HIV-1 non-macrophage-tropic R5 envelope glycoproteins are not more tropic for entry into primary CD4+ T-cells than envelopes highly adapted for macrophages

Abstract

Background: Non-mac-tropic HIV-1 R5 viruses are predominantly transmitted and persist in immune tissue even in AIDS patients who carry highly mac-tropic variants in the brain. Non-mac-tropic R5 envelopes (Envs) require high CD4 levels for infection contrasting with highly mac-tropic Envs, which interact more efficiently with CD4 and mediate infection of macrophages that express low CD4. Non-mac-tropic R5 Envs predominantly target T-cells during transmission and in immune tissue where they must outcompete mac-tropic variants. Here, we investigated whether Env+ pseudoviruses bearing transmitted/founder (T/F), early and late disease non-mac-tropic R5 envelopes mediated more efficient infection of CD4+ T-cells compared to those with highly mac-tropic Envs.

Results: Highly mac-tropic Envs mediated highest infectivity for primary T-cells, Jurkat/CCR5 cells, myeloid dendritic cells, macrophages, and HeLa TZM-bl cells, although this was most dramatic on macrophages. Infection of primary T-cells mediated by all Envs was low. However, infection of T-cells was greatly enhanced by increasing virus attachment with DEAE dextran and spinoculation, which enhanced the three Env+ virus groups to similar extents. Dendritic cell capture of viruses and trans-infection also greatly enhanced infection of primary T-cells. In trans-infection assays, non-mac-tropic R5 Envs were preferentially enhanced and those from late disease mediated levels of T-cell infection that were equivalent to those mediated by mac-tropic Envs.

Conclusions: Our results demonstrate that T/F, early or late disease non-mac-tropic R5 Envs do not preferentially mediate infection of primary CD4+ T-cells compared to highly mac-tropic Envs from brain tissue. We conclude that non-macrophage-tropism of HIV-1 R5 Envs in vitro is determined predominantly by a reduced capacity to target myeloid cells via low CD4 rather than a specific adaptation for T-cells entry that precludes macrophage infection.

Figure 1

Env+ pseudovirion infection of HeLa TZM-bl and macrophages. (A) Macrophage-tropic R5 Envs mediate 100-1000-fold higher infection of macrophages compared to T/F/acute and late disease non-mac-tropic R5 Envs. (B) Several late disease non-mac-tropic Envs and most T/F/acute Envs mediated lower infection of HeLa TZM-bl cells compared to macrophage tropic Envs. (C) Plotting macrophage infectivity titers as a percent of TZM-bl titers suggests that T/F/acute Envs are not as non-mac-tropic as late disease non-mac-tropic Envs. Significant differences were evaluated using Mann Whitney or Wilcoxon matched pair tests as described in Materials and Methods. Pseudoviruses carrying clade B Envs are represented by black symbols, those with clade C Envs by light gray symbols, clade A by hatched symbols and CRF A/C by dark gray. Median values are marked.

Figure 2

Infection of Jurkat/CCR5, primary CD4+ T-cells and primary MDDCs by pseudovirions carrying mac-tropic and non-mac-tropic R5 envelopes. Env+ GFP-reporter pseudovirus infection of Jurkat/CCR5 (A), primary CD4+ T-cells (B) and LPS matured MDDCs (C). Left panels shows FFU measured as GFP+ cells per ml of input virus. Right panels show infectivity as a percent of TZM-bl infectivity. Significant differences were evaluated using Mann Whitney or Wilcoxon matched pair tests as described in Methods. Only the p values that were significant are shown. Symbol colour designations are the same as described in Figure 1. Please also refer to Additional file 1: Figure S3, where FFU/ml for each Env+ pseudovirus is presented in bar graphs with standard deviation bars shown, while the column scatter plot of infectivity as a percent of TZM-bl infectivity is presented with each point labeled for Env used. Median values are marked.

Figure 3

DEAE dextran and spinoculation enhance infection for CD4+ T-cells but not for MDDCs. Infection in the presence and absence of DEAE dextran and spinoculation on CD4+ T-cells (A) and MDDCs (B). DEAE dextran and spinoculation enhance non-mac-tropic Envs and mac-tropic Envs to similar extents (C). Significant differences were evaluated using Mann Whitney or Wilcoxon matched pair tests as described in Materials and Methods. Figure 3 experiments were done in conjunction with the direct infections shown in Figure 2. Figure 3 data for infection without DEAE dextran and spinoculation is therefore the same as that shown in Figure 2. Only the p values relevant for DEAE dextran and spinoculation that show significance are shown. Symbol colour designations are the same as described in Figure 1. Please also refer to Additional file 1: Figure S4, where FFU/ml for each Env+ pseudovirus is presented in a bar graph with standard deviation bars shown, while the column scatter plot of infectivity as a percent of TZM-bl infectivity is presented with each point labeled for Env used. Median values are marked.

Figure 4

MDDC mediated trans-infection of CD4+ T-cells by Env+ pseudovirions. (A) Trans-infection of CD4+ T-cells following virion capture by MDDCs is substantially more efficient than cell free infection of T-cells or MDDCs alone. T/F/acute and late disease non-mac- and mac-tropic R5 Env+ pseudovirions are all enhanced. Note that for late disease non-mac-tropic and mac-tropic R5 Envs, trans-infectivity titers are not significantly different. Infectivities are shown as the number of FFU/ml (GFP+ cells per ml) of input virus. (B) Infectivity as a percent of infection for TZM-bl cells suggests that trans-infection preferentially enhances non-mac-tropic R5 Env+ pseudovirions, although differences are not significant. Symbol colour designations are the same as described in Figure 1. Please also refer to Additional file 1: Figure S5, where FFU/ml for each Env+ pseudovirus is presented in a bar graph with standard deviation bars shown, while the column scatter plot of infectivity as a percent of TZM-bl infectivity is presented with each point labeled for Env used. Median values are marked.