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Background and Aim Interferon‐stimulated gene 20 (ISG20) is an interferon‐inducible exonuclease that inhibits the replication of several RNA viruses. In patients with chronic hepatitis B, ISG20 expression is related to the interferon‐α treatment response. However, the molecular mechanism of ISG20‐mediated anti‐hepatitis B virus (HBV) activity is unclear. Methods We have investigated the effect of ISG20 on antiviral activity to address that. The life cycle of HBV was analyzed by the ectopic expression of ISG20 in HepG2 and HepG2‐NTCP cells. Finally, to provide physiological relevance of our study, the expression of ISG20 from chronic hepatitis B patients was examined. Results Interferon‐stimulated gene 20 was mainly induced by interferon‐β and dramatically inhibited HBV replication. In addition, ISG20 decreased HBV gene expression and transcription. Although ISG20 inhibited HBV replication by reducing viral enhancer activity, the expression of transcription factors that bind the HBV enhancer was not affected. Particularly, ISG20 suppressed HBV enhancer activity by binding to the enhancer II and core promoter (EnhII/Cp) region. Conclusion Our findings suggest that ISG20 exerts the anti‐HBV activity by acting as a putative repressor binding to the HBV EnhII/Cp region.

Hepatitis C virus (HCV) infection can lead to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV employs diverse strategies to evade host antiviral innate immune responses to mediate a persistent infection. In the present study, we show that nonstructural protein 5A (NS5A) interacts with an NF-κB inhibitor immunomodulatory kinase, IKKε, and subsequently downregulats beta interferon (IFN-β) promoter activity. We further demonstrate that NS5A inhibits DDX3-mediated IKKε and interferon regulatory factor 3 (IRF3) phosphorylation. We also note that hyperphosphorylation of NS5A mediats protein interplay between NS5A and IKKε, thereby contributing to NS5A-mediated modulation of IFN-β signaling. Lastly, NS5A inhibits IKKε-dependent p65 phosphorylation and NF-κB activation. Based on these findings, we propose NS5A as a novel regulator of IFN signaling events, specifically by inhibiting IKKε downstream signaling cascades through its interaction with IKKε. Taken together, these data suggest an additional mechanistic means by which HCV modulates host antiviral innate immune responses to promote persistent viral infection.

Background Integrase (IN) is an essential protein for HIV replication that catalyzes insertion of the reverse-transcribed viral genome into the host chromosome during the early steps of viral infection. Highly active anti-retroviral therapy is a HIV/AIDS treatment method that combines three or more antiviral drugs often formulated from compounds that inhibit the activities of viral reverse transcriptase and protease enzymes. Early IN inhibitors (INIs) mainly serve as integrase strand transfer inhibitors (INSTI) that disrupt strand transfer by binding the catalytic core domain of IN. However, mutations of IN can confer resistance to INSTI. Therefore, non-catalytic integrase inhibitors (NCINI) have been developed as next-generation INIs. Methods In this study, we evaluated and compared the activity of INSTI and NCINI according to the analysis method. Antiviral activity was compared using p24 ELISA with MT2 cell and TZM-bl luciferase system with TZM-bl cell. Each drug was serially diluted and treated to MT2 and TZM-b1 cells, infected with HIV-1 AD8 strain and incubated for 5 and 2 days, respectively. Additionally, to analyze properties of INSTI and NCINI, transfer inhibition assay and 3′-processing inhibition assay were performed. Results During screening of INIs using the p24 ELISA and TZM-bl luciferase systems, we found an inconsistent result with INSTI and NCINI drugs. Following infection of MT2 and TZM-bl cells with T-tropic HIV-1 strain, both INSTI and NCINI treatments induced significant p24 reduction in MT2 cells. However, NCINI showed no antiviral activity in the TZM-bl luciferase system, indicating that this widely used and convenient antiretroviral assay is not suitable for screening of NCINI compounds that target the second round of HIV-1 replication. Conclusion Accordingly, we recommend application of other assay procedures, such as p24 ELISA or reverse transcription activity, in lieu of the TZM-bl luciferase system for preliminary NCINI drug screening. Utilization of appropriate analytical methods based on underlying mechanisms is necessary for accurate assessment of drug efficacy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the cause of a present pandemic, infects human lung alveolar type 2 (hAT2) cells. Characterizing pathogenesis is crucial for developing vaccines and therapeutics. However, the lack of models mirroring the cellular physiology and pathology of hAT2 cells limits the study. Here, we develop a feeder-free, long-term, three-dimensional (3D) culture technique for hAT2 cells derived from primary human lung tissue and investigate infection response to SARS-CoV-2. By imaging-based analysis and single-cell transcriptome profiling, we reveal rapid viral replication and the increased expression of interferon-associated genes and proinflammatory genes in infected hAT2 cells, indicating a robust endogenous innate immune response. Further tracing of viral mutations acquired during transmission identifies full infection of individual cells effectively from a single viral entry. Our study provides deep insights into the pathogenesis of SARS-CoV-2 and the application of defined 3D hAT2 cultures as models for respiratory diseases., Graphical Abstract, Highlights • Long-term 3D culture of primary hAT2 cells in chemically defined conditions • SARS-CoV-2 infects in vitro hAT2 models with substantial ultrastructural changes • Single-cell RNA-seq finds two cellular states of distinct innate immune phenotypes • Mutation analysis reveals single SARS-CoV-2 entry to a hAT2 cell for full infection, To study infection response to SARS-CoV-2, Youk et al. developed a 3D culture model of human alveolar stem cells in chemically defined conditions. They reveal that SARS-CoV-2 productively infects alveolar type 2 cells, resulting in an innate immune response. Full infection of individual cells was exhibited from a single viral entry.

Background: Integrase (IN) is an essential protein for HIV replication that catalyzes insertion of the reverse-transcribed viral genome into the host chromosome during the early steps of viral infection. Highly active anti-retroviral therapy (HAART) is a HIV/AIDS treatment method that combines three or more antiviral drugs often formulated from compounds that inhibit the activities of viral reverse transcriptase and protease enzymes. Early IN inhibitors (INIs) mainly serve as integrase strand transfer inhibitors (INSTI) that disrupt strand transfer by binding the catalytic core domain (CCD) of IN. However, mutations of IN can confer resistance to INSTI. Therefore, non-catalytic integrase inhibitors (NCINI) have been developed as next-generation INIs. Methods: In this study, we evaluated and compared the activity of INSTI and NCINI according to the analysis method. Antiviral activity was compared using p24 ELISA with MT2 cell and TZM-bl luciferase system with TZM-bl cell. Each drug was serially diluted and treated to MT2 and TZM-b1 cells, infected with HIV-1 AD8 strain and incubated for 5 and 2 days, respectively. Additionally, to analyze properties of INSTI and NCINI, transfer inhibition assay and 3'-processing inhibition assay were performed. Results: During screening of INIs using the p24 ELISA and TZM-bl luciferase systems, we found an inconsistent result with INSTI and NCINI drugs. Following infection of MT2 and TZM-bl cells with T-tropic HIV-1 strain, both INSTI and NCINI treatments induced significant p24 reduction in MT2 cells. However, NCINI showed no antiviral activity in the TZM-bl luciferase system, indicating that this widely used and convenient antiretroviral assay is not suitable for screening of NCINI compounds that target the second round of HIV-1 replication. Conclusion: Accordingly, we recommend application of other assay procedures, such as p24 ELISA or reverse transcription activity, in lieu of the TZM-bl luciferase system for preliminary NCINI drug screening. Utilization of appropriate analytical methods based on underlying mechanisms is necessary for accurate assessment of drug efficacy.

Background: Integrase (IN) is an essential protein for HIV replication that catalyzes insertion of the reverse-transcribed viral genome into the host chromosome during the early steps of viral infection. Highly active anti-retroviral therapy (HAART) is a HIV/AIDS treatment method that combines three or more antiviral drugs often formulated from compounds that inhibit the activities of viral reverse transcriptase and protease enzymes. Early IN inhibitors (INIs) mainly serve as integrase strand transfer inhibitors (INSTI) that disrupt strand transfer by binding the catalytic core domain (CCD) of IN. However, mutations of IN can confer resistance to INSTI. Therefore, non-catalytic integrase inhibitors (NCINI) have been developed as next-generation INIs. Methods: In this study, we evaluated and compared the activity of INSTI and NCINI according to the analysis method. Antiviral activity was compared using p24 ELISA with MT2 cell and TZM-bl luciferase system with TZM-bl cell. Each drug was serially diluted and treated to MT2 and TZM-b1 cells, infected with HIV-1 AD8 strain and incubated for 5 and 2 days, respectively. Additionally, to analyze properties of INSTI and NCINI, transfer inhibition assay and 3'-processing inhibition assay were performed. Results: During screening of INIs using the p24 ELISA and TZM-bl luciferase systems, we found an inconsistent result with INSTI and NCINI drugs. Following infection of MT2 and TZM-bl cells with T-tropic HIV-1 strain, both INSTI and NCINI treatments induced significant p24 reduction in MT2 cells. However, NCINI showed no antiviral activity in the TZM-bl luciferase system, indicating that this widely used and convenient antiretroviral assay is not suitable for screening of NCINI compounds that target the second round of HIV-1 replication. Conclusion: Accordingly, we recommend application of other assay procedures, such as p24 ELISA or reverse transcription activity, in lieu of the TZM-bl luciferase system for preliminary NCINI drug screening. Utilization of appropriate analytical methods based on underlying mechanisms is necessary for accurate assessment of drug efficacy.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which is the cause of a present global pandemic, infects human lung alveolar cells (hACs). Characterising the pathogenesis is crucial for developing vaccines and therapeutics. However, the lack of models mirroring the cellular physiology and pathology of hACs limits the study. Here, we develop a feeder-free, long-term three-dimensional (3D) culture technique for human alveolar type 2 (hAT2) cells, and investigate infection response to SARS-CoV-2. By imaging-based analysis and single-cell transcriptome profiling, we reveal rapid viral replication and the increased expression of interferon-associated genes and pro-inflammatory genes in infected hAT2 cells, indicating robust endogenous innate immune response. Further tracing of viral mutations acquired during transmission identifies full infection of individual cells effectively from a single viral entry. Our study provides deep insights into the pathogenesis of SARS-CoV-2, and the application of long-term 3D hAT2 cultures as models for respiratory diseases.

The current coronavirus (COVID-19) pandemic is exacerbated by the absence of effective therapeutic agents. Notably, patients with COVID-19 and comorbidities such as hypertension and cardiac diseases have a higher mortality rate. An efficient strategy in response to this issue is repurposing drugs with antiviral activity for therapeutic effect. Digoxin (DIG) and ouabain (OUA) are FDA drugs for heart diseases that have antiviral activity against several coronaviruses. Thus, we aimed to assess antiviral activity of DIG and OUA against SARS-CoV-2 infection. The half-maximal inhibitory concentrations (IC50) of DIG and OUA were determined at a nanomolar concentration. Progeny virus titers of single-dose treatment of DIG, OUA and remdesivir were approximately 103-, 104- and 103-fold lower (> 99% inhibition), respectively, than that of non-treated control or chloroquine at 48 h post-infection (hpi). Furthermore, therapeutic treatment with DIG and OUA inhibited over 99% of SARS-CoV-2 replication, leading to viral inhibition at the post entry stage of the viral life cycle. Collectively, these results suggest that DIG and OUA may be an alternative treatment for COVID-19, with potential additional therapeutic effects for patients with cardiovascular disease.

The advent of the global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) necessitates a thorough study of the stability and transmissibility in the environment. We characterized the stability of SARS-CoV-2 in three water matrices: fresh, tap, and seawater. The minimum infective dose of SARS-CoV-2 in Vero cells was confirmed to be 10³ PFU/mL. The stability of SARS-CoV-2 varied according to the water matrix: infective SARS-CoV-2 was undetectable after treatment with fresh water and seawater, but remained detectable for 2 days in tap water, when starting with an initial concentration of 10⁴ PFU/mL. When the starting concentration was increased to 10⁵ PFU/mL, a similar trend was observed. In addition, viral RNA persisted longer than infectious virus in all water matrices. This study was conducted in stagnant water containing a significantly high titer of virus, thus, human-to-human transmission of SARS-CoV-2 through the actual aquatic environment is expected to be rare.

Human immunodeficiency virus (HIV) encodes a transcription trans-activator (Tat) with an essential role in the transcriptional elongation of viral RNA based on the viral promoter long terminal repeat (LTR). Tat-mediated transcription is conserved and can be distinguished from host transcription, so it is a therapeutic target for combating HIV replication. Traditional screening assays for Tat-mediated transcriptional inhibitors are based on the biochemical properties of Tat and transactivation-responsive RNA. We developed an inducible system based on two lentiviral expression cassettes for doxycycline (Dox)-inducible Tat and Renilla luciferase (R-Luc) using TZM-bl cells harboring LTR-driven firefly luciferase (F-Luc). The cells simultaneously expressed both Tat-induced F-Luc and R-Luc, so it was possible to recognize off-target effects in the presence of Dox. The system was validated with known inhibitors: CYC202 obtained high sensitivity and specificity, whereas 6Bio and DRB had off-target effects. The MTT-based cytotoxicity test indicated the resistance of the system even at concentrations with off-target effects. The specificity of the system was confirmed using antiretroviral drugs. Our dual reporter system can simply detect Tat inhibitory effects, as well as precisely discriminate between the inhibitory and off-target effects of inhibitors, and may be useful for the development of a therapeutic anti-HIV drug.

Background A reservoir of HIV-1 is a major obstacle in eliminating HIV-1 in patients because it can reactivate in stopping antiretroviral therapy (ART). Histone modifications, such as acetylation and methylation, play a critical role in the organization of chromatin domains and the up- or downregulation of gene expression. Although many studies have reported that an epigenetic mechanism is strongly involved in the maintenance of HIV-1 transcriptional latency, neither the epigenetic control of viral replication nor how HIV-1 latency is maintained is not fully understood. Results We re-analyzed a high throughput parallel DNA sequencing (ChIP-seq) data from previous work to investigate the effect of histone modifications, H3K4me3 and H3K9ac, on HIV-1 latency in terms of chromosome distribution. The outputs of ChIP-seq from uninfected CD4+ T cell lines and HIV-1 latently infected cells were aligned to hg18 using bowtie and then analyzed using various software packages. Certain chromosomes (16, 17, 19, and 22) were significantly enriched for histone modifications in both decreased and increased islands. In the same chromosomes in HIV-1 latently infected cells, 38 decreased and 41 increased islands from common islands of H3K4me3 and H3K9ac were selected for functional annotation. In Gene Ontology analysis, the 38 genes associated with decreased islands were involved in the regulation of biological process, regulation of cellular process, biological regulation, and purinergic receptor signaling pathway, while the 41 genes associated with increased islands were involved in nucleic acid binding, calcium-activated cation channel activity, DNA binding, and zinc ion binding. In Pathway Commons analysis, the 38 genes were strongly involved in the p63 transcription factor network, while the 41 genes were involved in the RNA polymerase III transcription termination pathway. Several genes such as Nuclear factor I X (NFIX) and TNF receptor association factor 4 (TRAF4) were selected as candidate genes for HIV latency. Especially, NFIX was highly expressed in HIV-1 latently infected cell lines and showed a dramatic reduction in expression after phorbol-13-myristate-12-acetate (PMA) treatment. Conclusions These results show that the unique enrichment of histone modifications and its linked genes in specific chromosomes might play a critical role in the establishment and maintenance of HIV-1 latency. Electronic supplementary material The online version of this article (doi:10.1186/s40246-017-0105-7) contains supplementary material, which is available to authorized users.

Trans-activator (Tat)-mediated human immunodeficiency virus type 1 (HIV-1) transcription is essential for the replication of HIV-1 and is considered a potent therapeutic target for HIV-1 inhibition. In this study, the Library of Pharmacologically Active Compounds (LOPAC1280) was screened using our dual-reporter screening system for repositioning as Tat-inhibitory compounds. Consequently, two compounds were found to be potent, with low cytotoxicity. Of these two compounds, Roscovitine (CYC202) is already known to be a Tat inhibitor, while gemcitabine has been newly identified as an inhibitor of Tat-mediated transcription linked to viral production and replication. In an additional screening using the ribonucleoside analogues of gemcitabine, two analogues (2'-C-methylcytidine and 3-deazauridine) showed a specific Tat-inhibitory effect linked to their anti-HIV-1 activity. Interestingly, these compounds did not affect Tat protein directly, while the mechanism underlying their inhibition of Tat-mediated transcription was linked to pyrimidine biosynthesis, rather than to alteration of the dNTP pool, influenced by the inhibition of ribonucleotide reductase. Taken together, the proposed functional screening system is a useful tool for the identification of inhibitors of Tat-mediated HIV-1 transcription from among a large number of compounds, and the inhibitory effect of HIV-1 transcription by gemcitabine and its analogues may suggest a strategy for developing a new class of therapeutic anti-HIV drugs.

Population-level phylogenetic patterns reflect both transmission dynamics and genetic changes, which accumulate because of selection or drift. In this study, we determined whether a longitudinally sampled dataset derived from human immunodeficiency virus (HIV)-1-infected individuals over a 14-year period (1999-2012) could shed light on the transmission processes involved in the initiation of the HIV-1 epidemic in Korea. In total, 927 sequences were acquired from 1999 to 2012; each sequence was acquired from an individual patient who had not received treatment. Sequences were used for drug resistance and phylogenetic analyses. Phylogenetic and other analyses were conducted using MEGA version 6.06 based on the GTR G+I parameter model and SAS. Of the 927 samples, 863 (93.1%) were classified as subtype B and 64 were classified as other subtypes. Phylogenetic analysis demonstrated that 104 of 927 patient samples (11.2%) were grouped into 37 clusters. Being part of a transmission cluster was significantly associated with subtype-B viruses, infection via sexual contact, and the infection of young males. Of all clusters, three (~8.1%) that comprised 10 individual samples (22.2% of 45 individuals) included at least one member with total transmitted drug resistance (TDR). In summary, HIV transmission cluster analyses can integrate laboratory data with behavioral data to enable the identification of key transmission patterns to develop tailored interventions aimed at interrupting transmission chains.

Humanized mouse models derived from immune-deficient mice have been the primary tool for studies of human infectious viruses, such as human immunodeficiency virus (HIV). However, the current protocol for constructing humanized mice requires elaborate procedures and complicated techniques, limiting the supply of such mice for viral studies. Here, we report a convenient method for constructing a simple HIV-1 mouse model. Without prior irradiation, NOD/SCID/IL2Rγ-null (NSG) mice were intraperitoneally injected with 1 × 10(7) adult human peripheral blood mononuclear cells (hu-PBMCs). Four weeks after PBMC inoculation, human CD45(+) cells, and CD3(+)CD4(+) and CD3(+)CD8(+) T cells were detected in peripheral blood, lymph nodes, spleen, and liver, whereas human CD19(+) cells were observed in lymph nodes and spleen. To examine the usefulness of hu-PBMC-inoculated NSG (hu-PBMC-NSG) mice as an HIV-1 infection model, we intravenously injected these mice with dual-tropic HIV-1DH12 and X4-tropic HIV-1NL4-3 strains. HIV-1-infected hu-PBMC-NSG mice showed significantly lower human CD4(+) T cell counts and high HIV viral loads in the peripheral blood compared with noninfected hu-PBMC-NSG mice. Following highly active antiretroviral therapy (HAART) and neutralizing antibody treatment, HIV-1 replication was significantly suppressed in HIV-1-infected hu-PBMC-NSG mice without detectable viremia or CD4(+) T cell depletion. Moreover, the numbers of human T cells were maintained in hu-PBMC-NSG mice for at least 10 weeks. Taken together, our results suggest that hu-PBMC-NSG mice may serve as a relevant HIV-1 infection and pathogenesis model that could facilitate in vivo studies of HIV-1 infection and candidate HIV-1 protective drugs.

Background & Aims Tenofovir disoproxil fumarate (TDF) is one the most potent nucleot(s)ide analogues for treating chronic hepatitis B virus (HBV) infection. Phenotypic resistance caused by genotypic resistance to TDF has not been reported. This study aimed to characterize HBV mutations that confer tenofovir resistance. Methods Two patients with viral breakthrough during treatment with TDF-containing regimens were prospectively enrolled. The gene encoding HBV reverse transcriptase was sequenced. Eleven HBV clones harboring a series of mutations in the reverse transcriptase gene were constructed by site-directed mutagenesis. Drug susceptibility of each clone was determined by Southern blot analysis and real-time PCR. The relative frequency of mutants was evaluated by ultra-deep sequencing and clonal analysis. Results Five mutations (rtS106C [C], rtH126Y [Y], rtD134E [E], rtM204I/V, and rtL269I [I]) were commonly found in viral isolates from 2 patients. The novel mutations C, Y, and E were associated with drug resistance. In assays for drug susceptibility, the IC50 value for wild-type HBV was 3.8 ± 0.6 µM, whereas the IC50 values for CYE and CYEI mutants were 14.1 ± 1.8 and 58.1 ± 0.9 µM, respectively. The IC90 value for wild-type HBV was 30 ± 0.5 µM, whereas the IC90 values for CYE and CYEI mutants were 185 ± 0.5 and 790 ± 0.2 µM, respectively. Both tenofovir-resistant mutants and wild-type HBV had similar susceptibility to the capsid assembly modulator NVR 3–778 (IC50 Conclusions Our study reveals that the quadruple (CYEI) mutation increases the amount of tenofovir required to inhibit HBV by 15.3-fold in IC50 and 26.3-fold in IC90. These results demonstrate that tenofovir-resistant HBV mutants can emerge, although the genetic barrier is high. Lay summary Tenofovir is the most potent nucleotide analogue for the treatment of chronic hepatitis B virus infection and there has been no hepatitis B virus mutation that confers >10-fold resistance to tenofovir up to 8 years. Herein, we identified, for the first time, a quadruple mutation that conferred 15.3-fold (IC50) and 26.3-fold (IC90) resistance to tenofovir in 2 patients who experienced viral breakthrough during tenofovir treatment.

Regarding the T cell function in HIV-1 infection, activation of T cells is enhanced in acutely HIV-1-infected T cells upon stimuli. However, T cell immune responses underlying the activation of T cell receptor (TCR) signaling molecules and interleukin (IL)-2 production in latently HIV-1-infected cells are poorly understood. The expression and activation of TCR components and its downstream molecules in acutely and latently HIV-1-infected T cells were compared using quantitative reverse transcription polymerase chain reaction (RT-PCR) for mRNA expression and enzyme-linked immunosorbent assay (ELISA) for levels of IL-2 in phytohemagglutinin M (PHA-M). The levels of T cell surface molecules and TCR signaling molecules in latently HIV-1-infected cells were greatly decreased without changes in their mRNA levels. In addition, downstream TCR-signaling molecules in latently HIV-1-infected cells were not activated even in the presence of PHA-M. The phosphorylation of mitogen-activated protein kinases (MAPKs) in the presence of PHA-M was weakly induced in latently HIV-1-infected cells but was greater in acutely HIVNL4-3-infected cells. Finally, the production of IL-2 was significantly decreased in latently HIV-1-infected cells compared with uninfected parent cells. Thus, IL-2-related immunological functions in latently HIV-1-infected T cells were markedly impaired even in the presence of stimuli.

ObjectivesTo estimate human immunodeficiency virus (HIV) incidence using HIV avidity assays in Korea, we established a serological testing method to differentiate recent HIV infections from long-standing ones.MethodsWe adopted two incidence assays, the BED HIV-1 incidence test (Calypte Biomedical) and an HIV avidity assay (using Abbott AxSYM HIV Antigen/Antibody Combo), and performed them on Korean HIV samples obtained from 81 HIV seroconverters (n = 193), 135 HIV-positive samples, and three HIV commercial incidence panels (PRB965, PRB933, and PRB601 from SeaCare). To determine the most optimal concentration of the chaotropic agent (Guanidine) and the cutoff value for the avidity assay, we evaluated the sensitivity and specificity of the assay at different concentration levels.ResultsWe determined that the concentration of Guanidine to be used in the avidity assay was 1.5M. The cutoff value of the avidity index (AI) was 0.8, and the sensitivity and specificity were 90.2% and 83.8%, respectively, under this condition. The gray zone for the avidity assay was 0.75–0.85 AI. The mean of coefficient of variation was low, at 5.43%.ConclusionAn optimized avidity assay for the diagnosis of recent HIV infections using Korean samples was established. This assay will be applied to investigate the level of recent infection and will provide basic data to the HIV prevention policy in Korea.

Epigenetic modification at CpG islands located on the promoter regions of tumor-suppressor genes has been associated with tumor development in many human cancers. Our study showed that the cell adhesion molecule 1 (CADM1) is downregulated in human papillomavirus (HPV)-infected cervical cancer cell lines via its hypermethylation and demethylation using 5-aza-2′-deoxycyticine (5-aza-dC) restored the expression of CADM1 protein. Overexpression of CADM1 inhibited cell proliferation. p53 was involved in the regulation of CADM1. Our results demonstrate that epigenetic alteration of CADM1 was more frequent in HPV-positive cervical cancers and that restoration of CADM1 expression may be a potential strategy for cervical cancer therapy.

Cytokines/chemokines play key roles in modulating disease progression in human immunodeficiency virus (HIV) infection. Although it is known that early HIV-1 infection is associated with increased production of proinflammatory cytokines, the relationship between cytokine levels and HIV-1 pathogenesis is not clear. The concentrations of 18 cytokines/chemokines in 30 HIV-1 negative and 208 HIV-1 positive plasma samples from Korean patients were measured by the Luminex system. Early HIV-1 infection was classified according to the Fiebig stage (FS) based on the characteristics of the patients infected with HIV-1. Concentrations of interleukin-12 (IL-12), interferon-inducible protein-10 (IP-10), macrophage inflammatory protein-1α (MIP-1α) and regulated upon activation, normal T cells expressed and secreted (RANTES) were increased significantly during the early stage of HIV-1 infection (FS II–IV) compared with the HIV-1-negative group. Of these cytokines, an elevated level of IP-10 was the only factor to be correlated positively with a higher viral load during the early stages of HIV-1 infection (FS II–IV) in Koreans (R = 0.52, P

A major obstacle in the treatment of human immunodeficiency virus type 1 (HIV-1) is its ability to establish latent infection. To find novel biomarkers associated with the mechanism of HIV-1 latent infection, we identified 70 candidate genes in HIV-1 latently infected cells through the integrated analysis in a previous study. It is important to select more effective biomarkers among 70 candidates and to verify the possibility of selected biomarkers for HIV-1 latency. We identified the 24 and 25 genes from 70 candidate genes in significantly enriched categories selected by Database for Annotation, Visualization and Integrated Discovery (DAVID) software and Gene Set Enrichment Analysis (GSEA) software, respectively. Also, we investigated genes regulated in both HIV-1 latently infected cell lines and PBMCs from HIV-1 infected patients and found the genes with a common pattern of expression levels in both cell lines and PBMCs. Consequently, we identified nine genes, APBB2, GMPR, IGF2BP3, LRP1, MAD2L2, MX1, OXR1, PTK2B, and TNFSF13B, via integrated analysis. Especially, APBB2 and MAD2L2 were identified in both DAVID and GSEA software. Our findings suggest that nine genes were identified via integrated analysis as potential biomarkers and in particular, APBB2 and MAD2L2 may be considered as more significant biomarkers for HIV-1 latency.

Chromosomal distribution of H3K9ac binding sites in HIV-1 latently infected cells. Enrichment pattern of decreased islands of H3K9ac among individual chromosomes is shown as bar chart. Percent of total H3K9ac islands (red bar) and what would be expected by random chance (blue bars) for each chromosome is shown. The value in parenthesis means p value.

Functional annotation of 38 decreased (A) and 41 increased (B) genes in chromosomes 16, 17, 19, and 22 of HIV-1 latently infected cells using WebGestalt. The x-axis values are â log10 of raw p values.

The ratio of percentage of total H3K4me3 or H3K9ac islands to percentage expected by random chance for each chromosome in three different HIV-1 latently infected cell lines.

Chromosomal distribution of H3K9ac binding sites in HIV-1 latently infected cells. Enrichment pattern of increased islands of H3K9ac among individual chromosomes is shown as bar chart. Percent of total H3K9ac islands (red bar) and what would be expected by random chance (blue bars) for each chromosome is shown. The value in parenthesis means p value.

Lists of 38 decreased genes and 41 increased genes identified in the co-occurrence of two histone modifications, H3K4me3 and H3K9ac.

Chromosomal distribution of H3K4me3 binding sites in HIV-1 latently infected cells. Enrichment pattern of increased islands of H3K4me3 among individual chromosomes is shown as bar chart. Percent of total H3K4me3 islands (red bar) and what would be expected by random chance (blue bars) for each chromosome is shown. The value in parenthesis means p value.

Background Highly active antiretroviral therapy (HAART) including protease inhibitors (PIs) has been used in South Korea since 1997. Currently, more than 20 types of antiretroviral drugs are used in the treatment of human immunodeficiency virus-infected/acquired immune deficiency syndrome patients in South Korea. Despite the rapid development of various antiretroviral drugs, many drug-resistant variants have been reported after initiating HAART, and the efficiency of HAART is limited by these variants. Objectives To investigate and estimate the annual antiretroviral drug resistance and prevalence of antiretroviral multi-class drug resistance in Korean patients with experience of treatment. Study design The amplified HIV-1 pol gene in 535 patients requested for genotypic drug resistance testing from 2004 to 2009 by the Korea Centers for Disease Control and Prevention was sequenced and analyzed annually and totally. The prevalence of antiretroviral drug resistance was estimated based on “SIR” interpretation of the Stanford sequence database. Results Of viruses derived from 787 specimens, 380 samples (48.3%) showed at least one drug class-related resistance. Predicted NRTI drug resistance was highest at 41.9%. NNRTI showed 27.2% resistance with 23.3% for PI. The percent of annual drug resistance showed similar pattern and slightly declined except 2004 and 2005. The prevalence of multi-class drug resistance against each drug class was: NRTI/NNRTI/PI, 9.8%; NRTI/PI, 21.9%; NNRTI/PI, 10.4%; and NRTI/NNRTI, 21.5%. Conclusions About 50% and less than 10% of patients infected with HIV-1 have multidrug and multiclass resistance linked to 16 antiretroviral drugs, respectively. The significance of this study lies in its larger-scale examination of the prevalence of drug-resistant variants and multidrug resistance in HAART-experienced patients in South Korea.

Here, we report a double-stranded, dual-anchored, fluorescent aptamer on reduced graphene oxide (rGO) for the sensitive, selective, and speedy detection of a target protein in biological samples. This nano detector is composed of a target protein-specific fluorescent aptamer with BHQ1 as one anchoring moiety that forms double-stranded sequences with a complementary oligonucleotide sequence with BHQ1 as the other anchoring moiety, anchored to rGO nanosheets. The double-stranded and dual-anchored aptamer on rGO nanosheets (DAGO) exhibited 7.3-fold higher fluorescence intensities compared to a single-stranded, single-anchored fluorescent aptamer on rGO. As a model target protein, interferon-γ was used. DAGO detected the target protein, with linearity over a five-orders-of-magnitude concentration range (0.1 ng/ml-10 μg/ml) in buffer and human serum. DAGO was highly specific for the target protein, exhibiting little changes in fluorescence intensity in response to the non-target proteins, interleukin-2 and tumor necrosis factor-α. Moreover, DAGO allowed rapid quantification of the target protein in human immunodeficiency virus-positive patient serum samples. DAGO-based detection was complete in less than 10 min. Our results indicate that the DAGO provides new opportunities for the rapid and specific detection of target proteins in biological samples and could be widely applied to quantitate various target proteins by replacing the aptamer sequences.

AIDS is a disease caused by HIV infection, which destroys the CD4 T cells responsible for cell-mediated immunity in the human body, thereby undermining the immune system. HIV infections have spread gradually since the first case was reported in the USA in 1981 (1). In Korea, the first patient was identified in 1985, with the number of HIV-infected patients rising to 8544 by 2011 (2). The increasing number of HIV-1-infected patients in Korea is causing novel clinical problems such as increased drug resistance in patients treated with highly active antiretroviral therapy (HAART), thus raising the need to develop more effective therapies for patients for whom antiretroviral treatment is or has been unsuccessful (3). Recently, the HIV-1 envelope glycoprotein was highlighted as an important alternative target for the development of new antiretroviral drugs and vaccines. The glycoprotein is expressed as a polyprotein, which is subsequently cleaved into two subunits, gp120 and gp41, by the host furin (subtilysin proteinase) (4). Despite the more stable genetic characteristics of gp41 compared with gp120, mutations in gp41 may affect HIV-1 entry, pathogenesis, escape from host immune pressure, and resistance to the fusion inhibitor T-20 (enfuvirtide, Fuzeon®). HIV-1 gp41 protein plays an essential role in virus-cell fusion and has a complex structure with three functionally critical domains. The fusion peptide (FP) at the amino (N) terminal of gp41 normally contains 20 amino acids, which attach directly to membrane of the host cell during the first step of fusion. The 16 residues of FP (AVGIGALFLGFLGAAG) are mainly hydrophobic, and facilitate attachment to the cell's membrane. In particular, the conserved FLGFL motif at positions 8-12 has been reported to be essential for fusogenic activities (5). Recent studies have shown that mutations with a polar residue inserted into this motif, such as V2E and L9R substitutions, reduced its fusogenic activity significantly (6). Other structures in gp41 that are important for viral fusion are two helical repeats, heptad repeat 1 (HR1) and HR2, which include hydrophobic regions that interact with each other during virus-cell fusion via the formation of a six-helix bundled structure (7). The integrity of both repeats is important for the formation of a functional fusogenic complex. Therefore, the HR1 and HR2 domains of gp41 have been considered as potential targets for anti-HIV-1 therapy. The HIV fusion inhibitor T-20, a synthetic 36-amino acid peptide, which is a homolog of amino acids 127-162 in the HR2 region, was approved as a new class of antiretroviral drug that targets the HR1 domain of gp41 (7). T-20 can restrict HIV-1 replication in an efficient manner (7), but several T-20 resistance mutations have been reported in HR1 as follows: G36D/S, I37V, V38A/M/E, Q39R, Q40H, N42S/T/D/E, N43D/K/S, L44M, L45M, G36S/L44M, G36S/V38M, V38A/N42D, V38A/N42T, V38E/N42S, N42T/N43S and N42T/N43K (8). Recently, it has also been reported that T-20 resistance is greatly increased by a combined mutation of HR1 with secondary and/or compensatory mutations in the HR2 region (N126K, E137K, S138A) (9). Although T-20 therapy is known to efficiently control viral load in HIV-1 infected patients, mutation associated with T-20 resistance was observed to allow persistent replication under T-20 treatment in a clinical study (10). In this study, we analyzed the distributions of HIV-1 gp41 polymorphisms and T-20 resistance mutations in order to determine whether T-20 is suitable for treating Korean patients infected with HIV-1. We studied HIV-1 isolates from 163 T-20-naive HIV-1 infected patients who were diagnosed in Korea National Institute of Health (KNIH) in the period 2009-2011. The median CD4+ count was 291 cells/µL, and fifteen patients received effective ART (9.2%). Peripheral blood mononuclear cells (PBMCs) were obtained from these patients. The HIV-1 gp41 genes were amplified by nested DNA PCR. The HIV-1 gp41 polymorphisms were analyzed using bioinformatics software DNASTAR® (DNASTAR Inc, Madison, Wisconsin, USA) (11) and compared with the gp41 regions of then reference strain HXB2. Previously, we reported that the dominant Korean HIV-1 subtype was Korean B subtype, which was classified into a separate branch compared with isolates obtained from North Americans and Europeans (12). In the present study, the distribution of the HIV-1 subtypes in our 163 HIV-1-infected patients comprised 148 patients with subtype B and 15 with non-B subtypes, which were identified as subtypes G (n=6), A (n=1), AB (n=1), AE (n=3), C (n=1), and E (n=3). In our FP analysis, we found that there were no variations in the FLGFL motif, but that there were many specific insertional mutations of polar uncharged (threonine: T) and non-polar hydrophobic amino acids (valine, V; alanine, A; isoluecine, I; and methionine, M) in position 4. Table 1 shows that the dominant mutation was a T-insertion mutation (n=122, 74.8%), while an L7M substitution near the FLGFL motif was also frequent in HIV-1-infected patients (n=139, 85.3%). Glycine insertions were observed in the reference J, K, and N subtypes obtained from the database of the Los Alamos National Laboratory (http://www.hiv.lanl.gov/content/index), but insertions of polar and other non-polar residues were detected for the first time in our study. In addition, many of the FPs in Korean HIV-1 isolates had L7M substitutions near the FLGFL motif. These mutated residues generally have hydrophobic side chains, with the exception of the fourth threonine insertion, but further investigations should be performed to determine the mutation-related fusion activities of Korean HIV-1 gp41 (Table 1). Table 1 Polymorphisms in the gp41 fusion peptide domain* Several in vitro studies have reported that the loss of T-20 susceptibility is closely related to mutations at positions 36-38 in the GIV motif, which is the T-20-binding domain in the HR1 region (13). Moreover, single-or double-amino acid mutations at positions 36-45 in the HR1 region reduced the susceptibility to T-20 by 5- to 100-fold, while the presence of more than three mutations in the HR1 region reduced the susceptibility by >400-fold (14). Under selective drug pressure, the low fidelity of HIV-1 reverse transcriptase could lead to the development of T-20 resistance mutations (15). Several mutations (N126K, E137K, and S138A) in the HR2 domain contributed to enhanced viral fusion activity and reduced the susceptibility to T-20 (16). The susceptibility was dramatically reduced by about 1,000-fold, and the structural stability of the HR/HR2 complex was improved when the HR1 resistance mutations were coupled with HR2 resistance mutations (16). In addition, minor mutations associated with reduced susceptibility to T-20 have been reported in several countries as follows: N42S (23%), L44M (2.5%), G36S (0.5%), and S138A (4%) in Spain (17); N43K (1.8%), L44M (2.5%), E137K (10%), and S138A (2.5%) in Brazil (18); N42S (16.4%) and E137K (25%) in Italy (19); and L44M (1.7%), N42S (16%), E137K (15.4%), S138A (8.6%), N42S/S137A (0.5%), and N42S/L44M/E137K (0.5%) in the USA (20). In our study, the naturally acquired T-20 resistance mutation was L45M substitutions (n=2, 1.2%); and the N42S substitution (n=21, 12.9%) associated with enhanced T-20 susceptibility was more frequently detected in the HR1 region of HIV-1 gp41 from 163 naive HIV-1 isolates (14). The N42S mutation has been detected more frequently in subtype non-B isolates than subtype B isolates (73.3% vs 6.8%; P< 0.001), whereas the L45M mutation has been detected only rarely in subtype B isolates (1.2%). The minor T-20 resistance mutations detected in the gp41 HR2 region were N126K (1.2%) and E137K (6.7%) in the HIV-1 B subtype only. By contrast, the major T-20 resistance mutations were not detected in the gp41 HR1 region, and the S138A substitution combined with mutations in the HR1 region was not detected in the HR2 regions of the Korean HIV isolates (Table 2). The frequencies of these mutations were also similarly observed in the ART experienced patients (for which the relevant data is not shown). These results demonstrate clearly that the naturally acquired T-20 resistance in the HR1 and HR2 regions of gp41 may be ignored in Korean HIV-1 isolates, while the geographic differences in T-20 resistance mutations are likely to be associated with HIV-1 evolution and HIV-1 subtype distributions in specific countries. Table 2 T-20 resistance-associated mutations in gp41 HR1 and HR2* Overall, our results suggest that the genetic variation of gp41 in specific countries may be provided as useful information to consider T-20 for studies considering the use of T-20 in the development of a more effective anti-retroviral treatment in Korea.

Objectives: Persistent HIV-1 infections are characterized by a long silent infection period in resting CD4+ T cells, which allows them to escape the host immune response. Several HIV-1 latency mechanisms have been reported, but the molecular mechanism underlying polycomb repressor complex (PRC)-mediated HIV-1 latency remains poorly understood. Methods: Expression of PRC proteins in latent cells was measured by Western blot. Knockdowns of PRC genes were conducted by the specific siRNA and methylations at H3K27 on the proviral LTR were investigated by ChIP assay. Results: PRC proteins (EED, BMI-1, and RNF2) were dramatically downregulated in latent cells after PMA treatment. The downregulation of PRC proteins was followed by a decrease in the methylation of H3K27 and ubiquitination of H2AK119 in the PMA-treated latent cells. siRNA knockdowns of EED and BMI-1 also enhanced HIV-1 reactivation significantly in latently infected cells. By contrast, proteasomal inhibitor MG132 successfully abrogated the PMA-induced downregulation of PRCs. In particular, di-/tri-methylations of histone-3 in the proviral LTR was absent from latent cells after PMA treatment. Conclusions: This study shows that PRC is strongly related to the control of HIV-1 latency and that PRC-breaking agents may be helpful for purging HIV-1 from latent reservoirs.

Objectives: Recent studies have reported that human immunodeficiency virus type 1 (HIV-1) proviruses are strongly suppressed in the unique epigenetic environments caused by chromatin modifications such as acetylation and methylation. Therefore, optimized therapeutic strategies directed against the virus reservoir using these epigenetic modifying agents (EMAs) should cure HIV infection. Methods: Cytotoxicity and HIV-1 reactivation were determined using the PrestoBlue™ Cell Viability Reagent and p24 HIV ELISA, respectively. Results: EMAs, including histone deacetylase inhibitors (VPA and SAHA), DNA methyltransferase inhibitor (5′-Aza-CdR), histone methyltransferase inhibitor (ADOX) and 12-O-tetradecanoylphorbol-13-acetate (TPA), were used to reactivate proviruses in HIV-1 latently infected cells. The effect of monotreatment with these EMAs on HIV-1 reactivation was VPA or SAHA > 5′-Aza-CdR > ADOX. Even though cotreatment with these potential HIV-1 reactivating agents did not show any significant reactivation effects in HIV-1 latently infected cells, employing SAHA under TPA treatment demonstrated a dramatic synergistic effect on purging HIV-1 proviruses in all HIV-1 latently infected cells via the ERK and AP-1 pathways. Conclusions: These results suggest that the combined approaches of EMAs, cotreatment of SAHA and TPA, could provide an effective way to lead a decline of HIV-1 reservoirs in patients.

Background Despite the successful inhibition of human immunodeficiency virus type 1 (HIV-1) replication by combination antiretroviral therapy, cells latently infected with HIV-1 remaining in patients are a major obstacle for eradication of HIV-1 infection. The tumor suppressor factor p53 is activated by HIV-1 infection, and restricts HIV-1 replication. However, a therapeutic strategy based on p53 activity has not been considered for elimination of latently infected cells. Methods Apoptotic cells were analyzed using flow cytometry with anti-annexin A5-FITC Ab and PI staining upon treatment of anticancer drugs. The expression and activation of p53 and apoptotic molecules in latently HIV-1-infected T cells were compared using Western blot analysis. The role of p53 in the anticancer drug treatment-induced apoptosis of cells latently infected with HIV-1 was determined by knock-down experiment using siRNA against p53. Results Upon treatment with 5-fluorouracil (5-FU), apoptosis was increased in latently infected ACH2 cells encoding competent p53 compared with uninfected parent A3.01 cells, while the apoptosis of latently infected p53 null J1.1 cells was less than that of uninfected cells. Treatment with 5-FU increased the levels of cleaved caspase-3 and PARP in ACH2 cells compared with uninfected and latently infected p53 null J1.1 cells. The levels of expression and activation of p53 were higher in both latently infected ACH2 and NCHA2 cells than in uninfected cells. Furthermore, the activation levels of p53 in both cells were further increased upon 5-FU treatment. Consistent with p53 status, apoptosis was markedly increased in ACH2 and NCHA2 cells compared with uninfected and latently infected J1.1 cells upon treatment with other anticancer drugs such as doxorubicin and etoposide. Inhibition of p53 in cells with latent HIV-1 infection diminished apoptosis upon 5-FU treatment. Conclusion Evidence described here indicate that when treated with anticancer drugs, apoptosis of cells with latent HIV-1 infection was increased via the p53 activation pathway and may provide information for application of anticancer drugs to selectively eliminate HIV-1 reservoirs.

HIV-1 reservoirs remain a major barrier to HIV-1 eradication. Although combination antiretroviral therapy (cART) can successfully reduce viral replication, it cannot reactivate HIV-1 provirus in this reservoir. Therefore, HIV-1 provirus reactivation strategies by cell activation or epigenetic modification are proposed for the eradication of HIV-1 reservoirs. Although treatment with the protein kinase A (PKA) activator cyclic AMP (cAMP) or epigenetic modifying agents such as histone deacetylase inhibitors (HDACi) alone can induce HIV-1 reactivation in latently infected cells, the synergism of these agents has not been fully evaluated. In the present study, we observed that treatment with 500μM of dibutyryl-cAMP, 1μM of vorinostat, or 1μM of trichostatin A alone effectively reactivated HIV-1 in both ACH2 and NCHA1 cells latently infected with HIV-1 without cytotoxicity. In addition, treatment with the PKA inhibitor KT5720 reduced the increased HIV-1 p24 level in the supernatant of these cells. After dibutyryl-cAMP treatment, we found an increased level of the PKA substrate phosphorylated cyclic AMP response element-binding protein. When we treated cells with a combination of dibutyryl-cAMP and vorinostat or trichostatin A, the levels of HIV-1 p24 in the supernatant and levels of intracellular HIV-1 p24 were dramatically increased in both ACH2 and NCHA1 cells compared with those treated with a single agent. These results suggest that combined treatment with a PKA activator and an HDACi is effective for reactivating HIV-1 in latently infected cells, and may be an important approach to eradicate HIV-1 reservoirs.

ObjectivesThe detection of HIV seroconverters increased annually since HIV antigen/antibody testing kits have been available widely in South Korea. This study aimed to identify the epidemiological characteristics of HIV seroconverters and their immune level at HIV diagnosis.MethodWe analyzed the epidemiological and immunological characteristics of 341 HIV seroconverters among 6,008 HIV-diagnosed individuals from 1999 and 2009. The analysis of immune level and epidemiological factors of HIV seroconverters was conducted by using chi-square test on SAS version 9.1.ResultsThe seroconverters among newly-identified HIV cases each year increased from 0.5% in 1999 to over 5% or in 2009. The sex ratio of seroconverters was 18:1 (male:female), and 33% were in their 30s, and 28% were in their 20s. Reasons for HIV testing were involvement in voluntary test due to risky behaviors (43%), and health check-up (36%). Discovery of HIV infection occurred primarily in hospitals (84%). Among seroconverters, 55 percent had a CD4 T-cell count of more than 350/μl.ConclusionKorean HIV seroconverters tended to be younger at diagnosis, diagnosed during a voluntary test, and their CD4+ T-cell counts at HIV diagnosis were higher than those of non-seroconverters all HIV-infected individuals. This study of HIV seroconverters will be important foundational in future studies on HIV incidence, disease progress, and survival rate.

It has been shown that porcine endogenous retrovirus (PERV) can infect human cells, indicating that PERV transmission poses a serious concern in pig-to-human xenotransplantation. A number of recent studies have reported on retrovirus interference by antiviral proteins. The most potent antiviral proteins are members of the APOBEC family of cytidine deaminases, which are involved in defense against retroviral attack. These proteins are present in the cytoplasm of mammalian cells and inhibit retroviral replication. To evaluate the inhibition of PERV transmission by human APOBEC3 proteins, we co-transfected 293T cells with a PERV molecular clone and human APOBEC3F or APOBEC3G expression vectors, and monitored PERV replication competency using a quantitative analysis of PERV pol genes. The replication of PERVs in cells co-expressing human APOBEC3s was reduced by 60-90% compared with PERV-only control. These results suggest that human APOBEC3G and APOBEC3F might serve a potential barrier function against PERV transmission in xenotransplantation.

Many human immunodeficiency virus 1 (HIV-1) researchers focus on the developing new anti-reservoir therapy to eradicate HIV-1 provirus from the HIV-1-infected patients. HIV-1 provirus is the major obstacle for effective HIV-1 treatment because it integrates into the host genome and can produce a virus progeny after stopping highly active antiretroviral therapy (HAART). We established two novel cell lines latently infected with HIV-1 by limiting dilution cloning of A3.01 cells infected with HIV-1. Analysis of the flanking sequence of HIV-1 proviral DNA integrated into chromosomal cellular DNA revealed that proviral DNA was inserted into different sites of different chromosomes in the two examined cell lines. In these lines, virus reactivation could be induced by a phorbol 12-myristate 13-acetate (PMA) treatment that resulted in a marked increase of the production HIV-1 p24 antigen and appearance of the infectious virus. The novel cell lines latently infected with HIV-1 represent further tool for the study of molecular mechanisms of viral latency and development of anti-reservoir therapy of HIV-1 infection.

Recent studies have reported that the distribution of HPV-16 sequence variation differs geographically, and more specifically that HPV-16 E6/E7 intratypic variants might carry a high risk for development of ICC (invasive cervical cancer) and CIN (cervical intraepithelial neoplasia) in a given population. To investigate the genetic diversities of HPV-16 E6/E7 oncogene by region, we collected nineteen HPV-16 isolates from sexually high-risk women in Busan, and analyzed the HPV-16 E6/E7 coding regions (nt 34 to 880) with HPV-16 E6/E7 specific PCR amplification. At the nucleotide levet eleven variants of the E6 genes and nine variants of the E7 genes were identified as follows: E6 T178G (n

consisted of 16 high-risk HPV genotypes (oncogenic genotypes) and 8 low-risk HPV genotypes. The overall prevalence of HPV infection in this study population was 39.1% (258/660) and the 20's showed the highest prevalence of HPV infection (51.5%). The dominant HPV genotypes including single or multiple HPV-infected women were resulted in HPV-16 (15.9%), -53 (10.2%), -58 (7.7%), -18 (5.2%) in case of high-risk HPV genotype and HPV-70 (10.4%), -6 (4.1%), - 11 (2.0%) in case of low-risk HPV genotypes. Remarkably, the proportion of women infected with high-risk HPV genotypes (62.0%) was almost four times higher than those of women infected with low- risk HPV genotypes (14.7%) and high/low-risk HPV genotypes (12.0%). Among the 258 HPV-infected women, single infection was 175, double infection 66, triple infection 12, quadruple infection 4, quintuple infection 1, respectively. Our finding suggests that the introduction and development of effec tive HPV vaccines should consider the current status of HPV genotypic infection in South Korean women.

Genetic polymorphisms of chemokine genes and chemokine-receptor genes in HIV-infected patients have been associated with delayed progression of this disease. The allelic frequencies of these genetic variants also differ between ethnic groups. To investigate the effects of the SDF1 and CCR2b genotypes on disease progression, survival of 200 HIV-infected persons for whom at least four subsequent immunologic data items had been collected was analyzed. A genotyping assay of SDF1 and CCR2b genes was carried out using polymerase chain reaction-restriction fragment length polymorphism analyses. HIV-infected persons heterozygous for the SDF1-3'A or CCR2b-64I alleles were included in the survival analysis, but homozygotes were excluded because of a very small sample number. Neither the CCR2b-+/64I allele nor the SDF1-+/3'A allele, separately or in combination, had a significant impact on survival during the asymptomatic period of HIV infection. However, CCR2b-+/64I alleles were associated with accelerated disease progression during the advanced period of HIV infection. The survival time of HIV-infected people with CCR2b-+/64I and SDF1-+/+ genotypes was significantly shorter than those of the other groups (p < 0.01), but this effect was not apparent in persons with CCR2b-+/64I alleles and SDF1-+/3'A genotypes. These results suggest that the effect of CCR2b-64I polymorphisms on disease progression may differ according to the stage of HIV infection and interactions with other gene variants.