Naf1 Regulates HIV-1 Latency by Suppressing Viral Promoter-Driven Gene Expression in Primary CD4+ T Cells.

HIV-1 latency is characterized by reversible silencing of viral transcription driven by the long terminal repeat (LTR) promoter of HIV-1. Cellular and viral factors regulating LTR activity contribute to HIV-1 latency, and certain repressive cellular factors modulate viral transcription silencing. Nef-associated factor 1 (Naf1) is a host nucleocytoplasmic shuttling protein that regulates multiple cellular signaling pathways and HIV-1 production. We recently reported that nuclear Naf1 promoted nuclear export of unspliced HIV-1 gag mRNA, leading to increased Gag production. Here we demonstrate new functions of Naf1 in regulating HIV-1 persistence. We found that Naf1 contributes to the maintenance of HIV-1 latency by inhibiting LTR-driven HIV-1 gene transcription in a nuclear factor kappa B-dependent manner. Interestingly, Naf1 knockdown significantly enhanced viral reactivation in both latently HIV-1-infected Jurkat T cells and primary central memory CD4+ T cells. Furthermore, Naf1 knockdown in resting CD4+ T cells from HIV-1-infected individuals treated with antiretroviral therapy significantly increased viral reactivation upon T-cell activation, suggesting an important role of Naf1 in modulating HIV-1 latency in vivo Our findings provide new insights for a better understanding of HIV-1 latency and suggest that inhibition of Naf1 activity to activate latently HIV-1-infected cells may be a potential therapeutic strategy. IMPORTANCE: HIV-1 latency is characterized mainly by a reversible silencing of LTR promoter-driven transcription of an integrated provirus. Cellular and viral proteins regulating LTR activity contribute to the modulation of HIV-1 latency. In this study, we found that the host protein Naf1 inhibited HIV-1 LTR-driven transcription of HIV genes and contributed to the maintenance of HIV-1 latency. Our findings provide new insights into the effects of host modulation on HIV-1 latency, which may lead to a potential therapeutic strategy for HIV persistence by targeting the Naf1 protein.

Screening of potential pseudo att sites of Streptomyces phage ΦC31 integrase in the human genome.

AIM: ΦC31 integrase mediates site-specific recombination between two short sequences, attP and attB, in phage and bacterial genomes, which is a promising tool in gene regulation-based therapy since the zinc finger structure is probably the DNA recognizing domain that can further be engineered. The aim of this study was to screen potential pseudo att sites of ΦC31 integrase in the human genome, and evaluate the risks of its application in human gene therapy. METHODS: TFBS (transcription factor binding sites) were found on the basis of reported pseudo att sites using multiple motif-finding tools, including AlignACE, BioProspector, Consensus, MEME, and Weeder. The human genome with the proposed motif was scanned to find the potential pseudo att sites of ΦC31 integrase. RESULTS: The possible recognition motif of ΦC31 integrase was identified, which was composed of two co-occurrence conserved elements that were reverse complement to each other flanking the core sequence TTG. In the human genome, a total of 27924 potential pseudo att sites of ΦC31 integrase were found, which were distributed in each human chromosome with high-risk specificity values in the chromosomes 16, 17, and 19. When the risks of the sites were evaluate more rigorously, 53 hits were discovered, and some of them were just the vital functional genes or regulatory regions, such as ACYP2, AKR1B1, DUSP4, etc. CONCLUSION: The results provide clues for more comprehensive evaluation of the risks of using ΦC31 integrase in human gene therapy and for drug discovery.

Association between the CCR2-Val64Ile polymorphism and susceptibility to HIV-1 infection: a meta-analysis.

Several studies have investigated whether the CCR2-Val64Ile polymorphism affects susceptibility to human immune deficiency virus type-1 (HIV-1), with inconclusive results. Here, we performed a meta-analysis of the literature aiming to clarify the relationship between the polymorphism of CCR2-Val64Ile and the risk of HIV-1 infection. Twelve studies with a total of 6,599 patients, including infants, were selected for inclusion in the analysis. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were assessed after the collected data were pooled for analysis. The risk estimates (OR) of HIV-1 infection were calculated in a homozygote comparison (OR=1.10, 95% CI 0.79-1.53), a heterozygote comparison (OR=0.98, 95% CI 0.70-1.37), a dominant model (OR=1.06, 95% CI 0.77-1.47) and a recessive model (OR=0.98, 95% CI 0.77-1.27, by random effects model) from among the total population. Taking into account the effect of sample size, ethnicity and control population, further stratified analyses were performed. The results showed no statistically significant difference in any genetic model, with the exception of the sub-analysis of mixed ethnicity (OR=0.33, 95% CI 0.11­0.98) using heterozygote comparison. The meta-analysis clarified that the CCR2-Val64Ile polymorphism has no effect on susceptibility to HIV-1 infection in the total population.

Histone deacetylase inhibitor Scriptaid reactivates latent HIV-1 promoter by inducing histone modification in in vitro latency cell lines.

Human immunodeficiency virus type 1 (HIV-1) latency remains a major problem for the eradication of viruses in infected individuals undergoing highly active anti-retroviral therapy. By inhibiting HIV-1 gene expression and virus production, histone deacetylase (HDAC) may contribute to the quiescence of HIV-1 within resting CD4+ T cells. A novel HDAC inhibitor, Scriptaid, has been found to have robust activity and lower toxicity compared to trichostatin A (TSA). We therefore investigated Scriptaid for its capability to reverse HIV-1 latency by inducing HIV-1 activation in the Jurkat T cell line containing latent HIV proviruses. We found that Scriptaid can activate HIV-1 gene expression in these latent infected cells by 2-15-fold over background levels, as analyzed by flow cytometry. Chromatin immunoprecipitation (ChIP) assays further revealed that the Scriptaid increased the acetylation level of histones H3 and H4 at the nucleosome 1 site of the HIV-1 long terminal repeat compared to mock treatment. In addition, Scriptaid can synergize with prostratin or tumor necrosis factor-alpha to activate the HIV-1 promoter, with relatively lower toxicity compared to TSA. These studies suggest the potential of Scriptaid in anti-latency therapies.

TAT-phiC31 integrase mediates DNA recombination in mammalian cells.

Streptomyces phage integrase phiC31 is capable of mediating site-specific insertions in mammalian genomes. To avoid potential toxicity of long-term expression of phiC31 in host cells, we developed a method employing a cell-permeable TAT-phiC31 integrase. His6-tagged phiC31 proteins with or without an HIV TAT intercellular transducing peptide were generated and purified. Both of them retained integrase activity in vitro. However, TAT-phiC31 but not phiC31 was able to mediate a specific integration between two att sites in the genome of 293-PB [EGFP] report cell line. Transduced TAT-phiC31 was mainly localized in the cytoplasm that is similar to the localization of phiC31 when expressed through cDNA transfection. Adding a nuclear localization signal (NLS) peptide to the C-terminus of TAT-phiC31 facilitated nuclear localization of the integrase with an increased efficiency of recombination in the reporter cell line. These results demonstrated that TAT can mediate a cell membrane entry of phiC31 protein to perform a site-specific integration in mammalian cells. This is a simple and possibly safer method of site-specific recombination for gene delivery.

Eliminating bacteria backbone of naked DNA enhanced hFIX expression and reduced inflammatory response in mice.

To test the hypothesis that the persistent high level of transgene expression of linear DNA eliminating bacterial backbone (LDEBB) results from less cytokine induction in vivo. We systematically investigated the effect of circular DNA (C DNA), linear DNA (L DNA) and LDEBB on gene expression in mice by hydrodynamics-based plasmid administration, and then determined serum cytokine levels in mice by enzyme linked immunosorbent assay (ELISA). The expression of human clotting factor IX (hFIX) gene in mice treated with LDEBB, L DNA or C DNA reached a maximum 1-day after injection (9809, 6447, 2368 ng/mL), respectively. Thirty days after injection, hFIX concentrations dropped to baseline in mice treated with C DNA group, while L DNA group and LDEBB group decreased to 207 and 377 ng/mL, respectively, at the same time-point. Mice receiving LDEBB encoding hFIX expressed approximately 1.5 to 20-fold more serum hFIX than mice injected with L DNA and C DNA for a period of 8 months, respectively. However, mice receiving LDEBB are much less inflammatory than L DNA and C DNA as shown by a 4-fold reduction in serum levels of both TNF-alpha and IL-12. These results demonstrate that LDEBB is not silenced and is capable of expressing persistently high levels of transgene in vivo, which result from less cytokine induction in vivo. LDEBB provides a promising approach and useful therapeutic strategy to improve naked DNA delivery.

DAXX interacts with phage PhiC31 integrase and inhibits recombination.

Phage PhiC31 integrase has potential as a means of inserting therapeutic genes into specific sites in the human genome. However, the possible interactions between PhiC31 integrase and cellular proteins have never been investigated. Using pLexA-PhiC31 integrase as bait, we screened a pB42AD-human fetal brain cDNA library for potential interacting cellular proteins. Among 61 positives isolated from 10(6) independent clones, 51 contained DAXX C-terminal fragments. The strong interaction between DAXX and PhiC31 was further confirmed by co-immunoprecipitation. Deletion analysis revealed that the fas-binding domain of DAXX is also the region for PhiC31 binding. Hybridization between a PhiC31 integrase peptide array and an HEK293 cell extract revealed that a tetramer, 451RFGK454, in the C-terminus of PhiC31 is responsible for the interaction with DAXX. This tetramer is also necessary for PhiC31 integrase activity as removal of this tetramer resulted in a complete loss of integrase activity. Co-expression of DAXX with PhiC31 integrase in a HEK293-derived PhiC31 integrase activity reporter cell line significantly reduced the PhiC31-mediated recombination rate. Knocking down DAXX with a DAXX-specific duplex RNA resulted in increased recombination efficiency. Therefore, endogenous DAXX may interact with PhiC31 causing a mild inhibition in the integration efficiency. This is the first time that PhiC31 was shown to interact with an important cellular protein and the potential effect of this interaction should be further studied.

Conditional gene modification in mouse liver using hydrodynamic delivery of plasmid DNA encoding Cre recombinase.

The success of Cre-mediated conditional gene targeting in liver of mice has until now depended on the generation of Cre recombinase transgenic mice or on viral-mediated transduction. Here, we sought to establish the feasibility of using hydrodynamic gene delivery of Cre recombinase into liver, using a ROSA26 EGFP mouse. The expression of EGFP and beta-galactosidase was exclusively detected in the liver of mice treated with hydrodynamic gene delivery of Cre recombinase, as assessed with fluorescence microscopy and X-Gal staining, respectively; Southern blotting also showed that Cre mediated recombination occurred specifically in the liver and not in other organs. The Cre mediated recombination reached about 61% of hepatocytes of mouse after repeated injection, as analyzed by flow cytometry. These results demonstrate that Cre recombinase can be transferred to the liver of mice through a simple hydrodynamic gene-delivery approach and can mediate efficient recombination in hepatocytes. Thus, hydrodynamic gene delivery of the Cre recombinase provides a valuable approach for Cre-loxP-mediated conditional gene modification in the liver of mice.

Efficient delivery of human clotting factor IX after injection of lentiviral vectors in utero.

AIM: To explore gene transfer feasibility for human clotting factor IX (hFIX) mediated by recombinant lentivirus in utero. METHODS: ICR mice fetus at 17-19 d gestation were received lentiviral vectors carrying hFIX cDNA under the control of liver specific promoter by intrahepatic injection. The expression and distribution of hFIX cDNA and possible immune responses against the hFIX were assessed by ELISA, PCR, RT-PCR, and immunohistochemistry, respectively. RESULTS: The serum hFIX protein were detected at different time points in all newborn mice, the highest level of hFIX was 50 microg/L and lasted for more than 30 d. Anti-hFIX antibody was not detected. hFIX cDNA was detected in liver, spleen, and heart. The expression of hFIX cDNA was only detected in liver. Besides, no germ line transmission was found at DNA and RNA levels, and no side effect associated with gene transfer was detected. CONCLUSION: The efficient delivery of hFIX can be achieved by prenatal gene transfer. It thus shows the feasibility of gene therapy for hemophilia in utero.

Enhancement of naked FIX minigene expression by chloroquine in mice.

AIM: To study the effect of chloroquine on the expression of human clotting factor IX (hFIX) in mice. METHODS: Hydrodynamics-based naked DNA plasmid administration was performed by tail vein injection of 10 microg of pCMV- hFIX and chloroquine (0, 100, 200, and 500 micromol/L) in 2.2 mL of Ringer's solution within 6-7 s, the level and stability of hFIX expression, liver damage and toxicity were then examined. RESULTS: The maximum expression of hFIX level was 4.4+/-1.8 mg/L at 8 h after injection, 9.7+/-1.6 mg/L at 24 h only existed in 200 micromol/L chloroquine-treated animals, which is 3-4 fold higher than that of control (P<0.01). There is no significant difference observed among all the treated groups, 3 d later. Transaminase level and liver histological study showed the damage of liver was not related to chloroquine (P>0.05). CONCLUSION: Chloroquine can enhance and sustain exogenous gene expression in vivo without side effect under our experimental conditions.

Generation and characterization of transgenic mice expressing tamoxifen-inducible cre-fusion protein specifically in mouse liver.

AIM: To establish transgenic mice expressing tamoxifen-inducible Cre-ERt recombinase specifically in the liver and to provide an efficient animal model for studying gene function in the liver and creating various mouse models mimicking human diseases. METHODS: Alb-Cre-ERt transgenic mice were produced by microinjecting the construct with Cre-ERt fusion gene of DNA fragments into fertilized eggs derived from inbred C57BL/6 strain. Transgenic mice were identified by using PCR and Southern blotting. Expression of Cre-ERt fusion gene was analyzed in the liver, kidney, brain and lung from F1 generation transgenic mice at 8 weeks of age by reverse transcription (RT)-PCR. RESULTS: Four hundred and fourteen fertilized eggs of C57 BL/6 mice were microinjected with recombinant Alb-Cre-ERt DNA fragments, and 312 survival eggs injected were transferred to the oviducts of 12 pseudopregnant recipient mice, 6 of 12 recipient mice became pregnant and gave birth to 44 offsprings. Of the 44 offsprings, two males and one female carried the hybrid Cre-ERt fusion gene. Three mice were determined as founders, and were back crossed to set up F1 generations with other inbred C57BL/6 mice. Transmission of Cre-ERt fusion gene in F1 offspring followed Mendelian rules. The expression of Cre-ERt mRNA was detected only in the liver of F1 offspring from two of three founder mice. CONCLUSION: Transgenic mice expressing tamoxifen-inducible Cre-ERt recombinase under control of the liver-specific promoter are preliminary established.

Conditional gene activation in cultured hepatocytes using a ligand-dependent chimeric Cre recombinase.

By combining liver-specific promoter and a chimeric Cre recombinase, conditional gene activation could be finely achieved in hepatocytes at selected time points. To this end, the expression vector of Cre-ERt under the control of the mouse albumin gene promoter/enhancer, alb-Cre-ERt, was constructed, and transfected into engineering BRL (Rat hepatocytes) and BRK (Rat kidney) reporter cells which carries a chromosomally integrated 'floxed' beta geo gene, which is inserted between the promoter and the human alkaline phosphatase( hAP) reporter gene, thereby preventing hAP reporter gene transcription, respectively. After treatment with 1 micromol/L 4-hydroxytamoxifen(4-OHT), a proportion of hAP staining positive cells were detected by hAP staining. It was further confirmed that 'floxed' beta geo cassette was removed by Cre excision by using PCR analysis of cellular DNA. No background recombinase activity could be detected in the absence of 4-OHT. Moreover, no hAP-positive cells could be detected in BHK cells untreated or treated with 4-OHT. These data suggested that alb-Cre-ERt expression vector was constructed successfully, and 4-OHT could induce Cre-mediated recombination only in hepatocytes expressing Cre-ERt, thereby activating a stably integrated hAP reporter gene. This provides a further foundation for producing transgenic mice expressing such an 4-OHT inducible Cre recombinase specifically in mouse liver.

Expression of glutathione S-transferase placental mRNA in hepatic preneoplastic lesions in rats.

AIM:To detect glutathione S-transferase placental (GST-P) mRNA expression in hepatic preneoplastic lesions in rats.METHODS: Using Solit-Farber model, the GST-P mRNA expression was observed in hepatic preneoplastic lesions induced by diethylnitrosamine (DEN) in rats and normal and regenerated hepatic tissues in the control group by in situ hybridization.RESULTS: GST-P mRNA was mainly expressed in altered hepatic foci (AHF) and some of the oval cells in hepatic preneoplastic lesions and the extent of its expression was different among various foci or/and positive cells in the same focus whereas no expression was observed in normal and regenerated hepatic tissues.CONCLUSION: Cells in AHF and oval cells may be the preneoplastic cells in the experimental hepatocellular carcinoma at the molecular level and heterogeneity exists in GST-P transcription levels.

Preliminary study on the production of transgenic mice harboring hepatitis B virus X gene.

AIM:To establish transgenic mice lineage harboring hepatitis B virus X gene and to provide an efficient animal model for studying the exact role of the HBx gene in the process of hepatocarcinogenesis.METHODS:The HBx transgenic mice were produced by microinjecting the construct with X gene of HBV (subtype adr) DNA fragment into fertilzed eggs derived from inbred C57BL/6 strain; transgenic mice were identified by using Nested PCR; expression and phenotype of HBx gene were analyzed in liver from transgenic mic at the age of 8 weeks by RT-PCR, pathologic examination and periodic acid-schiff staining (PAS), respectively.RESULTS:Five hundred and fourteen fertilized eggs of C57 BL/6 mice were microinjected with recombinant retroviral DNA fragment, and 368 survival eggs injected were transferred to the oviducts of 18 pseudopregnant recipient mice, 8 of them became pregnant and gave birth to 20 F1 offspring. Of 20 offsprings, four males and two females carried the hybrid gene (HBx gene). Four male mice were determined as founder, named X 1, X 5, X 9 and X 15. These founders were back crossed to set up F1 generations with other ibred C57BL/6 mice or transgenic littermates, respectively.Transmission of HBx gene in F1 offspring of X 1, X 5 and X 9 except in X 15 followed Mendelian rules. The expression of HBx mRNA was detected in liver of F1 offspring from the founder mice (X 1 and X 9), which showed vacuolation lesion and glycogen positive foci.CONCLUSION:Transgenic mice harboring HBx gene were preliminarily established.