Viral interactions in human lymphoid tissue: Human herpesvirus 7 suppresses the replication of CCR5-tropic human immunodeficiency virus type 1 via CD4 modulation

Abstract

Human immunodeficiency virus (HIV) infection is often accompanied by infection with other pathogens that affect the clinical course of HIV disease. Here, we identified another virus, human herpesvirus 7 (HHV-7) that interferes with HIV type 1 (HIV-1) replication in human lymphoid tissue, where critical events of HIV disease occur. Like the closely related HHV-6, HHV-7 suppresses the replication of CCR5-tropic (R5) HIV-1 in coinfected blocks of human lymphoid tissue. Unlike HHV-6, which affects HIV-1 by upregulating RANTES, HHV-7 did not upregulate any CCR5-binding chemokine. Rather, the inhibition of R5 HIV-1 by HHV-7 was associated with a marked downregulation of CD4, the cellular receptor shared by HHV-7 and HIV-1. HHV-7-induced CD4 downregulation was sufficient for HIV-1 inhibition, since comparable downregulation of CD4 with cyclotriazadisulfonamide, a synthetic macrocycle that specifically modulates expression of CD4, resulted in the suppression of HIV infection similar to that seen in HHV-7-infected tissues. In contrast to R5 HIV-1, CXCR4-tropic (X4) HIV-1 was only minimally suppressed by HHV-7 coinfection. This selectivity in suppression of R5 and X4 HIV-1 is explained by a suppression of HHV-7 replication in X4- but not in R5-coinfected tissues. These results suggest that HIV-1 and HHV-7 may interfere in lymphoid tissue in vivo, thus potentially affecting the progression of HIV-1 disease. Knowledge of the mechanisms of interaction of HIV-1 with HHV-7, as well as with other pathogens that modulate HIV-1 replication, may provide new insights into HIV pathogenesis and lead to the development of new anti-HIV therapeutic strategies.

FIG. 1.

Replication of HHV-7 in human lymphoid tissue ex vivo. Tissue blocks (27 from each donor) were inoculated with HHV-7. The culture medium was changed every 3 days, and HHV-7 replication was measured by real-time PCR (A) or flow cytometry (B). (A) Kinetics of HHV-7 replication. Shown is the HHV-7 genome accumulation in culture medium during 3 days between medium changes. Each datum point is the mean ± the SEM for experiments with tissues from nine donors. (B) Bivariate density plots of lymphocytes stained for CD3 and HHV-7 pp85. Lymphocytes were isolated after 12 days in culture from HHV-7-infected (right panel) and matched uninfected tissue (left panel). A sample representative of experiments with tissues from 10 donors is displayed.

FIG. 2.

R5_SF162 and X4_LAI.04 replication in HHV-7-coinfected human lymphoid tissues. Sets of matched tissue blocks (27 blocks for each experimental condition from each donor) were infected with one of the HIV-1 variants alone or together with HHV-7. HIV replication was evaluated by measurement of the p24 in culture medium. (A and B) Average kinetics of X4_LAI.04 (A) and R5_SF162 (B) replication in singly HIV-infected and HHV-7-coinfected tissues (mean ± the SEM, n = 8). The data are expressed as the percentage of the maximum p24 value in singly HIV-infected tissues to account for the variation in absolute replication levels in tissues from different donors. (C) Total production of R5_SF162 and X4_LAI.04 in HHV-7-coinfected tissues. Presented are the means ± the SEM for the total HIV-1 production over 12 days postinoculation for tissues from eight donors expressed as a percentage of the total HIV-1 production in matched singly infected tissues. An asterisk denotes a significant difference (P < 5 × 10⁻²). (D) Correlation between HHV-7 replication and HIV-1 inhibition in coinfected tissues. The data for HHV-7 replication are expressed as a log percentage of the maximum for each tissue; R5_SF162 HIV-1 inhibition is expressed as a percentage of the decrease in p24 relative to p24 production in matched singly HIV-infected tissue. The datum points from experiments with tissues from eight donors (days 6, 9, and 12 postinoculation) are plotted.

FIG. 3.

HHV-7 replication in human lymphoid tissues coinfected with R5_SF162 or X4_LAI.04. Sets of matched tissue blocks (27 blocks for each experimental condition from each donor) were infected with HHV-7 alone or together with one of the HIV-1 variants. HHV-7 replication was evaluated by measuring the number of HHV-7 genome equivalents accumulated in the culture medium. (A) Typical (n = 8) kinetics of HHV-7 replication in singly infected and HIV coinfected tissues. Shown are the means ± the SEM of the accumulation of HHV-7 genomes in culture medium during 3 days between medium changes. (B) Relative replication of HHV-7 in tissues coinfected with R5_SF162 and X4_LAI.04 Presented are the means ± the SEM of the production of HHV-7 genomes in culture medium over 12 days postinoculation for tissues from eight donors expressed as a percentage of the HHV-7 genome production in matched singly infected tissues. An asterisk denotes a significant difference (P < 5 × 10⁻²).

FIG. 4.

Phenotype of HHV-7-infected cells in human lymphoid tissues. Tissue blocks (27 blocks for each condition from each donor) were inoculated with HHV-7 and cultured for 12 days. Cells isolated from these blocks and from matched uninfected blocks were stained for lymphocytic markers and analyzed with flow cytometry. (A) Distribution of CD4 and CD8 in uninfected tissues and in matched HHV-7-infected tissue. Bars: □, T cells in uninfected tissue; ▪, uninfected T cells in HHV-7-infected tissue; ▤, HHV-7-infected T cells in infected tissue. Each bar represents the mean ± the SEM for tissues from nine donors. Asterisks denote significant differences: *, P < 10⁻²; and **, P < 10⁻³. (B) Bivariate density plot of T (CD3⁺) cells isolated from HHV-7-infected tissue on day 12 postinfection and stained for lymphocytic markers. Panels: left, distribution of T cells in uninfected tissue; middle, distribution of T cells in HHV-7-infected tissues; right, distribution of HHV-7-infected T cells (red dots) among T cells (gray) in HHV-7-infected tissues. A typical example of experiments with tissues from 10 donors is presented.

FIG. 5.

CD4 expression in T lymphocytes isolated from HHV-7-infected human lymphoid tissues. Cells from matched sets of uninfected or HHV-7-infected tissue blocks (27 blocks for each experimental condition from each donor) were stained for lymphocytic markers and for HHV-7 pp85 and then analyzed by flow cytometry. (A) Distribution of HHV-7⁺ CD3⁺ CD8⁻ and HHV-7⁻ CD3⁺ CD8⁻ cells isolated from uninfected tissues (left panel) and matched HHV-7-infected tissues (right panel) according to their CD4 fluorescence intensity. A typical bivariate dot plot for tissues from 11 donors is shown. (B) Distribution of CD4 fluorescence intensity in HHV-7⁺ (red histogram) and HHV-7⁻ (green histogram) cells from HHV-7-infected tissue blocks and in cells from matched uninfected control tissue (blue histogram). Presented are data for the same tissue as in panel A. A typical distribution for tissues from 11 donors is shown. (C) Cumulative frequency distribution of CD4 fluorescence intensities. The percentage of infected tissue HHV-7⁺ (red) and HHV-7⁻ (green) cells with the CD4 fluorescence below the median fluorescence of a matched uninfected control (blue) are indicated. Presented are data for the same tissue as in panels A and B. Typical for data from 11 donors are shown. (D) Downregulation of CD4 in HHV-7-infected and HHV-7-uninfected CD8⁻ T cells isolated from HHV-7-infected tissues and matched uninfected controls. Downregulation of CD4 was defined as the difference (percent) between the ratio of the numbers of cells with the CD4 fluorescence higher and lower than the median fluorescence for uninfected control tissue (by definition equals one) and a similar ratio for matched infected tissues. That is, D_CD4 = (Fl_h^ctrl/Fl_l^ctrl − Fl_h^inf/Fl_l^inf) × 100 = (1 − Fl_h^inf/Fl_l^inf) × 100, where D_CD4 is the downregulation of CD4 on the cell surface, Fl_l^inf and Fl_h^inf are the numbers of cells in infected tissues, with fluorescence lower and higher (respectively) than the median in an uninfected control, and Fl_h^ctrl and Fl_l^ctrl are similar parameters for a matched uninfected control. By definition, the median the ratio of Fl_h^cntrl/Fl_l^cntrl is equal to 1. Presented are means ± the SEM for tissues from 11 donors.

FIG. 5.

CD4 expression in T lymphocytes isolated from HHV-7-infected human lymphoid tissues. Cells from matched sets of uninfected or HHV-7-infected tissue blocks (27 blocks for each experimental condition from each donor) were stained for lymphocytic markers and for HHV-7 pp85 and then analyzed by flow cytometry. (A) Distribution of HHV-7⁺ CD3⁺ CD8⁻ and HHV-7⁻ CD3⁺ CD8⁻ cells isolated from uninfected tissues (left panel) and matched HHV-7-infected tissues (right panel) according to their CD4 fluorescence intensity. A typical bivariate dot plot for tissues from 11 donors is shown. (B) Distribution of CD4 fluorescence intensity in HHV-7⁺ (red histogram) and HHV-7⁻ (green histogram) cells from HHV-7-infected tissue blocks and in cells from matched uninfected control tissue (blue histogram). Presented are data for the same tissue as in panel A. A typical distribution for tissues from 11 donors is shown. (C) Cumulative frequency distribution of CD4 fluorescence intensities. The percentage of infected tissue HHV-7⁺ (red) and HHV-7⁻ (green) cells with the CD4 fluorescence below the median fluorescence of a matched uninfected control (blue) are indicated. Presented are data for the same tissue as in panels A and B. Typical for data from 11 donors are shown. (D) Downregulation of CD4 in HHV-7-infected and HHV-7-uninfected CD8⁻ T cells isolated from HHV-7-infected tissues and matched uninfected controls. Downregulation of CD4 was defined as the difference (percent) between the ratio of the numbers of cells with the CD4 fluorescence higher and lower than the median fluorescence for uninfected control tissue (by definition equals one) and a similar ratio for matched infected tissues. That is, D_CD4 = (Fl_h^ctrl/Fl_l^ctrl − Fl_h^inf/Fl_l^inf) × 100 = (1 − Fl_h^inf/Fl_l^inf) × 100, where D_CD4 is the downregulation of CD4 on the cell surface, Fl_l^inf and Fl_h^inf are the numbers of cells in infected tissues, with fluorescence lower and higher (respectively) than the median in an uninfected control, and Fl_h^ctrl and Fl_l^ctrl are similar parameters for a matched uninfected control. By definition, the median the ratio of Fl_h^cntrl/Fl_l^cntrl is equal to 1. Presented are means ± the SEM for tissues from 11 donors.

FIG. 6.

CD4 expression and HIV replication in CADA-treated human lymphoid tissues. Matched sets of tissue (27 blocks for each experimental condition from each of two donors) were incubated with CADA (1 or 2 μM) for 12 days or used as untreated controls. Some of these sets were infected with R5_SF162 and X4_LAI.04. (A) Cumulative frequency distribution of CD4 fluorescence intensities in T cells isolated from untreated (blue histogram) tissues and from tissues incubated with CADA at 1 μM (green histogram) and 2 μM (red histogram). The percentages of T cells with the CD4 fluorescence below the median fluorescence of matched uninfected control (blue) are indicated. The distributions for the tissues from three donors were similar, and only one is presented here. (B) Relative replication of R5_SF162 and X4_LAI.04 in tissues incubated with 1 or 2 μM CADA expressed as a percentage of p24 production in matched untreated tissues infected with HIV-1. Shown are means ± the SEM of the cumulative production of HIV over 12 days of infection.