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Journal of Virology, April 2005, p. 4347-4356, Vol. 79, No. 7
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.7.4347-4356.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Kinetic Factors Control Efficiencies of Cell Entry, Efficacies of Entry Inhibitors, and Mechanisms of Adaptation of Human Immunodeficiency Virus{dagger}

Emily J. Platt, James P. Durnin, and David Kabat*

Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon

Received 1 July 2004/ Accepted 1 November 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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-1JR-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 concentration-dependent 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 coreceptor-dependent 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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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 surfaces by a slow process that can be dramatically enhanced by spinoculation or by viral precipitation (39). On many cells, including HeLa-CD4 cells (38), initial attachment involves multiple weak bonds to abundant cell surface components, which enables the virus to move over the cell until the viral envelope glycoprotein gp120 subunit binds to the primary receptor CD4 (12, 24). This induces exposure of a site in gp120 for association with a coreceptor, which then induces subsequent steps that lead to membrane fusion (5, 15, 56, 60). Newly transmitted HIV-1 (R5 strains) exclusively use CCR5 as a coreceptor, whereas variants that use CXCR4 (X4 strains) often form during disease progression (2, 8, 10, 13, 19, 32, 37, 45, 47). These variants differ principally in the V3 loop domains of the viral gp120 envelope glycoprotein subunits, and the X4 variants have a broadened cellular tropism and usually are more syncytium inducing in peripheral blood mononuclear cells than the corresponding R5 strains (50, 51, 55, 57). The affinities of X4 viruses for CXCR4 are also generally very low (22).

In native HIV-1 virions, the envelope glycoproteins are heterotrimers with two subunits, surface gp120 subunits that bind CD4 and coreceptors and transmembrane gp41 subunits that mediate membrane fusion but are kinetically trapped in a metastable conformation (5, 15). The large activation energy barrier that prevents conformational rearrangement of gp41 is partially imposed by the gp120 cap, and this barrier is reduced but not eliminated by gp120 associations with CD4 and coreceptors, thereby enabling the gp41 subunits to irreversibly fold at an accelerated rate into the more stable fusogenic conformation (5, 15). This conformational rearrangement also occurs by a sequence of steps. The binding of gp120 to CD4 induces a conformational change in gp120 that exposes the coreceptor binding site (5, 15, 56, 60) and that enables the gp41 subunits within a trimer to collaboratively form a three-stranded coil that extends toward the cell surface and results in insertion of the hydrophobic gp41 amino termini into the cell membrane (5, 15). Although CD4 alone appears to be sufficient to induce a detectable amount of three-stranded coil formation in laboratory-adapted HIV-1 isolates that are relatively sensitive to inactivation by soluble derivatives of CD4, coreceptors substantially accelerate or enhance the efficiency of this conformational change (20). Coreceptors may be even more essential for this step in patient-derived HIV-1 isolates that are highly resistant to soluble CD4. Subsequently, the three-stranded coil conformation of gp41 is converted into a six-stranded trimer of hairpins that pulls the virus more tightly onto the cellular membrane (5, 15). Coreceptors are probably essential for this final conformational change, which leads to membrane fusion (5, 15).

Despite this detailed knowledge of the membrane fusion pathway, the factors that control efficiencies and rates of entry are largely unknown. Moreover, numerous changes in gp120 and gp41 occur during disease progression, yet the effects of these changes on the steps of infection are also substantially unknown. In addition, drugs that interfere with HIV-1 entry are being developed, and it is important to understand how they function and how adaptive viral mutations counteract their effects (16, 27, 30, 31, 33, 54, 58, 59). One limitation in many previous studies of HIV-1 entry is that the cells that were used had variable and often unknown amounts of CD4 and coreceptors, which made kinetic and inhibitor studies difficult to quantitatively interpret. This also has made it more difficult to understand effects of coreceptor mutations and of viral adaptations.

To begin to address these issues, we previously used panels of HeLa-CD4/CCR5 cell clones, in which the clones in a panel express the same quantity of CD4 but different amounts of CCR5 (23, 43). Similarly, we made clonal panels containing the low-affinity mutant coreceptors CCR5(G163R) and CCR5(Y14N) (28, 29, 52). R5 HIV-1 preparations were added to cultures of these clones, and the titers were measured 72 h later. This evidence implied that the efficiencies of infection might be principally controlled by kinetic competition between successful viral entry and a process of viral inactivation (28, 41). We now describe direct kinetic methods to test this interpretation and to measure the rates of the competing processes that control efficiencies of infectious HIV-1 entry into cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and viruses. HeLa-CD4 cells expressing low (6 x 103 molecules/cell, clone JC.10) or high (1.9 x 105 molecules/cell, clone JC.53) concentrations of CCR5 or panels that express varying, discrete concentrations of the mutant coreceptor CCR5(Y14N) or CCR5(G163R) were created and maintained as previously described (23, 28, 43). Viral stocks of the replication-competent R5 HIV-1JR-CSF molecular clone were created by transfection of the pYK-JRCSF plasmid (obtained from the National Institutes of Health [NIH] AIDS Research and Reference Reagent Program [ARRRP], contributed by I. Chen and Y. Koyanyagi) into HeLa cells as previously described (41). Viral titers were determined by using the focal infectivity assay of Chesebro et al. (6, 7) and were typically in the range of 0.5 x 106 to 1.0 x 106 focus-forming units per ml. HIV-gpt virions pseudotyped with CCR5(Y14N)- or CCR5(G163R)-adapted envelopes were produced as previously described (41).

Infectivity assays. Infectivity assays using replication-competent HIV-1JR-CSF or HIV-gpt virions pseudotyped with wild-type or mutant HIV-1JR-CSF envelopes were done as previously described (41, 43). Viral entry kinetics were measured by spinoculating viruses at 4°C onto replica HeLa-CD4/CCR5 cultures that contain either low or high concentrations of CCR5 and that were seeded at 5 x 103 cells/well in 48-well plates 24 h previously (39, 43). After the cultures were rinsed with fresh medium and warmed to 37°C, a completely inhibitory concentration of TAK-779, obtained from the ARRRP (1, 14) (25 µM), was added at zero time or at different later times (i.e., 5, 10, 15, 30, 60, 120, 240, and 360 min) to the wells to monitor viral completion of the CCR5-dependent steps of infection. Foci of viral infection were assayed at 72 h (6, 7). Relative infectivity values were obtained by dividing titers in each well by the titer at the final 360-min time point in the cells that contain a large excess of CCR5. In some assays, partially inhibitory amounts of TAK-779 or T-20 (obtained from the ARRRP and contributed by Roche) were added before the cultures were warmed to 37°C, in order to test their effects on the kinetic parameters.

Kinetic analysis. Here we derive a kinetic model of the HIV-1 entry pathway based on the simple idea that the viable virions adsorbed onto cell surfaces associate reversibly with CD4 and CCR5 to form an assemblage of complexes, some of which are competent to undergo subsequent steps that culminate in membrane fusion. Additionally, competing processes of viral inactivation or dissociation occur. These inactivation events can be considered to have a composite rate constant. We use HeLa-CD4/CCR5 cell clones that have either low or high concentrations of CCR5 for this analysis. It has been established that R5 HIV-1 virions bind reversibly to CD4 and CCR5 to form complexes with a requisite stoichiometry that are competent to undergo subsequent steps of the entry pathway (28, 43). Thus, we can write

{4347_eq_1}(1)
where V is the number of potentially viable virions on cell surfaces in the culture and ki is the composite rate constant for the subsequent coreceptor-dependent steps of the entry pathway. It is important to note that V is thus defined as the number of virions that would infect the culture if the entry process was perfectly efficient and if there was no competing process of viral inactivation. V is less than the total number of virions on the cells because many are inactive and because postentry steps such as reverse transcription are also not perfectly efficient. Because HIV-1 binds to CCR5 only after it has already adsorbed onto cells and associated with CD4 (56, 60), the numbers of virions that initially attach onto cells should be independent of the cellular concentrations of CCR5 (28, 43). In addition, HIV-1 attachment onto HeLa cells has been reported to be independent of whether the cells even contain CD4 or CCR5 (3, 25, 38, 48). Since the number of virion particles that attach to HeLa cells under the conditions of our spinoculation assays is at least two orders of magnitude lower than the numbers of CCR5 molecules on our cell clones, it is likely that the concentrations of free CCR5 are not substantially reduced by complexation with the virions that attach to each cell. In any case, at each CCR5 concentration a fraction, {alpha}, of the virions will be in competent complexes after the reversible assembly process has reached equilibrium. Thus, at very low CCR5 concentrations we expect that {alpha} will be low, whereas at high concentrations of wild-type CCR5 the efficiency of infection reaches a maximum, implying that the virions are saturated and that {alpha} is close to 1.0 (see below). Consequently, we infer from equation 1 that the rate of infection at any time is

(2)
V declines with time due to infection and to virus inactivation processes at a composite rate constant k2. Therefore,

(3)
Note that we are assuming that all of the potentially viable virions on the cell surfaces are susceptible to the inactivation process. As described below, this is consistent with our data and with previous evidence that HIV-1 virions are rapidly endocytosed by a constitutive process (3, 17, 21, 34, 35, 48). Alternatively, it could be assumed that the predominant viral inactivation process might depend on whether the virus is complexed or uncomplexed with coreceptors. These alternative hypotheses are mathematically evaluated in the supplemental material and are shown to be incompatible with our evidence. Integration of equation 3 gives

(4)
where V0 is the number of potentially viable virions on the cells (as defined above) of each culture at time zero. Substituting this in equation 2and integrating from time zero to time t gives

(5)
After a long time the viable virions have disappeared from the cell surfaces and the total number of infections has reached a final value ifinal, where

(6)
Consequently,

(7)
which implies that a semilog plot of log10f, as defined in equation 7, versus t should give a straight line with a slope of –({alpha}ki + k2)/2.3.

Our basic strategy is to compare the kinetics of infection of two cell clones, one with a low concentration of CCR5 (~6,000 molecules/cell) (low-CCR5 cells) and the other with a high concentration (~190,000 molecules/cell) (high-CCR5 cells). At different times after the virus is adsorbed by spinoculation onto multiple culture aliquots at 4°C and the cultures are then warmed to 37°C, a large excess of TAK-779 is added to block all virions that have not completed the CCR5-dependent steps of infection. Since the cell clones differ only in CCR5 concentrations, ki and k2 should be the same in both clones, whereas their {alpha} values should differ. Moreover, because relative infectivity values plateau at high CCR5 concentrations, we infer that {alpha} is ~1.0 on the high-CCR5 cells. On the low-CCR5 cells, {alpha} will be relatively low, so the infection kinetics will be more dominated by the inactivation processes. Therefore, the slope of the semilog plot obtained by using equation 7should be less steep on the low-CCR5 cells (~–k2/2.3) than on the high-CCR5 cells [~–(ki + k2)/2.3]. Using equation 6 and the ifinal values of the two cell clones, we can also calculate the fractions of the initially attached viable virions that successfully infect the cells and the value of {alpha} for the low-CCR5 cells.

It should be noted that the equations above assume that viral entry begins immediately after the cultures are warmed to 37°C. However, as demonstrated below, a lag phase, t*, precedes assembly of competent complexes and subsequent completion of the CCR5-dependent steps of entry. We presume that the viral inactivation process occurs during this lag, so that the number of viable virions on the cells becomes reduced to V0 exp(–k2 t*) before any have completed the CCR5-dependent steps of the entry pathway. This lag-phase correction would yield a variant of equation 7 with a term (t – t*) replacing the term t, but this would have no effect on the slopes of the resulting semilog plots or on our estimates of ki or k2. The lag, however, significantly affects estimates of ifinal/V0 obtained by using equation 6, which becomes

(8)
This correction is important because a portion of the initially attached virus is presumably inactivated during the lag phase before entry commences. Moreover, as shown below, t* differs for the cell clones being compared. This correction was used below in our estimates of {alpha}. Further discussion of these kinetic issues and the lag-phase corrections is in the supplemental material. Alternative kinetic models are also derived and evaluated in the supplemental material, and equations for T-20 sensitivities of HIV-1 infections are derived. This analysis suggests that T-20 sensitivities in cells that contain different amounts of CCR5 should be inversely proportional to their {alpha} values.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of factors that influence efficiencies of R5 HIV-1 infections. To identify factors that control efficiencies of R5 HIV-1 infections, we have used panels of HeLa-CD4/CCR5 cell clones in which the clones express a large uniform amount of CD4 but differ in their cell surface concentrations of wild-type CCR5 or of the mutant CCR5(G163R) or CCR5(Y14N), which bind R5 gp120s with low affinity (23, 28, 29, 43, 52). Because HIV-1 attachment onto HeLa-CD4/CCR5 cells is independent of the cell surface CCR5 concentrations, the virions bind to these clones equally (25, 28, 43). This is expected because HIV-1 associates with CCR5 only after binding to CD4 has already occurred (56, 60). Consequently, the relative viral titers on these cell clones are proportional to the efficiencies with which attached infectious virions successfully enter the cells. The titer on each cell clone was normalized relative to the titer of the same virus on a cell clone (JC.53) that expresses a high concentration of wild-type CCR5 and is highly susceptible to all of the viruses we have used. This normalization corrects for the fact that many virions are noninfectious and that intracellular steps such as reverse transcription are also inefficient. As described previously (28, 43), the infectivity curves of the viruses on each clonal panel have sigmoidal shapes that differ not only in their positions on the CCR5 concentration axis but also in their asymptotes on the relative infectivity axis. For example, as shown in Fig. 1A, wild-type HIV-1JR-CSF is highly infectious for cells with wild-type CCR5, even when the cell clones contain fewer than 10,000 CCR5 molecules/cell. In contrast, the lower-affinity CCR5(G163R) coreceptor is effective only at relatively high concentrations (i.e., its 50% effective concentration [EC50] is ~35,000 per cell). Similarly, the very low affinity CCR5(Y14N) coreceptor mediates infections by wild-type HIV-1 isolates only at high concentrations (EC50 of ~150,000 per cell), and even at saturatingly high CCR5(Y14N) concentrations, the asymptote on the relative infectivity axis for HIV-1JR-CSF plateaus at a low value of approximately 0.03 (Fig. 1A). In all of these assays the titers of the undiluted virus samples were sufficiently high to obtain accurate estimates of the EC50s.



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  		FIG. 1. Infectivities and T-20 sensitivities of wild-type and mutant CCR5(Y14N)-adapted HIV-1 in HeLa-CD4 cells expressing wild-type or mutant CCR5 coreceptors. (A) Infectivities of wild-type and CCR5(Y14N)-adapted HIV-1JR-CSF. The upper two curves show wild-type replication-competent HIV-1JR-CSF [JR-CSF(WT)] infectivity in HeLa-CD4 cells expressing wild-type CCR5 or in a HeLa-CD4 panel expressing varying discrete amounts of the mutant coreceptor CCR5(G163R) (28, 43). The lower three curves show infectivity data for HIV-gpt virions in a HeLa-CD4 panel expressing varying discrete amounts of the mutant coreceptor CCR5(Y14N) (28). The HIV-gpt virions were pseudotyped with either wild-type JR-CSF envelope [HIV-gpt(WT)] or JR-CSF envelopes that are moderately [HIV-gpt(N298, L313)] or highly [HIV-gpt(N298, N300, L313)] adapted to use CCR5(Y14N). Relative infectivity values for each virus were calculated by normalizing titers in a given cell clone to titers in HeLa-CD4 cells expressing a large amount of wild-type CCR5. Data are the averages from three to eight independent experiments, and error bars are standard errors of the means. (B) Efficiency of T-20 inhibition in HeLa-CD4/CCR5 and HeLa-CD4/CCR5(Y14N) cells. A HeLa-CD4/CCR5(Y14N) clone expressing large amounts of coreceptor was infected by HIV-gpt viruses pseudotyped with wild-type JR-CSF envelopes [HIV-gpt(WT)] or with envelopes adapted to use CCR5(Y14N) [(HIV-gpt(N298, N300, L313)] or [HIV-gpt(N298, L313)] in the presence of serial fivefold dilutions of T-20. The T-20 sensitivity of wild-type JR-CSF envelope-pseudotyped virions in HeLa-CD4 cells expressing a large amount of wild-type CCR5 [HIV-gpt(WT)] was also measured. Relative infectivity values were calculated for each virus by dividing titers generated in a given cell line at each T-20 concentration by titers obtained in the same cell line in the absence of inhibition. The averages of replicate experiments are displayed (n = 4); error bars are standard errors of the means.

 
As described previously (28, 41, 43) and diagrammed in equation 1, such titer results with our clonal panels imply that the virions on cell surfaces reversibly associate with CD4 and CCR5 to form complexes with a requisite stoichiometry that are competent to infect the cells. When virus-coreceptor affinities are low, higher coreceptor concentrations are required for infection (Fig. 1A). Presumably, according to this kinetic model, the fully assembled competent complexes of wild-type virus with CCR5(Y14N) have a low rate of entry (i.e., a low ki for equation 1). Thus, even at saturating concentrations this mutant coreceptor mediates entry slowly. However, a low rate of viral entry would result in a low efficiency of infection as seen in Fig. 1A only if there was a competing process of viral inactivation or dissociation. Otherwise, entry would proceed slowly to completion (28, 41). Therefore, we infer that HIV-1 entry efficiencies are strongly affected by three processes, i.e., a concentration-dependent association of virus with CD4 and coreceptors, a postassembly process that leads to membrane fusion, and a competing process of virus inactivation.

Although wild-type HIV-1JR-CSF uses CCR5(Y14N) inefficiently, we isolated adapted viral variants that replicate more efficiently in these cell clones (41). One variant that was selected for replication in a cell clone that expresses a high concentration of CCR5(Y14N) contained gp120 V3 loop mutations S298N and F313L. As shown in Fig. 1A, it infected the CCR5(Y14N) cells much more efficiently than did the wild-type virus. Specifically, its EC50 was approximately 120,000 CCR5(Y14N) per cell, and its asymptote on the relative infectivity axis was approximately 0.32. We then further selected this virus for replication in a cell clone that expresses only a low concentration of CCR5(Y14N), which yielded a variant with the additional V3 loop mutation N300Y. This triple mutant infected the CCR5(Y14N) clonal panel even more efficiently (EC50 of ~100,000/cell and maximum relative infectivity of ~0.56). Together, these V3 loop mutations reduced the EC50 only slightly, by 33 to 50%, whereas they increased the maximum relative infectivity by approximately 20-fold.

Based on the considerations described above, we hypothesized that these V3 loop mutations function principally by increasing the efficiency of the postassembly steps of infection rather than by increasing viral affinities for CCR5(Y14N). Consistent with this conclusion, wild-type virus infection of cells with CCR5(Y14N) was extremely sensitive to inhibition by T-20, which binds to the three-stranded conformational intermediate of gp41, whereas the adapted variant viruses were approximately 4- to 20-fold less sensitive, respectively (Fig. 1B). Furthermore, the wild-type virus was approximately 70-fold more sensitive to T-20 when it was saturated with CCR5(Y14N) than when it was saturated with wild-type CCR5. Since T-20 sensitivity is directly related to the lifetime of this conformational intermediate (46) (see below), we conclude that the adaptive V3 loop mutations accelerate resolution of this T-20-sensitive gp41 structure. The wild-type virus uses CCR5(Y14N) inefficiently, and this prolongs the lifetime of the T-20-sensitive intermediate, thereby enabling the viral inactivation process to predominate. This evidence supports the hypothesis that entry kinetics have a major effect on the efficiencies of HIV-1 infections and that changes in the V3 loop of gp120 can dramatically alter the lifetime of the T-20-sensitive conformational intermediate of gp41 while only slightly affecting the virus-coreceptor affinities.

Kinetic analysis of the infection pathway. We used the kinetic approach described in Materials and Methods to obtain further evidence concerning the factors that control the efficiencies of HIV-1 entry. In this method, a preparation of virions is adsorbed by spinoculation at 4°C onto multiple cultures of two clones of HeLa-CD4/CCR5 cells, one having ~6,000 CCR5 molecules/cell and the other having ~190,000 CCR5 molecules/cell. After the cell cultures are rinsed and warmed to 37°C, the rates at which infectious virions complete the CCR5-dependent steps of entry, as indicated by resistance to TAK-779, are measured. Figure 2 shows a kinetic analysis of the infection of these cell clones with the wild-type HIV-1JR-CSF (n = 11). The large number of assays was done to ensure maximum significance for our interpretations. The lag preceding onset of infection is clearly more prolonged for the cells with a low CCR5 concentration. The semilogarithmic plots of the data in Fig. 2B are linear, and the slopes of the lines differ for the two cell clones in a manner that is fully consistent with the predictions of equation 7. We believe that this strongly supports our hypothesis that a potent viral inactivation process competes with the entry pathway. The infectious virions are eliminated more rapidly from the surfaces of cells that have a high CCR5 concentration because both infection and inactivation occur, whereas they are eliminated from the surfaces of the low-CCR5 cells principally by the inactivation process. These data imply that the rate constant k2 for virus inactivation is ~0.47/h, that the rate constant ki at which fully assembled virus-CD4-CCR5 complexes complete the entry process is ~0.09/h, and that the proportion of virions that are fully assembled into competent complexes ({alpha}) is only ~0.23 (after the lag-phase correction) on the low-CCR5 cells. These results were somewhat surprising because the rate constant for viral inactivation is large compared to the rate constant for successful entry. Consequently, even for the optimally susceptible high-CCR5 cell clone, the viral inactivation process dominates the kinetics so that only approximately 15% of the initially attached viable virions successfully enter the cells. Because {alpha} is lower for the cells with a limiting CCR5 concentration, the viral inactivation process is even more predominant, and only ~3% of the initially adsorbed viable virions successfully enter the cells. An important implication of these results is that the kinetics of entry (i.e., the shapes of the plots in Fig. 2A) are determined principally by the rates of viral inactivation rather than by the rates of infection. Thus, the curves plateau rather rapidly, principally because of viral inactivation rather than because of successful entry. These results help to explain the selection pressures that induce adaptive changes in HIV-1 envelope glycoproteins during viral replication in cultured cells (18, 26, 40, 44). Very similar estimates of k2, the rate constant for viral inactivation, were obtained in numerous other experiments (results not shown).



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  		FIG. 2. Infection kinetics of HIV-1JR-CSF in HeLa-CD4/CCR5 cells. (A) Infection kinetic data in HeLa-CD4 cells expressing high or low concentrations of wild-type CCR5. Relative infectivity values were generated by dividing the titer from a given time point by the titer in HeLa-CD4 cells expressing a high concentration of CCR5 at the final time point (360 min). The averages of replicate experiments are displayed (n = 11), and error bars are standard errors of the means. (B) Mathematical analysis of the infection kinetic data. The data in panel A were analyzed according to equation 7 in Materials and Methods. Titers obtained at the final (360-min) time point were used as the ifinal value. The value for the final 360-min time point is not plotted because where ifinal = it, equation 7 is undefined. Therefore, the number of points is not the same as that in panel A, but all of the informative data available are represented. The intercept of the upper axis indicates the lag times for the two cell lines, with lags of ~7 min in cells expressing high concentrations of CCR5 and ~16 min in cells expressing low concentrations of CCR5. Measuring the slopes generated with HeLa-CD4 cells expressing high or low concentrations of CCR5 and then using equations 7 and 8 in Materials and Methods allows us to calculate the rate constant of viral inactivation, k2, as ~0.47/h and the rate constant at which completely assembled virus-CD4-CCR5 complexes complete cellular entry, ki, as ~0.09/h. The proportion of virions in competent assembly complexes, {alpha}, is ~0.23 in cells expressing low concentrations of CCR5.

 
As shown in Fig. 2, the lag phase is substantially longer at low CCR5 concentrations than at higher concentrations, suggesting that it may partly involve the concentration-dependent association of CCR5 with the virus. This lag is evident in the plots of relative infectivity versus time in Fig. 2A, and is also seen in Fig. 2B as the times at which the lines intersect the upper axis. To obtain further information about this lag, we compared the kinetics of infection in cell clones that express nearly identical quantities of either CCR5 (wild type) or the low-affinity CCR5(G163R) (Fig. 3). The lag was substantially longer on the cells that contain the low-affinity coreceptor. Similarly, as further discussed below, the lag phase was also prolonged by moderate concentrations of the competitive inhibitor TAK-779, which binds to CCR5 and reduces the pool available for association with HIV-1 (Fig. 4B). As expected, this lag-phase increase occurred when the CCR5 concentration was limiting but not when CCR5 was present in a large excess.



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  		FIG. 3. Infection kinetics of HIV-gpt virions in HeLa-CD4 cells expressing wild-type or mutant coreceptors. Infection kinetics of HIV-gpt virions pseudotyped with wild-type JR-CSF envelope were measured in HeLa-CD4 cells expressing CCR5(G163R) or in HeLa-CD4 cells expressing a similar amount of wild-type (WT) CCR5 (28, 43). Error bars are standard errors of the means for CCR5(G163R) (n = 4) or the range for wild-type CCR5 (n = 2). Relative infectivity values were obtained by dividing the titer from a given time point by the titer obtained in cells with wild-type CCR5 at the final time point (360 min). The data were analyzed according to equation 7, and lag times for the viruses were ~5 min in cells with wild-type CCR5 and ~48 min in cells with CCR5(G163R). It was not possible to calculate the other kinetic parameters since the ki values are different for the two coreceptors.

 


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  		FIG. 4. TAK-779 effects on infection efficiencies and infection kinetics. (A) Efficacy of TAK-779 inhibition. HeLa-CD4 cells expressing high or low concentrations of CCR5 were infected by wild-type HIV-1JR-CSF in the absence or presence of serial fivefold TAK-779 dilutions. Relative infectivities were calculated by dividing the titer in a given cell line at each inhibitor concentration by the titer in the same cell line in the absence of inhibitor. Error bars are standard errors of the means (n = 6). (B) Effect of TAK-779 on infection kinetics. A moderately inhibiting concentration (i.e., the 50% inhibitory concentration obtained in low-CCR5 cells in our inhibitor efficiency assays) of TAK-779 was included in medium added to HeLa-CD4 cells expressing low or high CCR5 concentrations after spinoculation at 4°C with wild-type HIV-1JR-CSF. Control cultures that lacked TAK-779 were analyzed in parallel. Relative infectivity values were generated by dividing the titer obtained at each time point by the titer obtained in the absence of inhibitor in HeLa-CD4 cells expressing a large amount of CCR5 at the final time point (360 min). Data points are the averages from a single representative experiment performed in duplicate, and error bars are the range.

 
Effects of entry inhibitors on efficiencies and kinetics of infections. To further characterize the inhibitors used for the studies described above, we employed our cell clones that express low or high concentrations of wild-type CCR5. As shown in Fig. 4A, TAK-779 is much more inhibitory on the cells with a limiting CCR5 concentration than on cells with an excess of CCR5. Moreover, the inhibition curves for these two cell clones are shifted by a constant amount of approximately 15-fold on the TAK-779 concentration axis. This is compatible with previous evidence of Reeves et al. (46) and Dragic et al. (14) that TAK-779 is a competitive inhibitor of virus binding. Our kinetic analysis supports the idea that it is a pure competitive inhibitor of CCR5 that reduces {alpha} specifically, because at a moderate concentration it increases the lag time and inhibits infection of the low-CCR5 cells but has no detectable effect on the high-CCR5 cells (Fig. 4B). If TAK-779 caused any reduction in ki, we would expect to see some inhibition for the high-CCR5 cells in Fig. 4B and a convergence of the inhibition curves at high TAK-779 concentrations in Fig. 4A. By these criteria, the 2D7 monoclonal antibody, which binds to CCR5, is a partial competitive inhibitor that also inhibits ki (results not shown).

In agreement with previous evidence (46) and with our kinetic model, T-20 sensitivity curves are shifted slightly (i.e., about fourfold) for the cell clones that have high or low CCR5 concentrations (Fig. 5A). This difference in T-20 sensitivity corresponds within experimental error to the relative magnitudes of the {alpha} factors in these cells as determined by our kinetic analysis in Fig. 2 (i.e., these data implied that {alpha} is ~1 for the high-CCR5 cells and ~0.23 for the low-CCR5 cells). This quantitatively supports previous evidence that T-20 is an irreversible inhibitor that specifically binds to the three-stranded prehairpin intermediate that transiently forms in gp41 and with the hypothesis that its potency may be proportional to the lifetime of this intermediate (46) (see equation 16 in the supplemental material). As implied by equation 2 of our kinetic model and by the reversibility of the association process, when the CCR5 concentration is limiting, it will intermittently dissociate from the intermediate, thereby prolonging its life span and slowing its conversion into the final fusion-active conformation (see Discussion) (36). This reversibility is strongly suggested by the fact that TAK-779 blocks viral infections rapidly and efficiently even long after the lag phase. Because of this continuing mobile equilibrium, each virus will spend only a portion, {alpha}, of its time in competent complexes that are capable of progressing through the CCR5-dependent entry steps, and the lifetimes of the intermediates will therefore be inversely proportional to {alpha} (see equation 16 in the supplemental material). As shown by our kinetic analysis in Fig. 5B and by similar studies at other T-20 concentrations, this inhibitor had no effect on the assembly process as indicated by its lack of a significant or reproducible influence on the lag phases preceding onset of infections. It inhibited infections somewhat more efficiently with the low-CCR5 cells than with the cells that had more CCR5. Thus, it inhibited the final infectivity values by ~30% for the high-CCR5 cells and by ~70% for the low-CCR5 cells (Fig. 5B). Similarly, in other conditions it reduced these infectivities by ~35% for the high CCR5 cells and by ~60% for the low CCR5 cells (results not shown). These relatively small effects are consistent with the magnitudes of the differences seen in Fig. 5A.



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  		FIG. 5. T-20 effects on infectivity efficiencies and infection kinetics. (A) Efficacy of T-20 inhibition. Infections of HeLa-CD4 cells expressing high or low concentrations of CCR5 were performed in the absence or presence of serial fivefold dilutions of T-20. Relative infectivities were calculated by dividing the titer from a given cell line at each T-20 concentration by the titer in the same cell line in the absence of T-20. Results of a representative experiment performed in duplicate are shown; error bars are the range. (B) Effect of T-20 on infection kinetics. A moderately inhibiting concentration of T-20 was included in the medium of cells expressing low or high concentrations of CCR5 after virus spinoculation and washing. Control cultures that lacked T-20 were analyzed in parallel. Relative infectivity values were obtained by dividing the titer at a given time point by the titer obtained in the absence of T-20 at the final time point (360 min) in cells expressing large amounts of CCR5. Data points represent the averages from a single representative experiment performed in duplicate, and error bars are the range.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Kinetic factors control efficiencies of HIV-1 entry into cells. Our studies using HeLa-CD4/CCR5 cell clones that have different amounts of wild-type or mutant CCR5 strongly support the hypothesis that the efficiencies of infectious R5 HIV-1 entry into cells are governed by three major kinetic processes that we can quantitatively measure (see equations 1 to 8). First, in an assembly process, the virus reversibly associates with CD4 and CCR5 to form an equilibrium assemblage of complexes. Depending on the concentration of CCR5 and its affinity for the virus, a certain proportion, {alpha}, of these complexes contain sufficient CCR5 at any moment to progress further along the entry pathway. We term these the competent complexes. This assembly process can also be competitively inhibited by TAK-779, which binds to CCR5 and reduces the pool available to HIV-1 (Fig. 4). Although the assembly process is a prerequisite for subsequent steps of infection, it is important to understand that it is a mobile equilibrium that can be rapidly and efficiently reversed at any time by adding TAK-779. The second process we call postassembly, although it clearly also requires reversible associations of CCR5 as indicated by its rapid blockade by additions of TAK-779. This process involves several irreversible conformational changes in gp41, including conversion of a three-stranded coil into a six-helix trimer of hairpins. The lifetime of the three-stranded prehairpin conformation closely correlates with the sensitivity of the infection to inhibition by T-20, which binds irreversibly to this intermediate (see Results and equation 16 in the supplemental material) (36, 46). This lifetime is also dramatically influenced by the gp120 V3 loop (Fig. 1). The third process is a competing process of viral inactivation. Our results suggest that the viral inactivation process is constitutive and independent of the CCR5 concentrations on the cells (see equations 1 to 8 and the supplemental material). Thus, the overall efficiency of successful viral entry appears to be kinetically determined by a competitive race between the membrane fusion pathway and a viral inactivation process.

Although it is formally convenient to describe the assembly and postassembly processes as distinct, it is critical to understand that the viral complexes are in a mobile equilibrium throughout their lifetimes on the cell surfaces. Consequently, even when the fraction {alpha} of viable virions that are in competent complexes at any instant is small (e.g., when the CCR5 concentration or its affinity for the virus is low), all of the virions spend a proportion, {alpha}, of their time in competent complexes, and during that interval they may enter into the postassembly stages. However, their progression through the postassembly steps will also be interrupted occasionally due to dissociation of CCR5. Thus, when {alpha} is low the virions all still progress through the postassembly steps, but their rate of progression will be lowered because they are in fully assembled competent complexes only a fraction {alpha} of the time. Therefore, the rates at which virions progress through the CCR5-dependent postassembly steps are expected to be proportional to {alpha}, as reflected in equation 2 of our derivation. Conversely, the lifetimes of the intermediates should be inversely proportional to {alpha}. This model is quantitatively consistent with the magnitude of the T-20 sensitivity increase that occurs when coreceptor concentrations are reduced (Fig. 5A; see equation 16 in the supplemental material), in agreement with previous reports (36, 46).

There were several surprising aspects of our kinetic results. One is the rapidity and predominance of the viral inactivation process, which occurs at a rate of ~0.47/h in HeLa-CD4/CCR5 cells. In contrast, the productive entry pathway occurs at a rate of only ~0.09/h when the CCR5 concentration is saturating. Consequently, we estimate that only ~15% of the potentially infectious HIV-1 virions that initially attach to these cells (as defined in Materials and Methods) escape the inactivation process and successfully complete the entry pathway and fuse with the cell surface membrane. At the lower CCR5 concentration in our assays, the entry rate is lower and only ~3% of the potentially viable virions successfully enter the cells. As we discuss below, these results are quantitatively consistent with the rapid rate of HIV-1 endocytosis, which was previously proposed to cause virus inactivation (3, 17, 21, 34, 35, 48). A second important feature of our results also concerns the low rate of productive HIV-1 entry. Specifically, as mentioned above, our data indicate that the composite rate constant for entry is ~0.09/h and thus that only ~9% of fully assembled competent complexes would successfully enter the cells per h if there were no competing inactivation process. In contrast, our kinetic assays also show that the lag time required to complete the CCR5-dependent steps of the entry process is only ~7 min when the CCR5 concentration is saturating (Fig. 2). Thus, the minimum time required to transit through the postassembly steps is short (i.e., no longer than ~7 min) despite the fact that only a small percentage of the saturated viral complexes complete this transit per hour. This difference strongly suggests that the assembly of competent complexes must lower but not eliminate a large activation energy barrier that limits a step in the postassembly process. Although only a small fraction of the fully saturated viral complexes overcome the residual barrier per hour at 37°C, the virions that overcome this barrier complete the CCR5-dependent steps within ~7 min. We conclude that there is a rate-limiting CCR5-dependent step in the entry pathway that is very slow even when CCR5 is present in saturating amounts. We believe that this insight is important for understanding the mechanisms of viral escape from entry inhibitors (see below).

A major advantage of our approach is that we are able to identify and to measure three major processes that control HIV-1 infection efficiencies in the context of infectious virions with trimeric envelope glycoproteins adsorbed onto cell surfaces. Furthermore, by using cells that contain different amounts of wild-type or mutant CCR5, we also can learn how entry inhibitors or mutations in the coreceptor or in the virus alter these three processes. We have recently exploited these advantages to investigate the mechanisms of adaptive viral mutations that enable HIV-1JR-CSF to efficiently use CCR5({Delta}18), a derivative that lacks the normal tyrosine sulfate-containing amino terminus of the coreceptor (42).

Despite these advantages, we emphasize that there are several complexities in our present analyses. For example, we reproducibly have found that the wild-type HIV-1 titers in our low-CCR5 cells are relatively more efficient when the virions diffuse slowly and asynchronously onto the cells at 37°C (as in Fig. 1) than when the adsorption occurs by spinoculation at 4°C (as in Fig. 2). One possible interpretation is that the rapid synchronous adsorption of multiple virions by spinoculation might reduce the concentration of free CCR5 on these cells. Alternatively, such a reduction might be caused by the centrifugation or by the temperature shift. An additional complexity occurs because some of the virions spinoculated onto the culture dishes might attach to the extracellular matrix or other sites that prevent their synchronous participation in the entry pathway after warming to 37°C. Subsequent migration of such virions onto the cell surfaces can apparently result in secondary slopes in our kinetic assays, but we have found that this can be minimized by rinsing the cultures after the spinoculation. We believe that the conclusions we have made would not be affected by any of these complicating factors. However, we are currently attempting to modify our methods to improve their accuracy and utility.

Adaptive viral mutations. Because the inactivation process occurs relatively rapidly, a small change in the rate of entry has a major effect on the efficiency of infection. In addition, our data imply that the efficiency of infectious HIV-1JR-CSF entry is severely limited due to a very slow CCR5-dependent postassembly step that probably involves a conformational change in gp41 from a three-stranded prehairpin into a six-helix bundle, also called a trimer of hairpins (see above). This step is very slow even when the virus is saturated with wild-type CCR5, and it is further slowed when the CCR5 concentration is subsaturating as described above. Therefore, we believe that this step is limiting in all of the diverse conditions we have studied. These considerations suggest that adaptive viral mutations are likely to be most effective in overcoming entry inhibitors or other limitations if they lower the activation energy barrier and therefore accelerate this rate-limiting step in entry. Moreover, our data suggest that single mutations in the V3 or V4 loop of gp120 can dramatically increase the rate of this entry step (Fig. 1B) (42). Wild-type HIV-1 utilization of the mutant coreceptor CCR5(Y14N) is extremely inefficient (Fig. 1A), and this inefficiency correlates with a large increase in sensitivity to T-20 (Fig. 1B). Adaptive mutations in the V3 loop of gp120 enhance utilization of CCR5(Y14N) and cause a decrease in T-20 sensitivity without substantially increasing the affinities of the virus for the mutant coreceptor (Fig. 1). The simplest interpretation of these results is that the V3 loop has a large effect on the activation energy barrier that limits the rate of this conformational change in gp41. Similarly, adaptive mutations that counteract effects of diverse entry inhibitors also seem to generally involve V3 loop mutations that may accelerate this postassembly step (30, 33). Previous evidence has focused on the role of the V3 loop in influencing gp120 binding to coreceptors and in controlling coreceptor choice (9, 11, 30, 53). Our results strongly suggest that the role of the V3 loop in coreceptor utilization and choice might principally involve the kinetic mechanism suggested by our results.

The virus inactivation process. Although our studies demonstrate the importance of a viral inactivation process in determining the efficiency of infectious HIV-1 entry into cells and provide a measurement of its high rate, we have not obtained specific information concerning the inactivation mechanism. Conceivably, several processes, including spontaneous inactivation and dissociation, could contribute. However, previous evidence suggests that the inactivation may principally involve endocytosis and lysosomal degradation of the virions (34, 48). Endocytosis is a kinetically prominent process that is believed to cause HIV-1 inactivation both in HeLa-CD4 cells and in T lymphocytes (34, 48, 49). Indeed, approximately 90% of the HIV-1 virions that attach to these cells are endocytosed (34, 49). Moreover, the endocytosis appears to be constitutive and independent of whether the cells contain coreceptors or whether the virions contain gp120-gp41 complexes (3, 34, 35, 48). Similarly, our results suggest that approximately 85% of HIV-1 virions that adsorb onto our optimally susceptible HeLa-CD4/CCR5 cell clone JC.53 become inactivated in a process that kinetically competes with successful infection. In a cell clone with a lower concentration of CCR5, approximately 97% of the infectious virions are inactivated. It should be noted, however, that most previous studies monitored HIV-1 particle endocytosis, whereas we are specifically investigating the factors that control entry of infectious virions. Further investigations will be needed to evaluate the role of endocytosis in the inactivation process implied by our experiments.


   ACKNOWLEDGMENTS
 
This research was supported by NIH grant CA67358.

We appreciate the supply of TAK-779 and T-20 from the NIH AIDS Research and Reference Reagent Program. We thank our colleagues Kristine Rose, Susan Kozak, Mariana Marin, and Sheetal Golem for encouragement and helpful suggestions.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97239-3098. Phone: (503) 494-8442. Fax: (503) 494-8393. E-mail: kabat{at}ohsu.edu. Back

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/. Back


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