The CDK Inhibitor p21 Cip1/WAF1 Is Induced by Fc (cid:1) R Activation and Restricts the Replication of Human Immunodeﬁciency Virus Type 1 and Related Primate Lentiviruses in Human Macrophages (cid:1)

Macrophages are major targets of human immunodeﬁciency virus type 1 (HIV-1). We have previously shown that aggregation of activating immunoglobulin G Fc receptors (Fc (cid:1) R) by immune complexes inhibits reverse transcript accumulation and integration of HIV-1 and related lentiviruses in monocyte-derived macrophages. Here, we show that Fc (cid:1) R-mediated restriction of HIV-1 is not due to enhanced degradation of incoming viral proteins or cDNA and is associated to the induction of the cyclin-dependent kinase inhibitor p21 Cip1/WAF1 (p21). Small interfering RNA-mediated p21 knockdown rescued viral replication in Fc (cid:1) R-activated macrophages and enhanced HIV-1 infection in unstimulated macrophages by increasing reverse transcript and integrated DNA levels. p21 induction by other stimuli, such as phorbol myristate acetate and the histone deacetylase inhibitor MS-275, was also associated with preintegrative blocks of HIV-1 replication in macro- phages. Binding of p21 to reverse transcription/preintegration complex-associated HIV-1 proteins was not detected in yeast two-hybrid, pulldown, or coimmunoprecipitation assays, suggesting that p21 may affect viral replication independently of a speciﬁc interaction with an HIV-1 component. Consistently, p21 silencing rescued viral replication from the Fc (cid:1) R-mediated restriction also in simian immunodeﬁciency virus SIV mac - and HIV-2-infected macrophages. Our results point to a role of p21 as an inhibitory factor of lentiviral infection in macrophages and to its implication in Fc (cid:1) R-mediated restriction. instructions. quantiﬁed by GeneQuant (Amersham), and equal amounts (1 (cid:4) g) were reverse transcribed with SuperScript II RT (Invitrogen). We used custom RT 2 Proﬁler PCR arrays (SABiosciences) to quantify TRIM transcrits, allowing us to detect TRIM5, TRIM11, TRIM19/PML, TRIM22, TRIM26, TRIM31, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) simultaneously. PCRs were performed with the RT 2 Realtime SYBR green PCR mix (SABiosciences) according to the manufacturer’s instructions on a Light-Cycler 480 (Roche Diagnostics). The ampliﬁcation program consisted of 10 min at 95°C, followed by 45 cycles at 95°C for 15 s and 60°C for 1 min. For other transcripts, PCR ampliﬁcation of cDNAs was carried out in duplicate in MicroAmp Optical 96-well reaction plates (30 (cid:4) l/well), using 25 (cid:4) l of Taq-Man Universal Master Mix, 0.2 mM TaqMan, and 1.5 (cid:4) l of Assays-on- Demand gene expression assay premade mix (GAPDH, Hs99999905_m1; p21, Hs00355782_m1; LEDGF/p75, Hs01045714_g1; BAF, Hs00427805_g1; Ini1, Hs00996890_m1; Gemin2, Hs01031721_m1; p53, Hs00153349_m1; and p27, Hs00153277_m1). The ampliﬁcation conditions were as follows: 50°C for 2 min and 95°C for 10 min, followed by 45 cycles at 95°C for 15 s and 60°C for 90 s, on an ABI Prism 7700 sequence detector (Applied Biosystems). The data were analyzed with the cycle threshold ( C T ) method, and the amount of target mRNA in each sample was normalized to GAPDH mRNA as an endogenous reference.