Stoichiometry for entry and binding properties of the Env protein of R5 T cell-tropic HIV-1 and its evolutionary variant of macrophage-tropic HIV-1

Abstract

Human immunodeficiency virus type 1 typically requires a high density of CD4 for efficient entry as a mechanism to target CD4+ T cells (T-tropic), with CCR5 being used most often as the coreceptor. When target T cells are limiting, the virus can evolve to infect cells with a low density of CD4 such as macrophages (M-tropic). The entry phenotype is known to be encoded in the viral Env protein on the surface of the virus particle. Using data showing a dose response for infectivity based on CD4 surface density, we built a model consistent with T-tropic viruses requiring multiple CD4 molecules to mediate infection, whereas M-tropic viruses can infect cells using a single CD4 receptor molecule interaction. We also found that T-tropic viruses bound to the surface of cells with a low density of CD4 are released more slowly than M-tropic viruses which we modeled to be due to multiple interactions of the T-tropic virus with multiple CD4 molecules to allow the initial stable binding. Finally, we found that some M-tropic Env proteins, as the gp120 subunit, possess an enhanced affinity for CD4 compared with their T-tropic pair, indicating that the evolution of macrophage tropism can be reflected both in the closed Env trimer conformation on the virion surface and, in some cases, also in the open confirmation of gp120 Env. Collectively, these studies reveal differences in the stoichiometry of interaction of T-tropic and M-tropic viruses with CD4 and start to identify the basis of binding differences at the biochemical level.

Importance: Human immunodeficiency virus type 1 normally targets CD4+ T cells for viral replication. When T cells are limiting, the virus can evolve to infect myeloid cells. The evolutionary step involves a change from requiring a high surface density of CD4 for entry to being able to infect cells with a low density of CD4, as is found on myeloid lineage cells such as macrophage and microglia. Viruses able to infect macrophages efficiently are most often found in the CNS late in the disease course, and such viruses may contribute to neurocognitive impairment. Here, we examine the CD4 binding properties of the viral Env protein to explore these two different entry phenotypes.

Keywords: CD4; human immunodeficiency virus; macrophage tropism; receptor; stoichiometry; virus entry.

Fig 1

Infection of Affinofile cells expressing low CD4 and high CCR5 levels of surface density. Affinofile cells expressing either a high or low density of CD4 were infected with a luciferase reporter virus with the indicated Env protein pseudotype. Infectivity levels were measured after 48 hours as luciferase activity in the cell lysate. The infectivity at low CD4 density is shown as the percentage of infectivity at high CD4. The experiment represents the result of an infection with two biological replicates for each individual virus.

Fig 2

Model of infectivity based on the number of CD4 molecules required. For T-tropic viruses (upper panels), infection is minimal at low CD4 densities, with significant infectivity apparent at high CD4 density and requiring three CD4 receptors (red lines) to enter cells. In contrast, M-tropic viruses (lower panels) can infect cells efficiently using only one CD4 receptor when CD4 density is low, and these viruses switch to using three CD4 receptors when CD4 density is very high. Values of infectivity from the model are shown using three CD4 molecules (red line) and one CD4 molecule (blue line) with the experimental data shown as points (black circles). A non-linear regression curve fit (black dotted line) through the experimental data points (black circles) is also shown. Total infectivity in the model is the sum of the red and blue lines.

Fig 3

Dissociation of M- and T-tropic viruses from cells expressing a low density of CD4. Infection of Affinofile cells expressing low densities of CD4 on their cellular surface was completed using two (4051 and 4059) M and T luciferase reporter virus pairs. The measured RLUs at the 0-hour time point were set to 100%, and infectivity thereafter for 2, 4, 8, and 24 hours was recorded. The percentage at each time point is a representation of the RLU recorded over the RLU 0-hour time point.

Fig 4

Dissociation model of M- and T-tropic viruses. Schematic (A) and kinetic scheme (B) of two CD4 receptors on an Affinofile cell bound to two trimers of a T-tropic virus, then releasing one trimer from one CD4, with a chance to rebind or subsequently release the second trimer. (C) The local volume of the hemisphere with radius r is used to compute the local concentration of trimer (D). The estimated relationship of the off rate of each trimer plotted as a function of the on rate, using a rapid equilibrium assumption, the apparent decay rate from the data, and the local concentration of trimer is computed in the text.

Fig 5

Analysis of gp120 binding affinity for four-domain CD4 using SPR. Four replicates were completed per sample where experiments were run in duplicate across 2 consecutive days. (A) Individual binding parameters are shown for each gp120 Env protein. (B) The K_D for each measurement of each gp120 Env protein is shown and compared between the M- and T-tropic pairs. Statistical significance testing was done using a Wilcoxon rank-sum test (**P < 0.001). gp120 proteins derived from the M-tropic viruses are in blue, and T-tropic viruses are in red.