Antiviral lectins from red and blue-green algae show potent in vitro and in vivo activity against hepatitis C virus.

Hepatitis C virus (HCV) infection is a significant public health problem with over 170,000,000 chronic carriers and infection rates increasing worldwide. Chronic HCV infection is one of the leading causes of hepatocellular carcinoma which was estimated to result in ∼10,000 deaths in the United States in the year 2011. Current treatment options for HCV infection are limited to PEG-ylated interferon alpha (IFN-α), the nucleoside ribavirin and the recently approved HCV protease inhibitors telaprevir and boceprevir. Although showing significantly improved efficacy over the previous therapies, treatment with protease inhibitors has been shown to result in the rapid emergence of drug-resistant virus. Here we report the activity of two proteins, originally isolated from natural product extracts, which demonstrate low or sub-nanomolar in vitro activity against both genotype I and genotype II HCV. These proteins inhibit viral infectivity, binding to the HCV envelope glycoproteins E1 and E2 and block viral entry into human hepatocytes. In addition, we demonstrate that the most potent of these agents, the protein griffithsin, is readily bioavailable after subcutaneous injection and shows significant in vivo efficacy in reducing HCV viral titers in a mouse model system with engrafted human hepatocytes. These results indicate that HCV viral entry inhibitors can be an effective component of anti-HCV therapy and that these proteins should be studied further for their therapeutic potential.

Reconstructing the epidemic history of HIV-1 circulating recombinant forms CRF07_BC and CRF08_BC in East Asia: the relevance of genetic diversity and phylodynamics for vaccine strategies.

HIV-1 CRF07_BC and CRF08_BC are closely related circulating recombinant forms (CRFs) with serious public health consequences in China. The temporal and spatial dynamics of these CRFs were determined by estimating their times of divergence, using phylogenetic and Bayesian coalescent methods. Studies of the timelines of CRF07_BC and CRF08_BC trace the expansion of these strains back their origins to Yunnan province. The present study highlights the relevance of incorporating evolutionary and molecular epidemiological analyses into an in-depth understanding of the genesis of HIV epidemic, providing information for determining regional and global public health policies, including future vaccine strategies.

Explosive HIV-1 subtype B' epidemics in Asia driven by geographic and risk group founder events.

We explored the timescale, spatial spread, and risk group population structure of HIV-1 subtype B', the cause of explosive blood-borne HIV-1 epidemics among injecting drug users (IDUs) and former plasma donors (FPDs) in Asia. Sequences from FPDs in China formed a distinct monophyletic cluster within subtype B'. Further analysis revealed that subtype B' was founded by a single lineage of pandemic subtype B around 1985. Subsequently, the FPD cluster appears to have derived from a single subtype B' lineage around 1991, corroborating the hypothesis that FPD outbreaks stemmed from the preceding epidemic among IDUs in Southeast Asia, most likely from the Golden-Triangle region.

Identification of a novel second-generation circulating recombinant form (CRF48_01B) in Malaysia: a descendant of the previously identified CRF33_01B.

A molecular epidemiological investigation conducted among injecting drug users in eastern Peninsular Malaysia in 2007 identified a cluster of sequences (n = 3) located outside any known HIV-1 genotype. Analyses of near full-length nucleotide sequences of these strains from individuals with no recognizable linkage revealed that they have an identical subtype structure comprised of CRF01_AE and subtype B', distinct from any known circulating recombinant forms (CRFs). This novel CRF, designated CRF48_01B, is closely related to CRF33_01B, previously identified in Kuala Lumpur. Phylogenetic analysis of multiple CRF48_01B genome regions showed that CRF48_01B forms a monophyletic cluster within CRF33_01B, suggesting that this new recombinant is very likely a descendant of CRF33_01B. CRF48_01B thus represents one of the first examples of a "second-generation" CRF, generated by additional crossover with pre-existing CRFs. Corroborating these results, Bayesian molecular clock analyses indicated that CRF48_01B emerged in approximately 2001, approximately approximately 8 years after the emergence of CRF33_01B.

[Recent advances in HIV vaccine research and development--newly unearthed challenges].

Historically, vaccines have been among the most effective public health interventions, preventing the spread of viral infections. However, HIV vaccine has thus far been extremely elusive. The recent failure of the first T-cell vaccine has led to a reexamination of the direction of HIV-vaccine field. An empirical approach that has been successfully applied to many human viruses appears not effective for HIV. Re-examination has pointed to a need to emphasize fundamental questions of HIV vaccine discovery. In particular, recent advances in the study on the earliest phase of HIV-1 infection unearthed novel challenges for HIV vaccine development.

Isolation and characterization of a replication-competent molecular clone of an HIV-1 circulating recombinant form (CRF33_01B).

A growing number of emerging HIV-1 recombinants classified as circulating recombinant forms (CRFs) have been identified in Southeast Asia in recent years, establishing a molecular diversity of increasing complexity in the region. Here, we constructed a replication-competent HIV-1 clone for CRF33_01B (designated p05MYKL045.1), a newly identified recombinant comprised of CRF01_AE and subtype B. p05MYKL045.1 was reconstituted by cloning of the near full-length HIV-1 sequence from a newly-diagnosed individual presumably infected heterosexually in Kuala Lumpur, Malaysia. The chimeric clone, which contains the 5' LTR (long terminal repeat) region of p93JP-NH1 (a previously isolated CRF01_AE infectious clone), showed robust viral replication in the human peripheral blood mononuclear cells. This clone demonstrated robust viral propagation and profound syncytium formation in CD4+, CXCR4-expressing human glioma NP-2 cells, indicating that p05MYKL045.1 is a CXCR4-using virus. Viral propagation, however, was not detected in various human T cell lines including MT-2, M8166, Sup-T1, H9, Jurkat, Molt-4 and PM1. p05MYKL045.1 appears to proliferate only in restricted host range, suggesting that unknown viral and/or cellular host factors may play a role in viral infectivity and replication in human T cell lines. Availability of a CRF33_01B molecular clone will be useful in facilitating the development of vaccine candidates that match the HIV-1 strains circulating in Southeast Asia.

Phylodynamic analysis of the dissemination of HIV-1 CRF01_AE in Vietnam.

To estimate the epidemic history of HIV-1 CRF01_AE in Vietnam and adjacent Guangxi, China, we determined near full-length nucleotide sequences of CRF01_AE from a total of 33 specimens collected in 1997-1998 from different geographic regions and risk populations in Vietnam. Phylogenetic and Bayesian molecular clock analyses were performed to estimate the date of origin of CRF01_AE lineages. Our study reconstructs the timescale of CRF01_AE expansion in Vietnam and neighboring regions and suggests that the series of CRF01_AE epidemics in Vietnam arose by the sequential introduction of founder strains into new locations and risk groups. CRF01_AE appears to have been present among heterosexuals in South-Vietnam for more than a decade prior to its epidemic spread in the early 1990s. In the late 1980s, the virus spread to IDUs in Southern Vietnam and subsequently in the mid-1990s to IDUs further north. Our results indicate the northward dissemination of CRF01_AE during this time.

Cadmium-induced alterations of gene expression in human cells.

We have reported the changes in gene expression in human HeLa cells exposed to a low concentration (5µM) of Cd. In the present study, cells exposed to a higher concentration of Cd were analyzed using a DNA microarray with 9182 human cDNA probes, in an attempt to obtain a comprehensive view on the biological effects of Cd. After a 6h exposure to 50µM Cd, 48 genes were up-regulated 2.5-fold or greater and 14 genes were down-regulated to 40% or less. Marked up-regulation of genes coding for metallothioneins, anti-oxidant proteins, and heat shock proteins was observed. Cd appeared to repress cell proliferation by modulating genes involved in multiple pathways. Cd also affected a number of genes related to apoptosis. Interestingly, it appeared that a series of genes were regulated to accelerate the intrinsic pathway of apoptosis, while others were directed to suppress the extrinsic pathway. Of these, rapid and transient induction of the TR3 gene was noted as a possible key process in Cd-induced apoptosis. Effects on several genes that may reflect mechanistic backgrounds of Cd toxicity were also observed. The present study disclosed a complex pleiotypic response of human cells to Cd, which was composed of a variety of changes in gene expression directed to defense, growth arrest, recovery from damage, apoptosis and so on.

Chronology of the HIV-1 CRF07_BC expansion in East Asia.

The HIV-1 epidemic among injecting drug users (IDU) in Taiwan is caused primarily by CRF07_BC infections. Evolutionary analyses, which utilize outgroup reference strains from northwestern China (Xinjiang), reveal that CRF07_BC was introduced into southern Taiwan in 1998-2001 and spread to central-northern Taiwan in 2001-2003, causing the largest HIV/AIDS epidemic in Taiwan. The separate introduction of CRF07_BC into Xinjiang occurred in 1992-1995. This study illustrates the temporal dynamics of CRF07_BC spread among IDU across east Asia.

Optimal design and validation of antiviral siRNA for targeting HIV-1.

We propose rational designing of antiviral short-interfering RNA (siRNA) targeting highly divergent HIV-1. In this study, conserved regions within HIV-1 genomes were identified through an exhaustive computational analysis, and the functionality of siRNAs targeting the highest possible conserved regions was validated. We present several promising antiviral siRNA candidates that effectively inhibited multiple subtypes of HIV-1 by targeting the best conserved regions in pandemic HIV-1 group M strains.

Transcriptional up-regulation of inhibitory PAS domain protein gene expression by hypoxia-inducible factor 1 (HIF-1): a negative feedback regulatory circuit in HIF-1-mediated signaling in hypoxic cells.

The inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS), a dominant negative regulator of hypoxia-inducible transcription factors (HIFs), is potentially implicated in negative regulation of angiogenesis in such tissues as the avascular cornea of the eye. We have previously shown IPAS mRNA expression is up-regulated in hypoxic tissues, which at least in part involves hypoxia-dependent alternative splicing of the transcripts from the IPAS/HIF-3alpha locus. In the present study, we demonstrate that a hypoxia-driven transcriptional mechanism also plays a role in augmentation of IPAS gene expression. Isolation and analyses of the promoter region flanking to the first exon of IPAS gene revealed a functional hypoxia response element at position -834 to -799, whereas the sequence upstream of the HIF-3alpha first exon scarcely responded to hypoxic stimuli. A transient transfection experiment demonstrated that HIF-1alpha mediates IPAS promoter activation via the functional hypoxia response element under hypoxic conditions and that a constitutively active form of HIF-1alpha is sufficient for induction of the promoter in normoxic cells. Moreover, chromatin immunoprecipitation and electrophoretic mobility shift assays showed binding of the HIF-1 complex to the element in a hypoxia-dependent manner. Taken together, HIF-1 directly up-regulates IPAS gene expression through a mechanism distinct from RNA splicing, providing a further level of negative feedback gene regulation in adaptive responses to hypoxic/ischemic conditions.

Cross talk of heat shock and heavy metal regulatory pathways.

Heavy metals induce transcription of human genes including those coding for metallothionein and heat shock protein 70 (HSP70). It has been suggested that these processes are mediated by metal-activated transcription factors, MTF-1 and HSF1, respectively, and are independent of each other. We raised an antibody against human MTF-1 which efficiently supershifts the protein-DNA complex formed by MTF-1 and its cognate binding sequence, MRE. We discovered that this antibody could also supershift complexes formed by HSF1 and its recognition sequence HSE, which suggested the involvement of MTF-1 in these complexes. This supershift was observed for HSF1/HSE complexes induced by Zn, Cd, Ag, and heat shock. Furthermore, overexpression of MTF-1 in HeLa cells markedly reduced metal-induced transcription from the hsp70-1 gene promoter which depends on HSF1. These data indicate that MTF-1 represses HSF1-mediated transcription probably through a direct protein-protein interaction, suggesting a cross talk of two lines of stress-responsive regulatory pathways.

Diesel exhaust particle-induced cell death of cultured normal human bronchial epithelial cells.

We investigated the effect of diesel exhaust particles (DEPs) on normal human bronchial epithelial (NHBE) cells. Inclusion of DEPs in culture media was lethal to NHBE cells. NHBE cells are more susceptible to DEPs than other normal human lung cells, normal human pulmonary artery endothelial cells and normal human embryonic lung fibroblasts. DEP-induced cell death was mainly due to necrosis. Using the fluorescence probes diacetoxymethyl 6-carboxy-3',6'-diacetoxy-2',7'-dichloro-3',6'-dideoxydihydrofluorescinate and 4,5-diaminofluorescein diacetate, it was observed that hydrogen peroxide and nitrogen monoxide, respectively, were generated within DEP-exposed NHBE cells. DEP cytotoxicity increased or decreased with an increase or decrease in the cellular level of reduced glutathione (GSH) by treatment with L-buthionine-(R,S)-sulfoximine or ethyl reduced glutathionate, respectively. In addition, DEPs themselves decreased the cellular level of GSH in a dose-dependent manner. Upon exposure of NHBE cells to high concentrations of DEPs, their cellular GSH was depleted almost throughout. Further, the following agents decreased DEP cytotoxicity: 1) antioxidants 2,2,5,7,8-pentamethylchroman-6-ol, ebselen, and N,N'-bis(salicylidene)ethylenediaminomanganese(II) dihydrate (EUK-8); 2) iron ion-chelating agents disodium bathophenanthrolinedisulfonate and desferrioxamine mesylate; 3) nitrogen monoxide synthase inhibitors N(G)-nitro-L-arginine methyl ester hydrochloride and N(G)-methyl-L-arginine acetate salt; and 4) an endocytosis inhibitor quinacrine. On the basis of these observations, the mechanism of DEP cytotoxicity toward NHBE cells is discussed.