In vitro infection of human primary adipose cells with HIV-1

We reassessed the infection ability of human primary preadipocytes. The use of X4, R5 or VSV-G pseudotyped viral particles indicated that viral entry is the limiting step. However, transfection with HIV-1 receptors restored efficient infection. Analyses of CD4, CXCR4 and CCR5 expression on preadipocytes and adipocytes revealed that receptor co-expression levels did not permit HIV-1 entry into adipose cells from all biopsies tested. We concluded that adipose tissue cannot be infected with HIV-1in vivo. The major target cells for HIV-1 are CD4-positive T lymphocytes and cells from the monocyte and macrophage lineages [1] that are potential reservoirs for HIV-1 in vivo. More particularly, HIV-1 can establish a latent infection in resting memory T cells and can be reactivated [2,3]. However, the amplitude of plasma viraemia rebounds after the discontinuation of highly active antiretroviral therapy cannot be explained exclusively by already known reservoirs, suggesting that HIV-1 persists within other reservoirs [4]. We recently reported that human adipose cells express HIV-1 receptors, and showed that these cells could be infected in vitro using high viral inputs and resulted in inefficient viral production [5]. Experiments performed on adipose cells from HIV-1-infected patients strongly suggested that viral DNA remained absent from this tissue in vivo [6]. The potential ability of the adipose tissue to behave as a new viral reservoir is thus an essential parameter to be determined, in the light of the lipodystrophy syndrome [7]. We therefore investigated whether the low viral production levels previously obtained [5] could be the result of low infection efficiency or low viral replication efficiency. We first transiently transfected HeLa cells or human primary preadipocytes with the pNL4.3-Δenv-Luc proviral plasmid. Luciferase activities and p24 production analyses revealed that proviral DNA could be expressed in preadipocytes and results in new viral particle release, indicating that there is no transcriptional block at the level of the HIV-1 promoter (not shown). This ruled out a post-integration restriction. In order to determine whether viral inhibition was caused by restricted viral entry, preadipocytes or 5-day differentiated adipocytes were infected with viral particles pseudotyped with an X4 (LAI), an R5 (ADA8) or an amphotropic (VSV-G) envelope glycoprotein. As the receptor for VSV-G is ubiquitous, this model allowed us to analyse the replication of HIV-1 after entry in preadipocytes and adipocytes even in the absence of HIV-1 receptors. No luciferase activity nor p24 production could be detected in HeLa cells (Fig. 1a) after infection with LAI and ADA8 pseudotyped viruses, whereas transfection of CCR5 in HeLa-CD4 cells (P4.2, which constitutively expresses CXCR4) (Fig. 1b) resulted in high luciferase activities [9, 6.105 and 9.105 normalized luciferase index (NLI) 72 h post-infection, respectively] and p24 production (100–200 pg/ml 72 h post-infection), as expected. When preadipocytes (Fig. 1d) and adipocytes (Fig. 1e) were infected with these pseudotypes, no luciferase activity nor p24 production could be detected. By contrast, infection of preadipocytes and adipocytes with the VSV-G pseudotyped viral particles resulted in strong luciferase signals (1, 5.107 NLI 72 h post-infection, Fig. 1d, and 3.107 NLI 72 h post-infection, Fig. 1e, respectively), and the production of p24 antigen reached 30 pg/ml in preadipocytes and 140 pg/ml in adipocytes, 72 h post-infection. Luciferase activities were equivalent or higher than those monitored in HeLa cells and were infected with the VSV-G pseudotype (3.106 NLI 72 h post-infection, Fig. 1a, and 1.107 NLI 72 h post-infection, Fig. 1b), and p24 levels were similar to those detected in HeLa cell supernatants (70 pg/ml). The same experiments were repeated in the PAZ6 human adipose cell line [5], and luciferase activity (6, 2.107 NLI 72 h post-infection, Fig. 1g) and p24 release (130 pg/ml 72 h post-infection) were detected after infection with VSV-G but not with LAI and ADA8 pseudotyped viruses. This demonstrated VSV-G pseudotyped virus entry, reverse transcription and expression, and that the entry of LAI and ADA8 pseudotyped viruses in adipose cells was, by contrast, restricted.Fig. 1.: Infection of preadipocytes and adipocytes with pseudotyped viruses. HeLa cells (a), HeLa-CD4 cells (P4.2, which constitutively express CXCR4) transfected with CCR5 (b), preadipocytes A2492 day 0 (d), 5-day differentiated adipocytes A2492 day 5 (e) and PAZ6 cells day 0 (g) were infected with 20 ng viral particles pseudotyped with either LAI (X4 tropism), ADA8 (R5 tropism) or VSV-G (extended tropism) envelope glycoproteins. HeLa cells (c), preadipocytes A2492 day 0 (f) and PAZ6 cells day 0 (h) were transfected or not (NT) either with CD4 and CXCR4 or with CD4 and CCR5, as indicated. Twenty-four hours post-transfection, cells were infected with 20 ng pseudotyped viral particles bearing either the LAI (for CD4-CXCR4 transfected cells) or the ADA8 (for CD4-CCR5 transfected cells) envelope glycoproteins, as indicated. Infection efficiencies were monitored by measuring the normalized luciferase index (NLI; defined as the ratio of luciferase activity to protein contents) 24, 48 and 72 h post-infection (p.i.). The background luciferase activity was not deducted from luciferase values; the threshold detection level was therefore 103 NLI. Results presented here are representative of three independent experiments.We thus assessed whether the expression of the HIV-1 receptors by adipose cells would render these cells susceptible to infection. We therefore transfected preadipocytes or PAZ6 cells with a combination of CD4/CXCR4 or CD4/CCR5 expression plasmids, and infected them 24 h post-transfection with viral particles pseudotyped with LAI or ADA8 envelope glycoproteins. A strong luciferase activity was detected in preadipocytes (5.104 NLI and 2, 4.104 NLI 72 h post-infection, Fig. 1f) or PAZ6 cells (4, 6.106 NLI and 5.105 NLI 72 h post-infection, Fig. 1h) previously transfected with CD4 and CXCR4 or CD4 and CCR5, and infected with LAI or ADA8 pseudotyped viruses, respectively, but not in non-transfected control cells. As compared with transfection efficiencies, these luciferase activities were similar to those obtained in HeLa cells transfected with the HIV-1 receptors (3, 4.105 NLI 72 h post-infection with LAI and 5.105 NLI 72 h post-infection with ADA8, Fig. 1c). In line with these observations, p24 antigen production could be detected 72 h post-infection with LAI or ADA8 in preadipocyte (20 pg/ml) or in PAZ6 (90 pg/ml) supernatants only in transfected cells. Therefore, infection with viral particles presenting X4 or R5 tropisms could be efficiently restored after the transfection of all necessary HIV-1 entry receptors, confirming that viral entry is the limiting step in adipose cell infection. As HIV-1 viral entry seems to be a limiting step for HIV-1 replication in adipose cells, we thus reassessed the in-vitro expression of HIV-1 entry receptors on adipose cells. We tested four biopsies from different donors for the expression of CD4, CXCR4 and CCR5 at different timepoints and differentiation stages by microarray experiments. CD4 and CCR5 were never detected on these different biopsies, whereas CXCR4 expression fluctuated depending on cell culture conditions and on the differentiation stage on two out of four biopsies tested (not shown). This was confirmed by semi-quantitative reverse transcriptase–polymerase chain reaction experiments (not shown). Interestingly, the co-expression of the three receptors at the same timepoint could not be observed on the same cell batch in adipose cells from any of four different donors. Our data strongly suggest that HIV-1 receptor co-expression at high levels is a rare event in adipose cells, which may explain why viral entry is not efficient. These results clearly demonstrate that infection with HIV-1 was not productive in primary adipose cells, as a result of the absence of all necessary entry receptors. Moreover, once the entry step is bypassed, viral replication takes place efficiently. We thus propose that although adipose cells can be infected in vitro, infection occurs only rarely. In the light of recently published in-vivo data [6], adipose tissue cannot be considered a target tissue for HIV-1 in vivo.