TRIM22 can activate the noncanonical NF-κB pathway by affecting IKKα.

Tripartite motif 22 (TRIM22) is involved in various cellular processes. It has been reported that TRIM22 can activate nuclear factor-κB (NF-κB) pathway, but the precise mechanism remains unclear. In this study, we explored the exact role of TRIM22 in activating the NF-κB pathway. Different to tumor necrosis factor-α (TNF-α) induction, we found that the overexpression of TRIM22 could induce the processing of p100 to p52 in HEK293T cells. Furthermore, based on the results of co-immunoprecipitation and co-localization experiments, we demonstrated that TRIM22 could interact with IκB kinase (IKK)α but not IKKß and could increase the level and phosphorylation of IKKα through its really interesting new gene (RING) and spla-ryanodine receptor (SPRY) domains. These results suggest that TRIM22 is able to activate the noncanonical but not the canonical NF-κB pathway by activating IKKα. This finding will aid our understanding of the biological function of TRIM22.

Associated changes in the transcription levels of IL-17A and tight junction-associated genes in the duodenal mucosa of rhesus macaques repeatedly exposed to simian/human immunodeficiency virus.

BACKGROUND: Mucosal barrier dysfunction might play a key role in HIV/AIDS, yet the early effects of HIV-1 on intestinal mucosal barrier, especially tight junctions (TJ) have not been well addressed. AIMS: To investigate the effects of acute HIV-1 infection on the expression of intestinal IL-17A and TJ-associated genes using an NHP-AIDS model. METHODS: TaqMan probe real-time RT-PCR methods were established and claudin-1, claudin-3, occludin and zonula occluden-1 (ZO-1) mRNA levels in the duodenal biopsies of rhesus macaques collected before and after rectal exposures to SHIV-SF162P4 were examined and compared with that of IL-17A, IL-6, TGF-ß, RORγt, T-bet, Foxp-3 and GATA-3. RESULTS: The mRNA levels of TJ-associated genes were statistically significantly reduced soon after viral exposures and the mRNA levels of claudin-1, occludin and ZO-1 in viral positive tissues (from Group I) were lower than that in viral negative tissues (from Group II) after viral exposure. IL-17A mRNA levels were also decreased and positively correlated with the mRNA levels of the TJ-associated genes after viral exposure or infection, although the levels of IL-6, TGF-ß and RORγt mRNA showed no statistical difference. The levels of GATA-3 mRNA in tissues collected before viral exposure were statistically different between Group I and Group II animals. The balance between T-bet and GATA-3 mRNA levels in Group II was markedly altered and statistically significantly different from that in Group I. CONCLUSIONS: Acute SHIV, and by extension HIV infection could affect the expression of TJ-associated genes, probably through IL-17A and other immune alterations.

DNA methyltransferases 1 and 3B are required for hepatitis C virus infection in cell culture.

DNA methyltransferases (DNMTs) are responsible for establishing and maintaining DNA methylation, which are dysregulated in hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC). In this report, using lentivirus-mediated shRNA interference technology, we identified DNMT1 and DNMT3B as host factors involved in HCV propagation. Our results demonstrated that down-regulation of DNMT1 or DNMT3B expression in Huh7.5.1 cells severely impaired cell culture-produced HCV (HCVcc) infection. Furthermore, knockdown of DNMT1 or DNMT3B did not affect HCV entry and internal ribosome entry site (IRES)-directed translation but did inhibit subgenomic replication. In contrast, knockdown of DNMT3A had no significant effect on HCV infection, entry, translation, or replication, which suggested that DNMT3A did not play a significant role in HCV life cycle. Moreover, we showed that DNMT inhibitors 5-Aza-C and 5-Aza-dC significantly suppressed HCVcc infection, viral RNA replication, and protein expression. These results suggest that DNMTs are critical for HCV replication and may represent potent targets for the treatment of HCV infection.

Characterization of TRIM62 as a RING finger E3 ubiquitin ligase and its subcellular localization.

TRIM62, also named DEAR1, is a member of the TRIM/RBCC family, which includes proteins with conserved RING finger, B-box and coiled-coil domains. Several reports have identified a role for this family in cancer, retroviral infection and innate immunity. In this study, the E3 ubiquitin ligase activity and subcellular localization of TRIM62 were characterized. TRIM62, in association with the E2 enzyme UbcH5b, was found to catalyze self-ubiquitination in vitro, a process that required an intact RING finger domain. A ubiquitination assay performed in HEK293T cells further confirmed the E3 ubiquitin ligase activity and self-ubiquitination activity of TRIM62 and the requirement of the RING finger domain. Importantly, the treatment of HEK293T cells with a proteasome inhibitor stabilized poly-ubiquitinated TRIM62, indicating that self-ubiquitination promoted the proteasomal degradation of TRIM62. Additionally, TRIM62 and its two mutants were distinctly localized in the cytoplasm in both HEK293T and HeLa cells. Collectively, our data indicate that TRIM62, a cytoplasmic protein, is a RING finger domain-dependent E3 ubiquitin ligase that catalyzes self-ubiquitination both in vitro and in vivo.

(-)-Epigallocatechin-3-gallate inhibits the replication cycle of hepatitis C virus.

(-)-Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea. In this study, we found that hepatitis C virus (HCV) infection was significantly suppressed by EGCG in an HCV cell culture (HCVcc) system using a JFH1-GFP chimeric virus, with a 50 % effective concentration (EC(50)) of 17.9 µM. The inhibitory activity of EGCG was confirmed by monitoring HCV RNA and protein expression levels in Huh7.5.1 cells infected with the JFH1 virus. Moreover, we demonstrated that the inhibitory mechanisms of EGCG were attributable to the suppression of both the HCV entry and RNA replication steps, although EGCG had little effect on translation directed by the viral internal ribosome entry site (IRES). Furthermore, HCV could be rapidly eliminated from cell cultures after two and five passages in the presence of 50 and 25 µM EGCG, respectively. These results indicate that EGCG is a potential candidate as a preventive and antiviral drug for HCV infection.

Emerging trends of drug-resistant HIV-1 among drug-treated patients in former blood donors in Hubei, China: a three-year surveillance from 2004 to 2006.

This study aimed to evaluate emerging trends of drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) among 290 former blood donor HIV-1 infected patients in Hubei, China, from 2004 to 2006, all of whom had received anti-HIV-1 therapy. The presence of NRTI- and NNRTI-associated mutations were established by sequencing; genotypic and predicted phenotypic drug resistance were evaluated using HIVdb Program version 5.0.1 (http://hivdb.stanford.edu/pages/algs/HIVdb.html ). Genotypic drug resistance analysis showed significant increases in percentages of patients carrying HIV-1 strains with M41L, T215Y/F, D67N, K103N, G190A/S, Y181C/F or L210W mutations. Of the variants' predicted phenotypic drug resistance, highly significant increases were detected in percentages of patients carrying HIV-1 with high resistance to zidovudine (AZT) or stavudine (D4T) in NRTIs, and to delavirdine (DLV), efavirenz (EFV) or nevirapine (NVP) in NNRTIs; intermediate resistance to abacavir (ABC), AZT, D4T, didanosine (DDI) or tenofovir disoproxil fumarate (TDF) in NRTIs, and to etravirine (ETR) in NNRTIs; and low and potential low resistance to lamivudine (3TC), ABC, emtricitabine (FTC) or TDF in NRTIs, and to ETR in NNRTIs.

Rhesus monkey TRIM5α represses HIV-1 LTR promoter activity by negatively regulating TAK1/TAB1/TAB2/TAB3-complex-mediated NF-κB activation.

TRIM5α has been identified as the main restriction factor responsible for resistance of Old World monkey cells to HIV-1 infection. The precise mechanism of viral inhibition by TRIM5α remains elusive but appears to occur in multiple ways. Here, we report that rhesus monkey TRIM5α (TRIM5α(rh)) can represses HIV-1 LTR promoter activity by negatively regulating TAK1/TAB1/TAB2/TAB3-complex-mediated NF-κB activation when TRIM5α(rh) is overexpressed. We show that the overexpressed TRIM5α(rh) can interact with the TAK1/TAB1/TAB2/TAB3 complex by binding to TAB1 and promotes the degradation of TAB2 within the complex via the lysosomal degradation pathway. Subsequently, TRIM5α(rh) lowers the IKKα protein level and inhibits NF-κB p65 phosphorylation, and knockdown of TRIM5α(rh) expression by small interfering RNA in TRIM5α(rh)-overexpressing cells can abolish this inhibition. Finally, the inhibition of p65 phosphorylation results in the repression of HIV-1 LTR promoter activity. Taken together, these findings indicate that TRIM5α(rh) plays a previously unrecognized role in repressing HIV-1 transcription by inhibiting TAK1/TAB1/TAB2/TAB3-complex-mediated NF-κB activation when TRIM5α(rh) is overexpressed.

Rhesus monkey TRIM5α has distinct HIV-1 restriction activity among different mammalian cell lines.

Rhesus monkey TRIM5α (TRIM5α(rh)), a member of the tripartite motif (TRIM) family, was identified as the main restriction factor responsible for resistance of old world monkey cells to HIV-1 infection. However, the precise mechanism of HIV-1 infection inhibition by TRIM5α remains elusive and appears to be related to some cellular cofactors. Here we reported that TRIM5α(rh) can significantly reduce the infection efficiency of VSV-G pseudotyped HIV-1/MA-YFP virus in human epithelial carcinoma (HeLa) cells, moderately reduce in porcine kidney (PK-15) cells and have no effect on the pseudotyped virus infection in Madin-Darby canine kidney (MDCK) cells. Furthermore, we found that the different HIV-1 restriction activities have no relation with the intracellular localization of TRIM5α(rh). These results indicate that the cellular environment is very important for the efficient anti-HIV-1 activity of TRIM5α(rh). We speculate that some unknown factors required for HIV-1 infection inhibition activity are adequately expressed in HeLa cells, inadequately expressed in PK-15 cells and absent in MDCK cells.

Down-regulation of HIV-1 infection by inhibition of the MAPK signaling pathway.

The human immunodeficiency virus type 1 (HIV-1) can interact with and exploit the host cellular machinery to replicate and propagate itself. Numerous studies have shown that the Mitogen-activated protein kinase (MAPK) signal pathway can positively regulate the replication of HIV-1, but exactly how each MAPK pathway affects HIV-1 infection and replication is not understood. In this study, we used the Extracellular signal-regulated kinase (ERK) pathway inhibitor, PD98059, the Jun N-terminal kinase (JNK) pathway inhibitor, SP600125, and the p38 pathway inhibitor, SB203580, to investigate the roles of these pathways in HIV-1 replication. We found that application of PD98059 results in a strong VSV-G pseudotyped HIV-1(NL4-3) luciferase reporter virus and HIV-1(NL4-3) virus inhibition activity. In addition, SB203580 and SP600125 also elicited marked VSV-G pseudotyped HIV-1(NL4-3) luciferase reporter virus inhibition activity but no HIV-1(NL4-3) virus inhibition activity. We also found that SB203580 and SP600125 can enhance the HIV-1 inhibition activity of PD98059 when cells were treated with all three MAPK pathway inhibitors in combination. Finally, we show that HIV-1 virus inhibition activity of the MAPK pathway inhibitors was the result of the negative regulation of HIV-1 LTR promoter activity.

Activity of superior interferon α against HIV-1 in severe combined immunodeficient mice reconstituted with human peripheral blood leukocytes.

BACKGROUND: Interferon (IFN) can inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro and in clinic. However, IFN therapy for HIV infection was limited by its moderate antiviral efficacy and its frequent adverse effects. In the present study we evaluated the anti-HIV efficacy of a novel synthesized superior interferon α (sIFNα). METHODS: We performed in vitro experiments with HIV-1 IIIB infected MT4 cells, and evaluated in vivo anti-HIV efficacy of sIFNα in severe combined immunodeficient (SCID) mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID mice). RESULTS: We found that the 50% effective concentrations (EC(50)) of sIFNα against the replication of HIV-1 in MT4 cells was 0.06 ng/ml, representing stronger antiviral activity than interferon-α in vitro. In the hu-PBL-SCID mice, a dose-dependent protection pattern was observed: with 0.45 µg and 1.35 µg sIFNα daily treatments, parts of SCID mice were protected from HIV infection, whereas 2.25 µg sIFNα daily treatments resulted in a terminally complete protection. CONCLUSIONS: sIFNα shows good anti-HIV activity both in vitro and in SCID mice, may be a promising anti-HIV agent deserving clinical investigation, especially considering the potential of IFN-α to inhibit HIV replication in patients infected with drug-resistant variants or co-infected with hepatitis C virus (HCV).

Human immunodeficiency virus-1 genotypic drug resistance among volunteer blood donors in Yunnan, China.

BACKGROUND: Drug resistance profiles of human immunodeficiency virus-1 (HIV-1) in treatment-naïve infections have been reported in developed countries. However, little is known in developing countries, including China, especially in treatment-naïve volunteer blood donors. STUDY DESIGN AND METHODS: Fifty-two HIV-1-positive samples of blood donors were collected from 2005 to 2006 in Yunnan, China. Recent and long-term infections were distinguished by the HIV-1 subtypes B, E, and D immunoglobulin G-capture enzyme immunoassay assay. The nucleotide sequences of pol genes were amplified and sequenced. Phylogenetic tree and drug resistance analyses were performed. RESULTS: Of 49 samples successfully analyzed, circulating strains were circulating recombinant form (CRF)08_BC (51.0%), CRF07_BC (24.5%), CRF01_AE (20.4%), and B (4.1%). No protease inhibitors (PI) major drug resistance mutation (DRM) was detected. Six samples (12.2%) displayed seven minor PI DRMs. Nine samples (18.4%) displayed 10 nucleoside reverse transcriptase inhibitor DRMs, and DRMs to nonnucleoside reverse transcriptase inhibitors were present in one sample (2.0%). There was only one sample of the 49 (2.0%) in which the DRMs were of sufficient magnitude to result in a clinical change to drug susceptibility, but even in this sample, the clinical effect of these DRMs was predicted to be low. Significant differences were not observed between the long-term and recent infected population. Differences in DRMs were not observed between peripheral blood mononuclear cells and plasma within an individual. CONCLUSIONS: CRF_BC was the dominant subtype circulating in HIV-1-infected donors in Yunnan. Prevalence of genotypic drug resistances among donors in Yunnan was low in this study. Surveillance on HIV-1 infections among blood donors should be continued in China.

One-step label-free optical genosensing system for sequence-specific DNA related to the human immunodeficiency virus based on the measurements of light scattering signals of gold nanorods.

A one-step label-free optical genosensing method has been developed in this contribution by taking short DNA target with its sequence related to the human immunodeficiency virus type 1 (HIV-1) as an example. By employing anisotropic nonspherical and positively charged gold nanorods (Au-NRs) as the recognition platform, which show high stability against aggregation under high ionic strength conditions without any additional stable reagent, we found that the addition of target DNA to the mixture of nonmodified Au-NRs suspension and label-free probe DNA in high ionic strength buffer leads to a color change from red to light purple in less than 5 min, displaying strong plasmon resonance light scattering (PRLS) signals. Mechanism investigations showed that the strong PRLS signals should be ascribed to the aggregation of Au-NRs induced by the formed double-stranded oligonucleotides (dsDNA) from the hybridization of target DNA with probe DNA. With the PRLS signals, we monitored the hybridization process of a 21-mer single-stranded oligonucleotide (ssDNA) from the HIV-1 U5 long terminal repeat (LTR) sequence with its complementary oligonucleotide and detected the effect of single-base-pair mismatches. Two polymerase chain reaction (PCR) amplicon artificial samples derived from Mycobacterium tuberculosis glmS and genes encoding for Bacillus glucanase and an HIV-1 LTR sample isolated from HIV-1-positive blood were detected with satisfactory results, showing that the present method has simplicity, sensitivity, specificity, and reliability for sequence-specific DNA detection related to the HIV gene.

[Comparative proteome analysis of human papillomavirus-infected cervical specimens and the difference between the high- and low-risk genotypes of human papillomavirus].

OBJECTIVE: To perform an comparative proteome analysis of human papillomavirus-infected cervical specimens and to investigate different expressions between high- and low-risk genotypes. METHODS: The cervical specimens were divided into two groups (cervical intraepithelial neoplasia group and condyloma acuminatum group) according to their genotypes. Using comparative proteome technology, high-risk human papillomavirus-infected cervical intraepithelial neoplasia, low-risk human papillomavirus-infected condyloma acuminatum, and normal cervical intraepithelial tissue were compared. The differential expression protein spots were identified by mass spectrometry. RESULTS: Totally 26 differential spots were selected and analyzed, and 22 peptide mass fingerprints (PMF) maps were obtained by MALDI-TOF-MS. Eighteen proteins were preliminarily identified after searching the NCBInr database. The function information of these 18 proteins mainly involved cell metabolism, signal transduction, cell secretion, cell cytoskeleton construction, cell proliferation, and apoptosis. CONCLUSION: The proteomic expressions after the cervical infection of high- or low-risk genotype of human papillomavirus are obviously different.

[Study on genotypic resistance mutations to antiretroviral drugs on HIV strains of treated and treatment-naive HIV-1 infectious patients in Hubei province].

OBJECTIVE: To study the drug resistance status on HIV-1 patients who had been treated with highly active antiretroviral therapy (HAART) and those treatment-naive ones in Hubei province. METHODS: Nested polymerase chain reaction (PCR) was used to amplify 2 kb DNA fragment in HIV pol gene from peripheral blood of the HIV infected patients and the PCR products were sequenced. The sequences were compared to the Stanford HIV drug resistance database. RESULTS: Nineteen patients were treated with regimens composed of two Nucleoside Reverse Transcriptase Inhibitors (NRTIs) and one Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTI), with 25 patients as treatment-naive. Some protease (PR) drug-resistant mutations were found in these samples, such as D30N (2.27%), D30G (2.27%), M46I (4.55%), M46N (2.27%), I47V (4.55%), I84V (4.55%), I84L (2.27%), N88S (2.27%) and L90S (2.27%) that all belonged to major drug-resistant but A71T (29.55%) belonged to minor resistance mutations Five treated patients were detected having mutations associated RT drug resistance: M41L (5.26%), A62V (5.26%),D67N (5.26%), L210W (5.26%), T215Y (15.79%); K103E (5.26%), K103N (10.53%), Y181C (5.26%), G190A (5.26%), K238N (5.26%), while five treatment-naive patients were detected to have had mutations associated RT drug resistance M184V (4%), K65N (4%), Y115M (4%), F116L (4%), M184I (4%), V179D (4%), G190R (4%).Some additional mutations were detected in RT whose role involve in drug resistance still remained unknown. F214L was positively associated with HAART treatment (P = 0.03). CONCLUSION: Significant differences were found between drug resistance mutations to RTIs in treated and treat-naive patients in Hubei province,indicating that drugs had affected the occurrence of drug resistance mutations. At the same time, novel RT mutations F214L might be associated with HAART or some other drugs.

Snapshot of HIV pathogenesis in China.

Several reviews have focused on the nature of HIV infection and its spread in various geographical regions of China. In contrast, this review provides a comprehensive update on the prevalence of multiple HIV-1 subtypes, consequent emergence of recombinant and novel forms of HIV-1 in China, and the implications this may have on HIV diversity and the development of effective vaccines. In addition it also examines the dissemination of primary drug resistance in therapy naïve patients, as well as co-infections with two other important viruses-hepatitis B and C. The main purpose of this review is to provide a current snapshot of HIV-1 pathogenesis in China and possibly shed some light on the future of HIV evolution, and potential challenges for future vaccine and anti-retroviral therapeutics against HIV strains in this area.