Membrane-fusing capacity of the human immunodeficiency virus envelope proteins determines the efficiency of CD+ T-cell depletion in macaques infected by a simian-human immunodeficiency virus

Abstract

The mechanism of the progressive loss of CD4+ T lymphocytes, which underlies the development of AIDS in human immunodeficiency virus (HIV-1)-infected individuals, is unknown. Animal models, such as the infection of Old World monkeys by simian-human immunodeficiency virus (SHIV) chimerae, can assist studies of HIV-1 pathogenesis. Serial in vivo passage of the nonpathogenic SHIV-89.6 generated a virus, SHIV-89.6P, that causes rapid depletion of CD4+ T lymphocytes and AIDS-like illness in monkeys. SHIV-KB9, a molecularly cloned virus derived from SHIV-89.6P, also caused CD4+ T-cell decline and AIDS in inoculated monkeys. It has been demonstrated that changes in the envelope glycoproteins of SHIV-89.6 and SHIV-KB9 determine the degree of CD4+ T-cell loss that accompanies a given level of virus replication in the host animals (G. B. Karlsson et. al., J. Exp. Med. 188:1159-1171, 1998). The envelope glycoproteins of the pathogenic SHIV mediated membrane fusion more efficiently than those of the parental, nonpathogenic virus. Here we show that the minimal envelope glycoprotein region that specifies this increase in membrane-fusing capacity is sufficient to convert SHIV-89.6 into a virus that causes profound CD4+ T-lymphocyte depletion in monkeys. We also studied two single amino acid changes that decrease the membrane-fusing ability of the SHIV-KB9 envelope glycoproteins by different mechanisms. Each of these changes attenuated the CD4+ T-cell destruction that accompanied a given level of virus replication in SHIV-infected monkeys. Thus, the ability of the HIV-1 envelope glycoproteins to fuse membranes, which has been implicated in the induction of viral cytopathic effects in vitro, contributes to the capacity of the pathogenic SHIV to deplete CD4+ T lymphocytes in vivo.

FIG. 1

Structure and biological properties of the HIV-1 envelope glycoproteins used in this study. The envelope glycoprotein ectodomains of the SHIV variants used in this study are depicted. The gp41 ectodomain is shaded gray. The vertical bars represent the positions of amino acids that are altered in the pathogenic SHIV-KB9, compared with the parental SHIV-89.6 (31). The residue number is indicated beneath the diagram, with numbering according to recommended convention (37). The gp120 variable (V1 to -4) and conserved (C2) regions in which the residues are located are indicated. The SHIV-89.6, SHIV-89.6*, and SHIV-KB9ct envelope glycoprotein ectodomains are identical, as are those of SHIV-KB9 and SHIV-KB9ecto. The relative membrane-fusing ability of the envelope glycoproteins was determined by cocultivating cells expressing comparable levels of surface envelope glycoproteins with CEMx174 lymphocytes for 6 h at 37°C. The number of syncytia formed was counted and normalized to that seen for the 89.6 envelope glycoproteins (15). The relative ability of the gp120 glycoproteins to bind to cells expressing the CCR5 chemokine receptor in the presence of soluble CD4 was determined as described previously (15, 32). The binding obtained for the 89.6 gp120 glycoprotein was assigned a value of 1. Relative CD4-binding ability was evaluated as described previously (32) and was normalized to that of the 89.6 gp120 glycoprotein.

FIG. 2

Syncytium-forming ability of the HIV-1 envelope glycoprotein variants. COS-1 cells expressing equivalent surface levels of the indicated HIV-1 envelope glycoproteins were cocultivated at 37°C with CEMx174 CD4⁺ lymphocytes, and syncytia were scored after 6 h as described previously (15). Control COS-1 cells were transfected with the ΔKS plasmid, which does not express functional envelope glycoproteins. Means and standard deviations of duplicate experiments are indicated.

FIG. 3

CD4⁺ T-lymphocyte counts in rhesus monkeys infected with SHIV variants. Two rhesus monkeys were inoculated intravenously with SHIV-89.6* (▿, ▾), SHIV-KB9 (○, ●), or SHIV-89.6(+225–567) (□, ■). The absolute CD4⁺ T-lymphocyte counts in the peripheral blood of the infected monkeys are shown, with normal values in uninfected animals ranging from 1,000 to 1,400 cells per microliter.

FIG. 4

Relationship between viremia and CD4⁺ T-lymphocyte counts in monkeys infected with SHIV variants. The set point value for the peripheral blood CD4⁺ T-cell counts was plotted against the cumulative p27 antigenemia for each infected animal. The curves were fitted to the data using a nonlinear exponential decay model with a viral group-replication interaction term (23, 67). A single curve was fitted to the data for SHIV-KB9 and SHIV-KB9ecto, which both contain KB9 envelope glycoprotein ectodomains; likewise, a single curve was fitted to the data for SHIV-89.6* and SHIV-KB9ct, which contain the 89.6 envelope glycoprotein ectodomains.

FIG. 5

Ratio of viremia to the number of virus-producing cells in macaques infected with SHIV variants differing in membrane fusogenicity. The ratio of the cumulative antigenemia to the number of viral RNA-positive cells measured in the lymph nodes on day 10 after infection is shown for macaques infected with SHIV variants with less fusogenic envelope glycoproteins [89.6*, KB9ct, KB9(−225), and KB9(−305)], compared with that for SHIV variants with more fusogenic envelope glycoproteins (KB9 and KB9ecto). In the three instances in which viral RNA-positive cells in the lymph nodes were undetectable, the lowest detectable value, 0.12, was used to calculate the ratio. The median values for the two groups are 1.96 and 0.32, respectively (P = 0.0006; Wilcoxon rank sum test).