yet the mechanisms of coreceptor function and their stoichiometries in the infection pathway remain substantially unknown. To address these issues, we studied the effects of CCR5 concentrations on HIV-1 infections using wild-type CCR5 and two attenuated mutant CCR5s, one with the mutation Y14N at a critical tyrosine sulfation site in the amino terminus and one with the mutation G163R in extracellular loop 2. The Y14N mutation converted a YYT sequence at positions 14 to 16 to an NYT consensus site for N-linked glycosylation, and the mutant protein was shown to be glycosylated at that position. The relationships between HIV-1 infectivity values and CCR5 concentrations took the form of sigmoidal (S-shaped) curves, which were dramatically altered in different ways by these mutations. Both mutations shifted the curves by factors of approximately 30-to 150-fold along the CCR5 concentration axis, consistent with evidence that they reduce affinities of virus for the coreceptor. In addition, the Y14N mutation specifically reduced the maximum efficiencies of infection that could be obtained at saturating CCR5 concentrations. The sigmoidal curves for all R5 HIV-1 isolates were quantitatively consistent with a simple mathematical model, implying that CCR5s reversibly associate with cell surface HIV-1 in a concentration-dependent manner, that approximately four to six CCR5s assemble around the virus to form a complex needed for infection, and that both mutations inhibit assembly of this complex but only the Y14N mutation also significantly reduces its ability to successfully mediate HIV-1 infections. Although several alternative models would be compatible with our data, a common feature of these alternatives is the cooperation of multiple CCR5s in the HIV-1 infection pathway. This cooperativity will need to be considered in future studies to address in detail the mechanism of CCR5-mediated HIV-1 membrane fusion.

The TZM-bl cell line that is commonly used to assess neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) was recently reported to be contaminated with an ecotropic murine leukemia virus (MLV) (Y. Takeuchi It was recently reported that TZM-bl cells, which are commonly used to assess neutralizing antibodies (Abs) against human immunodeficiency virus type 1 (HIV-1), are contaminated with an ecotropic murine leukemia virus (MLV) (22). TZM-bl (also called JC.53bl-13) is a HeLa cell derivative that was engineered by amphotropic retroviral transduction to express CD4 and CCR5 (17) and was further engineered with an HIV-1-based vector to contain Tat-responsive reporter genes for firefly luciferase (Luc) and Escherichia coli ␤-galactosidase (24). These engineered features made TZM-bl cells highly susceptible to HIV-1 infection in a readily quantifiable assay for neutralizing Abs. Many published studies used this cell line for assessments of HIV-1 neutralization; these include several recent reports describing the magnitude, breadth, and epitope specificity of the neutralizing Ab response in infected individuals (14,(18)(19)(20), neutralization escape (25), and the neutralization phenotype of transmitted/founder viruses (10). TZM-bl cells are also gaining popularity for assessments of vaccineelicited neutralizing Ab responses (13). The validity of these and other published results, together with a rationale for the continued use of TZM-bl cells in assessing neutralizing Abs against HIV-1, are very dependent on establishing to what extent, if any, MLV contamination affects the outcome of the assay.It was suggested that ecotropic MLV entered TZM-bl cells via the progenitor JC.53 cell line as an amphotropic MLV pseudotype (22). In this regard, JC.53 cells were constructed from HeLa cells in two stages by using ping-pong technology to amplify the pSFF vector derived from the replication-defective and highly truncated Friend spleen focus-forming virus (3).When used with this vector, this procedure has previously resulted in stable vector expression (17) without formation of replication-competent MLV recombinants (8, 11). A panel of HeLa-CD4 clones was made that express different amounts of CD4 and where the high-expression HI-J clone was used to make a derivative panel of clones (termed JC), including JC.53, that expressed diverse levels of CCR5 (9,16,17). In addition, the HeLa-CD4 clone HI-R that expressed low levels of CD4 was used to make another panel of CCR5-expressing clones (termed RC). To investigate this newly reported issue, cell extracts from these clonal panels and from TZM-bl cells were analyzed for MLV Gag antigens by Western immunoblotting. Representative data, as shown in Fig. 1A, confirm that JC.53 and TZM-bl cells express MLV Gag antigens, whereas the progenitor HI-J clone of HeLa-CD4 cells and many but not all of the other HeLa-CD4/CCR5 clones in the JC panel lack MLV antigens.Initial studies of HI-R cells and other clonal panels that were made using these methods also suggested a lac...

Replication of human immunodeficiency virus type 1 (HIV-1) in diverse conditions limiting for viral entry into cells frequently leads to adaptive mutations in the V3 loop of the gp120 envelope glycoprotein. This has suggested that the V3 loop limits the efficiencies of HIV-1 infections, possibly by directly affecting gp120-coreceptor affinities. In contrast, V3 loop mutations that enable HIV-1 JR-CSF to use the low-affinity mutant coreceptor CCR5(Y14N) are shown here to have negligible effects on the virus-coreceptor affinity but to dramatically accelerate the irreversible conformational conversion of the envelope gp41 subunits from a three-stranded coil into a six-helix bundle. This slow step is blocked irreversibly by the inhibitor T-20. To further evaluate the role of entry rates in controlling infection efficiencies and viral adaptations, we developed methods to quantitatively measure viral entry kinetics. The virions were adsorbed by spinoculation at 4°C onto HeLa-CD4/CCR5 cell clones that either had limiting or saturating concentrations of CCR5. After warming to 37°C, the completion of entry was monitored over time by the resistance of infections to the competitive CCR5 inhibitor TAK-779. Our results suggest that the efficiency of entry of cell-attached infectious HIV-1 is principally controlled by three kinetic processes. The first is a lag phase that is caused in part by the concentrationdependent reversible association of virus with CD4 and CCR5 to form an equilibrium assemblage of complexes. Second, this assembly step lowers but does not eliminate a large activation energy barrier for a rate-limiting, CCR5-dependent conformational change in gp41 that is sensitive to blockage by T-20. The rate of infection therefore depends on the fraction of infectious virions that are sufficiently saturated with CCR5 to undergo this conformational change and on the magnitude of the activation energy barrier. Although only a small fraction of fully assembled viral complexes overcome this barrier per hour, the ensuing steps of entry are rapidly completed within 5 to 10 min. Thus, this barrier limits the overall flow rate at which the attached virions enter cells, but it has no effect on the lag time that precedes this entry flow. Third, a relatively rapid and kinetically dominant process of viral inactivation, which may partly involve endocytosis, competes with infectious viral entry. Our results suggest that the V3 loop of gp120 has a major effect on the rate-limiting coreceptordependent conformational change in gp41 and that adaptive viral mutations, including V3 loop mutations, function kinetically by accelerating this inherently slow step in the entry pathway.Human immunodeficiency virus type 1 (HIV-1) invasion of cells involves a sequence of steps that ultimately cause fusion of the viral membrane with the cell surface membrane and entry of viral cores into the cytosol (3,4,17,34,48), but the rates of these steps and of the competing viral inactivation processes are unknown. Initially, the virus diffuses onto cell ...

By using immunofluorescence microscopy to observe and analyze freshly made HIV-1 virions adsorbed onto cells, we found that they are inherently highly infectious, rather than predominantly defective as previously suggested. Surprisingly, polycations enhance titers 20-to 30-fold by stabilizing adsorption and preventing a previously undescribed process of rapid dissociation, strongly implying that infectivity assays for many viruses are limited not only by inefficient virus diffusion onto cells but also by a postattachment race between entry and dissociation. This kinetic competition underlies inhibitory effects of CCR5 antagonists and explains why adaptive HIV-1 mutations overcome many cell entry limitations by accelerating entry.It is widely believed that retroviruses, including HIV-1, are predominantly defective, with fewer than 0.1% in plasma or culture media being infectious (4,23,24,30,45). However, other evidence indicates that diffusion severely limits virion contact with cultured cells and that forcing virions onto cells by spinoculation or magnetic methods greatly increases titers (6,17,25,36,45). Additionally, polycations, including DEAEdextran and polybrene, substantially increase titers (5,12,13,20,25,47). Reverse transcription is also somewhat inefficient, depending on the virus isolate and cells used. To reconcile these observations and more accurately measure infectivities, we adsorbed HIV-1 onto highly susceptible JC.53 cells (33, 42) at 4°C by spinoculation or brief incubation with concentrated virus, and we analyzed cell-attached virions by immunofluorescence and deconvolution microscopy (29,39). Figure 1A shows fields of adsorbed HIV-1. Cells were fixed for 10 min with 3.7% paraformaldehyde in phosphate-buffered saline (PBS) containing 2% sucrose and then rinsed and incubated for 10 min in 0.5% NP-40 in PBS containing 10% sucrose, and virions were stained using anti-p24 Gag mouse hybridoma 183 (from the NIH AIDS Research and Reference Reagent Program; provided by B. Chesebro). The Z-stack of deconvoluted images was compressed to show all virions. The fluorescent foci had similar intensities, implying that they were single virions. Accordingly, their staining required extraction of lipids with NP-40 (Fig. 1A, lower panels), and the numbers of foci were directly proportional to virus concentrations in the media (Fig. 1A, inset). Similar results were obtained using HIV-1 labeled with Vpr-green fluorescent protein (GFP), and correlative electron microscopy confirmed that these fluorescent foci were single virions (results not shown).We measured the infectivities of cell-attached HIV-1 following spinoculation (4°C, 30 min, 188 ϫ g) onto cultures pretreated with