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. 2001 Sep;75(18):8498-506.
doi: 10.1128/jvi.75.18.8498-8506.2001.

Human immunodeficiency virus type 1 IIIB selected for replication in vivo exhibits increased envelope glycoproteins in virions without alteration in coreceptor usage: separation of in vivo replication from macrophage tropism

E D Miller  1 , K M Duus, M Townsend, Y Yi, R Collman, M Reitz, L Su
Affiliations

Human immunodeficiency virus type 1 IIIB selected for replication in vivo exhibits increased envelope glycoproteins in virions without alteration in coreceptor usage: separation of in vivo replication from macrophage tropism

E D Miller et al. J Virol. 2001 Sep.

Abstract

Analysis of viral replication and pathogenicity after in vivo selection of human immunodeficiency virus type 1 (HIV-1) attenuated in vitro will help to define the functions involved in replication and pathogenesis in vivo. Using the SCID-hu Thy/Liv mouse and human fetal thymus organ culture as in vivo models, we previously defined HIV-1 env determinants (HXB2/LW) which were reverted for replication in vivo (L. Su et al., Virology 227:46-52, 1997). In this study, we examined the replication of four highly related HIV-1 clones directly derived from Lai/IIIB or after selection in vivo to investigate the envelope gp120 determinants associated with replication in macrophages and in the thymus models in vivo. The LW/C clone derived from the IIIB-infected laboratory worker and HXB2/LW both efficiently infected monocyte-derived macrophages (MDM) and the human thymus. Although the laboratory worker (LW) isolates showed altered tropism from IIIB, they still predominantly used CXCR4 as coreceptors for infecting peripheral blood mononuclear cells, macrophages, and the thymus. Interestingly, a single amino acid mutation in the V3 loop associated with resistance to neutralizing antibodies was also essential for the replication activity of the LW virus in the thymus models but not for its activity in infecting MDM. The LW virions were equally sensitive to a CXCR4 antagonist. We further demonstrated that the LW HIV-1 isolate selected in vivo produced more infectious viral particles that contained higher levels of the Env protein gp120. Thus, selection of the laboratory-attenuated Lai/IIIB isolate in vivo leads to altered tropism but not coreceptor usage of the virus. The acquired replication activity in vivo is correlated with an early A-to-T mutation in the V3 loop and increased virion association of HIV-1 Env gp120, but it is genetically separable from the acquired replication activity in macrophages.

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Figures

FIG. 1
FIG. 1
Replication of LW clones in MDM. (A) Genomic organization of HXB2 and LW-derived HIV-1 clones. In panel a, the HXB2 HIV-1 genome has nonsense mutations within the vpr, vpu, and nef ORFs, denoted by asterisks. A six-amino-acid sequence within the V3 loop is highlighted (white segment). In panel b, HXB2/LW was created by replacing the SalI-BamHI fragment from LW12.3 (striped segment) into an HXB2 background. The V3 loop is in white, with the amino acid change to Thr (T) in bold. In panel c, LW/C is a full-length macrophage-tropic clone derived from the LW isolate LW12.3. LW/C contains repaired vif, vpr, and nef accessory ORFs (28). In panel d, LW/C IIIB is identical to LW/C except for the T-to-A (back to HXB2) change at the V3 loop. (B) Replication of the LW clones in MDM. Monocytes were cultured and infected with each HIV-1 stock. HIV-1 replication was monitored by measuring virion-associated RT activity (3H activity, in counts per minute per milliliter) in the culture medium. Experiments were repeated with independent PBMC donors with similar results.
FIG. 2
FIG. 2
Replication of LW clones in the human thymus. (A) Replication of LW clones in HF-TOC. Shown is the level of p24 present in HF-TOC at 10 to 12 days postinfection (dpi), with HXB2, HXB2/LW, LW/C, LW/CIIIB, NL4-3, and JRCSF. Error bars are standard deviations based on three or more independent experiments. (B) Replication of LW clones in SCID-hu Thy/Liv mice. Shown is the level of p24 present in the SCID-hu Thy-Liv organ at 4 and 6 weeks postinfection (wpi) with HXB2, HXB2/LW, LW/C, LW/CIIIB, and NL4-3. Error bars represent standard deviations.
FIG. 3
FIG. 3
HXB2/LW, LW/C, and LW/CIIIB utilize the same coreceptor (CXCR4) as HXB2. (A) The LW viruses used CXCR4 but not CCR5 as coreceptor for entry. HIV-1 viral supernatants were used to infect the CXCR4- or CCR5-MAGI cells. Each virus was scored for the number of blue cells per milliliter for both cell lines. HXB2, HXB2/LW, LW/C, and LW/CIIIB utilized CXCR4 (gray) as the primary coreceptor. JRCSF, a macrophage-tropic virus, utilized CCR5 (black) for entry. (B) HXB2/LW only utilized CXCR4 for entry into PBMCs. The bicyclam AMD3100, a specific inhibitor of HIV-1 using the CXCR4 coreceptor, was added (15 ng/ml) to the media for the PBMC culture. Shown is AMD3100 inhibition in PBMC cultures at 9 dpi. HIV-1 replication for each virus in the absence of AMD3100 is normalized to 100% (wild-type; black bar). Error bars represent standard deviations. Three independent experiments were performed with similar results. JRCSF was used as a CCR5-tropic HIV-1 resistant to AMD3100 inhibition. (C) HXB2/LW only used CXCR4 as a coreceptor for infection in the human thymus. Shown is the relative replication (based on picograms of p24 per 106 T cells) of each virus with AMD3100 in HF-TOC. HIV-1 replication for each virus in the absence of AMD3100 is normalized to 100% (wild-type; black bar). Relative replication of NL4-3 (checked bar), HXB2/LW (gray bar), and JRCSF (striped bar) in the presence of AMD3100 is presented. Error bars represent standard deviations. Three independent experiments were performed with similar results. JRCSF was used as a CCR5-tropic HIV-1 resistant to AMD3100 inhibition. (D) HXB2/LW Env used only CXCR4 as a coreceptor for infecting macrophages. env genes from HX/LW or the CXCR4-dependent macrophage-tropic strain UG024 were expressed in 293T cells and tested for the ability to mediate fusion with primary human MDM or with U87 cells expressing CD4 alone or CD4 in conjunction with CCR5 or CXCR4. Macrophages were pretreated for 1 h with or without the CXCR4 antagonist AMD3100 (10 μg/ml). Data are expressed as the percentage of fusion relative to untreated MDM or relative to U87/CD4/CXCR4 cells. Error bars are the standard deviations of three independent experiments.
FIG. 4
FIG. 4
HXB2/LW Env shows normal affinity for CXCR4 coreceptor and fusion activity. (A) Serial dilutions of AMD3100 were used to determine the inhibitory dose for HXB2 (black line) and HXB2/LW (dashed line) to infect CXCR4-MAGI cells. Results are indicated as the percentage of replication compared to replication in the absence of AMD3100. Error bars represent standard deviations. (B) The HXB2 env and LW env showed similar fusogenic activity. The IG5 Jurkat cell line with the LTR-luciferase gene was cocultured with 293T cells transfected with HIV-1 Tat and one of the HIV-1 env genes (HXB2, HXB2/LW, or NL4-3). Tat-mediated LTR activation after fusion was measured by luciferase activity (in relative light units [RLU]). Three independent experiments were performed with similar results.
FIG. 5
FIG. 5
HXB2/LW produced more infectious virions with higher levels of Env gp120. (A) The HXB2/LW viral stock contained higher fractions of infectious virions. The infectious virions and total virions from HXB2 or HXB2/LW viral stocks prepared from PBMCs were measured. The relative infectious virions (IU/RT) are presented. Standard deviations are shown as error bars. Three independent experiments were analyzed with similar results. (B) The HXB2/LW and LW/C virions contained higher levels of gp120. Pelleted virions were analyzed by Western blotting with HIV-positive human patient sera. The gp120, gp41, and major gag proteins are indicated. The level of gp120 or gp41for each virus relative to p24 capsid protein was calculated (gp120/p24 or gp41/p24; Table 1).
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