Increased BST2 expression during simian immunodeficiency virus infection is not a determinant of disease progression in rhesus monkeys

Abstract

Background: Bone marrow stromal cell antigen 2 (BST2), also known as tetherin, HM1.24 or CD317 represents a type 2 integral membrane protein, which has been described to restrict the production of some enveloped viruses by inhibiting the virus release from the cell surface. This innate antiviral mechanism is counteracted by the HIV-1 viral factor Vpu, targeting BST2 for cellular degradation. Since antiviral BST2 activity has been mainly confirmed by in vitro data, we investigated its role in vivo on the disease progression using the SIV/macaque model for AIDS. We determined BST2 expression in PBMC and leukocyte subsets of uninfected and SIV-infected rhesus macaques by real-time PCR and flow cytometry and correlated it with disease progression and viral load.

Results: Compared to pre-infection levels, we found increased BST2 expression in PBMC, purified CD4(+) lymphocytes and CD14(+) monocytes of SIV-infected animals, which correlated with viral load. Highest BST2 levels were found in progressors and lowest levels comparable to uninfected macaques were observed in long-term non-progressors (LTNPs). During acute viremia, BST2 mRNA increased in parallel with MX1, a prototype interferon-stimulated gene. This association was maintained during the whole disease course.

Conclusion: The detected relationship between BST2 expression and viral load as well as with MX1 indicate a common regulation by the interferon response and suggest rather limited influence of BST2 in vivo on the disease outcome.

Fig. 1

Whole blood BST2 mRNA levels in SIV-infected macaques. BST2 mRNA levels in whole blood of 17 rhesus macaques were determined using PAXgene Blood RNA Kit and real time PCR before and at 24 weeks after infection (wpi). a BST2 mRNA levels were compared between uninfected (circles) and SIVmac251 infected animals (squares). BST2 mRNA levels are shown in copy numbers per 100 copies of GAPDH. Horizontal lines within the clusters are depicting the median. Group comparisons were calculated using two-tailed Mann–Whitney’s U test. BST2 mRNA levels at 24 wpi (b) and BST2 mRNA levels after normalization to individual pre-infection values (c) were correlated with plasma viral load. Viral load is displayed as log-transformed RNA copies per millilitre (ml) plasma. r Spearman’s correlation coefficient; regression line is shown; p, p value. Each data point represents one individual animal

Fig. 2

BST2 mRNA and protein expression in uninfected and SIVmac251-infected macaques. Representative flow cytometric analyses of BST2 surface expression of an uninfected animal shown as histogram for leukocyte populations (a) and histogram for lymphocyte subsets including a fluorescence minus one (FMO) control (b). BST2 surface expression, displayed as median fluorescence intensity (MFI) on all leukocytes (c), on CD4⁺ lymphocytes (e) and on CD14⁺ monocytes (g) is illustrated for 12 uninfected and 32 SIVmac251 infected animals at 24 wpi. Box plots depict median and quartiles while whiskers show range. Group comparisons were calculated using two-tailed Mann–Whitney’s U test. d Whole blood BST2 mRNA levels correlate with BST2 surface protein expression. BST2 mRNA levels are depicted as copy numbers per 100 copies of GAPDH. BST2 surface expression on CD4⁺ lymphocytes (f) and CD14⁺ monocytes (h) correlates with plasma viral load. Each data point represents one individual animal. Regression lines are depicted; r Spearman’s correlation coefficient; p, p value

Fig. 3

BST2 mRNA induction by type I interferon. a Fold induction of relative BST2 mRNA in PBMC from three uninfected rhesus macaques after stimulation with human Interferon Alpha A (Alpha 2a) for 16 h. Data are expressed as fold increase over baseline after normalization to pre-treatment values. Error bars represent standard deviation, b relative mRNA copies of BST2 in PBMC (shown in copy numbers per 100 copies of GAPDH) are illustrated in relation to plasma IFN-alpha levels from blood samples of 18 uninfected rhesus macaques 24 h after inoculation of replication incompetent adenovirus or fowl pox vectors. The black dashed line indicates the detection limit of the ELISA and c whole blood MX1 mRNA levels correlate with BST2 mRNA determined in 38 SIVmac251 infected rhesus macaques at 24 wpi. Relative mRNA levels are depicted as log-transformed copy numbers per 100 copies of GAPDH. Each data point represents one animal. Regression line is shown; r, Spearman’s correlation coefficient; p, p value

Fig. 4

BST2 and MX1 mRNA levels in uninfected and SIV-infected macaques with different disease progression. Relative BST2 (left panels) and relative MX1 (right panels) mRNA levels were determined in uninfected (triangles), SIV-infected asymptomatic progressors (empty circles), progressors with AIDS-like symptoms (filled circles) and LTNPs (squares). Relative BST2 and MX1 mRNA levels are shown in copy numbers per 100 copies of GAPDH in PBMC (a, b), CD4⁺ lymphocytes (c, d) and CD14⁺ monocytes (e, f). Each data point represents one individual animal. Horizontal lines within the clusters are depicting the median. Group comparisons were calculated using Kruskal–Wallis test with Dunn’s multiple comparison analysis; p, p value; ns not significant

Fig. 5

Kinetics of RNA plasma viral load and mRNA levels of BST2 and MX1 in SIV-infected macaques. Plasma viral load as well as relative BST2 and MX1 mRNA levels in PBMC were determined longitudinally in seven macaques before and after inoculation with SIVmac251 MPBMC (a–c). Viral load is depicted as log-transformed RNA copies per millilitre (ml) plasma (a). Relative BST2 mRNA (b) and relative MX1 mRNA (c) in PBMC were calculated as copy numbers per 100 copies of GAPDH. Data are expressed as fold increase over baseline after normalization to the mean of three pre-infection values. Fine grey lines with symbols represent individual infected animals. Fine black lines with closed triangles depict the one animal inoculated but not infected. Bold lines show mean values of infected animals. p value show a significant difference to the mean of the three pre-infection values calculated by Mann–Whitney’s U test. d Relationship between relative BST2 and MX1 mRNA levels in PBMC from rhesus macaques during acute SIV infection (10–14 dpi). Line shows linear regression; r, Spearman’s correlation coefficient; p, p value. Samples from LTNPs (filled triangles) and progressor (open circles) rhesus macaques were analysed for viral load (e), relative BST2 (f) and relative MX1 (g) RNA levels at 2 wpi. h Relationship between pre-infection BST2 mRNA levels and viral load at 2 wpi. Each data point represents one individual animal. Horizontal lines within the clusters are depicting the median. Group comparisons were calculated using Mann–Whitney’s U test