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. 1999 Feb;73(2):976-84.
doi: 10.1128/JVI.73.2.976-984.1999.

Changes in human immunodeficiency virus type 1 envelope glycoproteins responsible for the pathogenicity of a multiply passaged simian-human immunodeficiency virus (SHIV-HXBc2)

M Cayabyab  1 , G B Karlsson, B A Etemad-Moghadam, W Hofmann, T Steenbeke, M Halloran, J W Fanton, M K Axthelm, N L Letvin, J G Sodroski
Affiliations

Changes in human immunodeficiency virus type 1 envelope glycoproteins responsible for the pathogenicity of a multiply passaged simian-human immunodeficiency virus (SHIV-HXBc2)

M Cayabyab et al. J Virol. 1999 Feb.

Abstract

In vivo passage of a poorly replicating, nonpathogenic simian-human immunodeficiency virus (SHIV-HXBc2) generated an efficiently replicating virus, KU-1, that caused rapid CD4(+) T-lymphocyte depletion and AIDS-like illness in monkeys (S. V. Joag, Z. Li, L. Foresman, E. B. Stephens, L.-J. Zhao, I. Adany, D. M. Pinson, H. M. McClure, and O. Narayan, J. Virol. 70:3189-3197, 1996). The env gene of the KU-1 virus was used to create a molecularly cloned virus, SHIV-HXBc2P 3.2, that differed from a nonpathogenic SHIV-HXBc2 virus in only 12 envelope glycoprotein residues. SHIV-HXBc2P 3.2 replicated efficiently and caused rapid and persistent CD4(+) T-lymphocyte depletion in inoculated rhesus macaques. Compared with the envelope glycoproteins of the parental SHIV-HXBc2, the SHIV-HXBc2P 3.2 envelope glycoproteins supported more efficient infection of rhesus monkey peripheral blood mononuclear cells. Both the parental SHIV-HXBc2 and the pathogenic SHIV-HXBc2P 3.2 used CXCR4 but none of the other seven transmembrane segment receptors tested as a second receptor. Compared with the parental virus, viruses with the SHIV-HXBc2P 3.2 envelope glycoproteins were more resistant to neutralization by soluble CD4 and antibodies. Thus, changes in the envelope glycoproteins account for the ability of the passaged virus to deplete CD4(+) T lymphocytes rapidly and specify increased replicative capacity and resistance to neutralization.

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Figures

FIG. 1
FIG. 1
Sequence of the KU-1 and SHIV-HXBc2P 3.2 viruses. (A) The predicted amino acid sequence of the envelope glycoproteins of the KU-1 virus, based on the consensus sequence of the env fragment amplified from KU-1-infected CEMx174 cells, is aligned with the HXBc2 sequence. The predicted sequence of the molecularly cloned HXBc2P 3.2 envelope glycoproteins is also shown. Residues in which differences are found between HXBc2 and the KU-1/HXBc2P 3.2 sequences are highlighted. The positions of the gp120 and gp41 boundaries are shown, as are the locations of the gp120 variable regions. Sequences in which the KU-1-associated changes result in the acquisition (∗) or loss (∗∗) of potential N-linked glycosylation are indicated. (B) The Vpu+ SHIV-HXBc2 genome is shown, with the sequences derived from HIV-1 (HXBc2 strain) in white and those from SIVmac239 in black. The positions of the 12 amino acid changes in the SHIV-HXBc2P 3.2 provirus are indicated by black arrows.
FIG. 2
FIG. 2
Replication of viruses in activated rhesus monkey PBMC and blood-derived macrophages. (A) Activated rhesus monkey PBMC were either mock infected or infected with the uncloned KU-1 virus or the molecularly cloned SHIV-HXBc2 or SHIV-HXBc2P 3.2 viruses. Culture supernatants were harvested on the days indicated and assayed for reverse transcriptase activity. The results shown are representative of two independent experiments. (B) Blood-derived macrophages from a rhesus monkey were cultured for 2 weeks as described in Materials and Methods and then infected with the indicated SHIV and SIV viruses. Culture supernatants were harvested and assayed for p27 protein. Similar results were obtained in two independent experiments, one of which is shown here. (C) The CAT activity in the lysates of activated rhesus monkey PBMC infected with recombinant SHIV-CAT containing either no envelope glycoproteins (ΔK/S) or the HXBc2 or HXBc2P 3.2 envelope glycoproteins. Two different amounts of the latter viruses were used for infection. The amount (either 10,000 or 30,000 reverse transcriptase units) of each virus used for infection is indicated.
FIG. 3
FIG. 3
Coreceptor use by the HXBc2 and HXBc2P 3.2 envelope glycoproteins. (A) The figure shows the CAT activity in the lysates of Cf2Th canine thymocytes expressing CD4 only, both CD4 and CCR5, or both CD4 and CXCR4 after incubation with recombinant SHIV-CAT containing the HXBc2, HXBc2P 3.2 or SIVmac239 envelope glycoproteins. The percentage of chloramphenicol acetylated in each experiment is indicated. (B) The amount of CAT activity in the lysates of rhesus PBMC after incubation with viruses containing the HXBc2 or HXBc2P 3.2 envelope glycoproteins is shown. The target PBMC were incubated in the absence or presence of 5 μg of SDF-1 per ml. Similar results were obtained with PBMC from three different monkey donors.
FIG. 4
FIG. 4
Infection of rhesus macaques with SHIV-HXBc2P 3.2. The average amount of p27 antigen in the plasma of rhesus macaques inoculated intravenously with SHIV-HXBc2 and SHIV-HXBc2P 3.2 is shown for the first 5 weeks after infection. The average absolute number of CD4+ T lymphocytes in the peripheral blood of SHIV-HXBc2- and SHIV-HXBc2P 3.2-infected macaques is shown. Results obtained in four SHIV-HXBc2-infected (previously shown in reference 41) and three SHIV-HXBc2P 3.2-infected monkeys are indicated. Error bars show the standard deviation of the combined animal experiments.
FIG. 5
FIG. 5
Sensitivity of viruses with the HXBc2 and HXBc2P 3.2 envelope glycoproteins to neutralization by sCD4 and antibodies. In panel A, the level of infectivity of recombinant viruses containing either HXBc2 or HXBc2P 3.2 into CEMx174 cells is shown as the percentage of chloramphenicol conversion. Recombinant viruses containing either the HXBc2 or HXBc2P 3.2 envelope glycoproteins were incubated with sCD4 (B), the F105 antibody (C), the IgG1b12 antibody (D), or the AG1121 antibody (E) at the indicated concentrations prior to infection of CEMx174 cells. The results shown are representative of two independent experiments. In panel F, lysates of COS-1 cells expressing either the HXBc2 or HXBc2P 3.2 envelope glycoproteins were precipitated with pooled sera from HIV-1-infected individuals (PS) or with monoclonal antibodies. The 97.4 and 200-kDa protein molecular size markers (M) are shown. The gp160 and gp120 envelope glycoproteins are indicated.
FIG. 5
FIG. 5
Sensitivity of viruses with the HXBc2 and HXBc2P 3.2 envelope glycoproteins to neutralization by sCD4 and antibodies. In panel A, the level of infectivity of recombinant viruses containing either HXBc2 or HXBc2P 3.2 into CEMx174 cells is shown as the percentage of chloramphenicol conversion. Recombinant viruses containing either the HXBc2 or HXBc2P 3.2 envelope glycoproteins were incubated with sCD4 (B), the F105 antibody (C), the IgG1b12 antibody (D), or the AG1121 antibody (E) at the indicated concentrations prior to infection of CEMx174 cells. The results shown are representative of two independent experiments. In panel F, lysates of COS-1 cells expressing either the HXBc2 or HXBc2P 3.2 envelope glycoproteins were precipitated with pooled sera from HIV-1-infected individuals (PS) or with monoclonal antibodies. The 97.4 and 200-kDa protein molecular size markers (M) are shown. The gp160 and gp120 envelope glycoproteins are indicated.
FIG. 5
FIG. 5
Sensitivity of viruses with the HXBc2 and HXBc2P 3.2 envelope glycoproteins to neutralization by sCD4 and antibodies. In panel A, the level of infectivity of recombinant viruses containing either HXBc2 or HXBc2P 3.2 into CEMx174 cells is shown as the percentage of chloramphenicol conversion. Recombinant viruses containing either the HXBc2 or HXBc2P 3.2 envelope glycoproteins were incubated with sCD4 (B), the F105 antibody (C), the IgG1b12 antibody (D), or the AG1121 antibody (E) at the indicated concentrations prior to infection of CEMx174 cells. The results shown are representative of two independent experiments. In panel F, lysates of COS-1 cells expressing either the HXBc2 or HXBc2P 3.2 envelope glycoproteins were precipitated with pooled sera from HIV-1-infected individuals (PS) or with monoclonal antibodies. The 97.4 and 200-kDa protein molecular size markers (M) are shown. The gp160 and gp120 envelope glycoproteins are indicated.
FIG. 5
FIG. 5
Sensitivity of viruses with the HXBc2 and HXBc2P 3.2 envelope glycoproteins to neutralization by sCD4 and antibodies. In panel A, the level of infectivity of recombinant viruses containing either HXBc2 or HXBc2P 3.2 into CEMx174 cells is shown as the percentage of chloramphenicol conversion. Recombinant viruses containing either the HXBc2 or HXBc2P 3.2 envelope glycoproteins were incubated with sCD4 (B), the F105 antibody (C), the IgG1b12 antibody (D), or the AG1121 antibody (E) at the indicated concentrations prior to infection of CEMx174 cells. The results shown are representative of two independent experiments. In panel F, lysates of COS-1 cells expressing either the HXBc2 or HXBc2P 3.2 envelope glycoproteins were precipitated with pooled sera from HIV-1-infected individuals (PS) or with monoclonal antibodies. The 97.4 and 200-kDa protein molecular size markers (M) are shown. The gp160 and gp120 envelope glycoproteins are indicated.
FIG. 5
FIG. 5
Sensitivity of viruses with the HXBc2 and HXBc2P 3.2 envelope glycoproteins to neutralization by sCD4 and antibodies. In panel A, the level of infectivity of recombinant viruses containing either HXBc2 or HXBc2P 3.2 into CEMx174 cells is shown as the percentage of chloramphenicol conversion. Recombinant viruses containing either the HXBc2 or HXBc2P 3.2 envelope glycoproteins were incubated with sCD4 (B), the F105 antibody (C), the IgG1b12 antibody (D), or the AG1121 antibody (E) at the indicated concentrations prior to infection of CEMx174 cells. The results shown are representative of two independent experiments. In panel F, lysates of COS-1 cells expressing either the HXBc2 or HXBc2P 3.2 envelope glycoproteins were precipitated with pooled sera from HIV-1-infected individuals (PS) or with monoclonal antibodies. The 97.4 and 200-kDa protein molecular size markers (M) are shown. The gp160 and gp120 envelope glycoproteins are indicated.
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