Polyubiquitination of APOBEC3G Is Essential for Its Degradation by HIV-1 Vif ▿

Proteasomal degradation of APOBEC3G is a critical step for human immunodeficiency virus type 1 (HIV-1) replication. However, the necessity for polyubiquitination of APOBEC3G in this process is still controversial. In this study, we showed that although macaque simian immunodeficiency virus (SIVmac) Vif is more stable than HIV-1 Vif in human cells, SIVmac Vif induces degradation of APBOEC3G as efficiently as HIV-1 Vif. Overexpression of APOBEC3G or lysine-free APOBEC3G stabilized HIV-1 Vif, indicating that APOBEC3G degradation is independent of the degradation of Vif. Furthermore, an in vivo polyubiquitination assay showed that lysine-free APOBEC3G was also polyubiquitinated. These data suggest that polyubiquitination of APOBEC3G, not that of HIV-1 Vif, is crucial for APOBEC3G degradation

Medroxyprogesterone Acetate (MPA) Enhances HIV-1 Accumulation and Release in Primary Cervical Epithelial Cells by Inhibiting Lysosomal Activity

Medroxyprogesterone acetate (MPA) is one of the most widely used contraceptives in the world. Epidemiologic studies have uncovered a possible link between the use of MPA and an increased risk of HIV-1 transmission. However, the understanding of the mechanism is still limited. Our previous publication demonstrated that the lysosomal activity in human vaginal epithelial cells attenuated the trafficking of viral particles during HIV-1 transcytosis. In this study, we show that treating human primary cervical epithelial cells with MPA led to a reduction in lysosomal activity. This reduction caused an increase in the intracellular HIV-1 accumulation and, consequently, an increase in viral release. Our study uncovers a novel mechanism by which MPA enhances HIV-1 release in primary cervical epithelial cells, thus providing vital information for HIV intervention and prevention

The active CC5A molecule inhibits HIV-1 replication. (A) THP-1/IIIB cells were treated with PBS or CC5A/PBS for 16 h.

<p>Post-treatment, THP-1 cells were washed twice by PBS and cultured for 24 h. The cells were then washed with PBS and cultured for another 24 h. The culture supernatants were collected for infectivity assays. The amount of input virus for the infectivity assay was normalized by p24 ELISA. (B) THP-1 cells were pre-treated overnight with CC5A/PBS or PBS before IIIB infection. After 3 h of infection, the cells were washed twice with PBS and put back into culture for eight days. Viral samples were collected every other day and viral replication was monitored by p24 assay.</p

A3F and A3G are regulated through the MEK1/2 dependent pathway.

<p>(<b>A</b>) Prior to treatment with CC5A/PBS for 16 h, THP-1 cells were pre-treated by DMSO alone or the ERK1/2 inhibitor U0126 in DMSO for 1 h. The induction of A3G and A3F was measured by qRT-PCR. (B) Lysates from THP-1 cells pre-treated DMSO or U0126 in DMSO, following by CC5A/PBS (40 µg) are subjected to Western blot analysis. Phosphorylated ERK1/2 and ERK1/2 were probed by anti-p-ERK1/2 and anti-ERK2, respectively.</p

CC5A/PBS and sonicated CC5A do not cause cytotoxicity in THP-1 cells.

<p>The cytotoxicity of CC5A/PBS and sonicated CC5A treatment was measured by a Live/Dead Cell Vitality Assay Kit (Invitrogen). THP-1 cells were treated with PBS, 40 µg CC5A/PBS and 25 µg sonicated CC5A for 16 h then subjected to the Live/Dead Cell Vitality Assay Kit analysis.</p

CC5A/PBS induces A3G and A3F expression in human oral keratinocyte (OKF6) and human vaginal VK2 epithelial cells.

<p>(<b>A</b>) CC5A/PBS (5 µg) was used to treat OKF6 cells. The cells were harvested at the indicated time points, and qRT-PCR was used to measure the relative expression of A3F and A3G mRNA. The expression levels shown represent the ratio of CC5A/PBS treated cells compared to the untreated cells at each given time point. (<b>B</b>) Different doses (0–5 µg) of CC5A/PBS were used to treat OKF6 cells for 72 h, and A3G expression was measured by Western-blot analysis. (<b>C</b>) VK2 cells were treated with 40 µg CC5A/PBS or vehicle for the time periods shown. qRT-PCR was used to measure the relative expression of A3F and A3G mRNA in these cells. The expression levels shown represent the ratio of the levels found in CC5A/PBS treated cells compared to untreated cells at each time point. (<b>D</b>) Different doses (0–40 µg) of CC5A/PBS were used to treat VK2 cells for 48 h. A3G protein expression was measured by Western-blot analysis.</p

A molecule derived from oral <i>Streptococcus cristatus</i> induces APOBEC3F/G expression.

<p>(A) Cell extracts from NC3 (<i>Actinomyces naeslundii</i> NC-3), DL1 (<i>Streptococcus gordonii</i> DL1) and DH5a, CC5A (<i>S. cristatus</i> CC5A), A.V (<i>Actinomyces viscosus</i>), 33277 (<i>Porphyromonas gingivalis</i> 33277) and S.MU (<i>S. mutans</i> KPSK2) were used to treat THP-1 cells. THP-1 cells treated by PBS were used as a control. After 16 h of exposure, total RNA was isolated and the expression of A3G and A3F quantified by qRT-PCR. (B) CC5A and different strains of <i>S. cristatus</i> (pSH11b, pSH11a, CR311, CR3, and CH34110) were used to treat THP-1. The induction of A3G and A3F was measured by qRT-PCR. (C) Different doses (0–40 µg) of CC5A/PBS were used to treat THP-1 cells for 16 h. A3G and A3F expression was measured by qRT-PCR. (D) A3G and A3F expression following a 48 h exposure to 0–10 µg CC5A was tested by Western blot assay. Values shown in (A), (B) and (C) are given as means ± SD of three independent experiments compared to normalized to 1.0 for controls. The Western-blot data is representative of three independent experiments.</p