Table 1.

Effects of Wild-type and Mutant CCR5s on Activation Energy Barriers that Limit Infections of HIV-1 Isolates.

Virus	CCR5	Maximum Relative Infectivity(irel max)*1	$\mathbf{\Delta }{\mathbf{G}}_{\mathbf{0}\mathbf{rel}}^{\ne }$	$\mathbf{\Delta }{\mathbf{G}}_{\mathbf{1}\mathbf{rel}}^{\ne }$	$\mathbf{\Delta }{\mathbf{G}}_{\mathbf{2}\mathbf{rel}}^{\ne }$	$\mathbf{\Delta }{\mathbf{G}}_{\mathbf{3}\mathbf{rel}}^{\ne }$ *2
Wild-type	wild-type	1.0	(>4.9)*3	--	--	0
Wild-type	HHMH	0.00032	(>4.9)*3	--	--	4.9
F313L	HHMH	0.025	--	--	--	2.3
F313L/N403S	HHMH	0.286	4.0	1.2	0.77	0.77
Fully adapted (S298N/F313L/N403S/A428T)	HHMH	0.43	2.8	1.0	0.59	0.53
Fully adapted	Δ18+ amino terminal peptide	0.51	2.7	0.96	0.40	0.40

^*1i_{rel max} is the maximum infectivity of each virus at saturating concentrations of the CCR5 used relative to the titer in JC.53 cells that express an optimally saturating amount of wild-type CCR5.

^*2 $\mathbf{\Delta }{\mathbf{G}}_{\mathbf{3}\mathbf{rel}}^{\ne }$ is the difference in kcal/mole between the activation free energy barrier that limits infection when the virus is saturated with the test CCR5 and the corresponding barrier when the virus is saturated with three wild-type CCR5s. This value was estimated from the i_{rel max} values for each virus-coreceptor combination as described in Experimental Procedures. Based on a previous estimate that the efficiency of infection by wild-type HIV-1_JRCSF in JC.53 cells is ~0.2 ⁶, we infer that the absolute $\mathbf{\Delta }{\mathbf{G}}_{\mathbf{3}}^{\ne }$ for infection is ~ 1 kcal/mole larger than the relative values recorded in this column. For example, we estimate that there is a residual activation energy barrier of ~1 kcal/mole when the wild-type virus is saturated with wild-type CCR5 and ~5.9 kcal/mole when it is saturated with CCR5(HHMH). Similarly, $\mathbf{\Delta }{\mathbf{G}}_{\mathbf{0}\mathbf{rel}}^{\ne },\mathbf{\Delta }{\mathbf{G}}_{\mathbf{1}\mathbf{rel}}^{\ne }$, and $\mathbf{\Delta }{\mathbf{G}}_{\mathbf{2}\mathbf{rel}}^{\ne }$ are the free energy barriers in kcal/mole when the gp120-CD4 fusion complexes contain 0, 1, or 2 molecules of the test coreceptor. These values were estimated by fitting the data to equation 4 using Kaleidagraph, and using the resulting best-fit parameters in these estimates. Values for these sequential energy barriers were not obtained for the unadapted and poorly adapted viruses because they bind CCR5s in a highly concerted manner that was not amenable to allosteric analysis (see text). The f₁, f₂, f₃, and k curve fitting values derived from use of equation 4 were as follows: 0.01, 0.5, 1.0, and 0.154 ×10⁵/cell for the F313L/N403S virus using CCR5(HHMH), 0.05, 0.5, 0.9, and 0.154 ×10⁵/cell for the fully adapted virus using CCR5(HHMH). The f₁, f₂, f₃ values were 0.06, 0.40, and 1.0 for the fully adapted virus using the peptide plus CCR5(δ18). In that case, the apparent k values were 3.39μM and 8.13μM for the cells with high or lower amounts of CCR5(Δ18), respectively, consistent with the 2.4-fold difference predicted.

^*3This is a minimum estimate of ${\mathbf{G}}_{\mathbf{0}\mathbf{rel}}^{\ne }$ for the wild-type virus-CD4 complex lacking CCR5s. Since the complex saturated with CCR5(HHMH) has a residual activation energy of 4.9 kcal/mole, the unliganded complex must have a barrier at least that large.