Fangchinoline Inhibits Human Immunodeficiency Virus Type 1 Replication by Interfering with gp160 Proteolytic Processing

The introduction of highly active antiretroviral therapy has led to a significant reduction in the morbidity and mortality of acquired immunodeficiency syndrome patients. However, the emergence of drug resistance has resulted in the failure of treatments in large numbers of patients and thus necessitates the development of new classes of anti-HIV drugs. In this study, more than 200 plant-derived small-molecule compounds were evaluated in a cell-based HIV-1 antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (fangchinoline). Fangchinoline, a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae, exhibited antiviral activity against HIV-1 laboratory strains NL4-3, LAI and BaL in MT-4 and PM1 cells with a 50% effective concentration ranging from 0.8 to 1.7 µM. Mechanism-of-action studies showed that fangchinoline did not exhibit measurable antiviral activity in TZM-b1 cells but did inhibit the production of infectious virions in HIV-1 cDNA transfected 293T cells, which suggests that the compound targets a late event in infection cycle. Furthermore, the antiviral effect of fangchinoline seems to be HIV-1 enve1ope-dependent, as the production of infectious HIV-1 particles packaged with a heterologous envelope, the vesicular stomatitis virus G glycoprotein, was unaffected by fangchinoline. Western blot analysis of HIV envelope proteins expressed in transfected 293T cells and in isolated virions showed that fangchinoline inhibited HIV-1 gp160 processing, resulting in reduced envelope glycoprotein incorporation into nascent virions. Collectively, our results demonstrate that fangchinoline inhibits HIV-1 replication by interfering with gp160 proteolytic processing. Fangchinoline may serve as a starting point for developing a new HIV-1 therapeutic approach

Kaposi's Sarcoma-Associated Herpesvirus-Encoded LANA Down-Regulates IL-22R1 Expression through a Cis-Acting Element within the Promoter Region

Kaposi's sarcoma-associated herpesvirus (KSHV) is considered to be a necessary, but not sufficient, causal agent of Kaposi's sarcoma (KS). All forms of KS are characterized by the proliferation of spindle-shaped cells, and most (>90%) spindle cells from KS lesions are latently infected with KSHV. During KSHV latency, only a few viral genes are expressed. Among those latent genes, the ORF 73 gene encodes the latency-associated nuclear antigen (LANA), which is critical for the establishment and maintenance of the latent KSHV infection. Much evidence suggests that many cytokines can increase the frequency and aggressiveness of KS. In this study, a microarray analysis of KS and normal tissues revealed that multiple cytokines and cytokine receptors are regulated by KSHV latent infection. Of special interest, IL-22R1 transcript level was found to be down-regulated in the KS tissue. To study the possible regulation of IL-22R1 by LANA, the IL-22R1 promoter was constructed and found to contain a LANA-binding site (LBS). LANA was demonstrated to down-regulate IL-22R1 expression via direct binding to the LBS located within the IL-22R1 promoter region. Furthermore, KSHV latently infected cells showed an impaired response to IL-22 stimulation. These results suggest that LANA can regulate host factor expression by directly binding to a cis-acting element within the factor's promoter to benefit latent viral infection and suppression of the antiviral immune response

KSHV-infected cells show impaired response to IL-22 stimulation.

<p>293T and 293T-BAC36 cells are stimulated with 100 ng/ml of rIL-22 for 0, 5, 15, or 30 min. The cells were lysed and subjected to immunoblot analysis to detect the phosphorylation of STAT3 and ERK.</p

LANA interacts with the IL-22R1 promoter in vivo.

<p>(<b>A</b>) Schematic diagram showing the locations of the two pairs of primers used in the ChIP assay. (<b>B</b>) Formaldehyde cross-linked chromatin was prepared from 293T cells that were transfected with pFLAG, pFLAG-LANA, pFLAG-LANA_1–939 or pFLAG-LANA_933–1162, and immunoprecipitated with anti-FLAG antibody. PCR was performed with primer pair 1 to amplify a 197 bp DNA fragment containing LBS or with primer pair 2 to amplify a 283 bp DNA fragment, approximately 2 kb upstream of LBS-like sequence in IL-22R1 promoter. A sample representative of 5% the total input chromatin was included in the PCR analysis. (<b>C</b>) The expression levels of LANA and its truncated mutants were detected in cell lysates by western blotting using anti-FLAG antibody and anti-actin served as a loading control.</p

Sequences of the primers used for the generation of luciferase reporter plasmids.

<p>Restriction enzyme sites are underlined.</p

Expression levels of cytokines and cytokine receptors in KS lesions vs. normal tissues.

<p>A cDNA microarray analysis was performed to compare the gene expression between KS tissue and normal tissue. Differentially expressed interleukin-associated genes are listed (Ratio ≥2 or ≤0.5 and p value of log-ratio <0.05).</p

LANA binds to the IL-22R1 promoter in vitro.

<p>(<b>A</b>) The aligned sequences of DNA fragments from TR DNA, wild type and mutant IL-22R1 promoters which contain LBS, the wild type LBS-like sequence and scrambled-mutant DNA sequence (underlined) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019106#pone.0019106-Garber2" target="_blank">[38]</a>. The nucleotide sequences represent the probe sequences used in the EMSA and were labeled with biotin. (<b>B</b>) LANA can bind to the LBS-like DNA sequence in IL-22R1 promoter. Probes as indicated were incubated for 20 min with or without purified LANA-C (a.a. 951–1162). NC, a DNA fragment derived from the sequence in M13 DNA, used as a negative control for LANA binding. (<b>C</b>) The binding of LANA to the wild type LBS-like sequence is specific. A 10-fold excess of cold competitor or mutant competitor DNA was added to compete the reaction between WT LBS-like and LANA. The upper and lower arrows indicate the LANA-specific binding band and free probe.</p

LANA down-regulates IL-22R1 promoter activity in a dose-dependent manner.

<p>(<b>A</b>) Schematic of pIL22R1 (−2139 to +39) and a series of deletion mutants (pIL22R1-D1, D2, D3, and D4). (<b>B</b>) LANA down-regulates IL-22R1 promoter (−2139 to +39) activity. pIL22R1 (−2139 to +39) was co-transfected with increasing amounts (0, 0.1, 0.2 or 0.4 µg) of LANA into 293T cells. At 48 h post-transfection, cells were lysed and assayed for luciferase activity. The expression levels of LANA were detected in cell lysates by western blotting using anti-LANA-C antibody (top panel). The regulation of the IL-22R1 promoter (−2139 to +39) by LANA was also analyzed in HUVEC cells (lower panel).</p

The LBS-like sequence is required for LANA to down-regulate IL-22R1 expression.

<p>(<b>A</b>) Schematic of pIL22R1 (−2139 to +39), pIL22R1ΔLBS-like and pIL22R1mLBS-like DNA sequences. (<b>B</b>) Increasing amounts of pcDNA-LANA expressing full-length LANA were co-transfected with either pIL22R1 (−2139 to +39) or its mutants (pIL22R1ΔLBS-like and pIL22R1mLBS-like) into 293T cells. At 48 h post-transfection, cells were harvested and assayed for luciferase activity. (<b>C</b>) Increasing amounts of pcDNA-LANA-C-expressing carboxyl-terminal domain (amino acids 951–1162) of LANA were co-transfected with either pIL22R1 (−2139 to +39) or its mutants (pIL22R1ΔLBS-like and pIL22R1mLBS-like) into 293T cells. At 48 h post-transfection, cells were harvested and assayed for luciferase activity. (<b>D</b>) Increasing amounts of pcDNA-LANA expressing full-length LANA were co-transfected with either pIL22R1 (−2139 to +39) or its mutants (pIL22R1ΔLBS-like and pIL-22R1mLBS-like) into HUVEC cells. At 48 h post-transfection, cells were harvested and assayed for luciferase activity.</p

<i>In vitro</i> antiviral activities of fangchinoline against different HIV-1 laboratory strains.

<p>Antiviral assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039225#s2" target="_blank">Materials and Methods</a>. Values represent average of at least three independent experiments.</p>a<p>Compound concentration required to reduce the production of p24 antigen by 50%.</p>b<p>Compound concentration required to reduce mock-infected cell viability by 50%.</p>c<p>Selective index, ratio of CC₅₀/EC₅₀.</p