Involvement of the 3’ Untranslated Region in Encapsidation of the Hepatitis C Virus

Although information regarding morphogenesis of the hepatitis C virus (HCV) is accumulating, the mechanism(s) by which the HCV genome encapsidated remains unknown. In the present study, in cell cultures producing HCV, the molecular ratios of 3’ end- to 5’ end-regions of the viral RNA population in the culture medium were markedly higher than those in the cells, and the ratio was highest in the virion-rich fraction. The interaction of the 3’ untranslated region (UTR) with Core in vitro was stronger than that of the interaction of other stable RNA structure elements across the HCV genome. A foreign gene flanked by the 3’ UTR was encapsidated by supplying both viral NS3-NS5B proteins and Core-NS2 in trans. Mutations within the conserved stem-loops of the 3’ UTR were observed to dramatically diminish packaging efficiency, suggesting that the conserved apical motifs of the 3´ X region are important for HCV genome packaging. This study provides evidence of selective packaging of the HCV genome into viral particles and identified that the 3’ UTR acts as a cis-acting element for encapsidation.     

Does Phospholipid Methylation Play a Role in the Primary Mechanism of Action of Nerve Growth Factor?

Nerve growth factor (NGF)-untreated (naive) and neurite-bearing NGF-treated ("primed") PC12 rat pheochromocytoma cells were used as model system to study the role of phospholipid methylation in the NGF mechanism of action. The neurite-bearing cultures were deprived of NGF for 3 h before experimentation. Under both experimental conditions, the cells were labelled with [methyl-3H]methionine and then challenged with NGF for time periods ranging from 5 s to 30 min. Methylated phospholipids were extracted and then resolved and identified by TLC as phosphatidyl mono-, di-, and trimethyl ethanolamine. Quantification of the amount of radioactivity incorporated into each of the phospholipids indicated that NGF does not significantly alter phospholipid methylation either in naive or in neurite-bearing cells. Furthermore, using a methyltransferase inhibitor, it was found that neurite outgrowth still occurs when phospholipid methylation is almost completely blocked. These results indicate that phospholipid methylation does not play a primary role in the mechanism of action of NGF.     

Polymorphism of the 5′ Untranslated Region of NHE1 Gene Associated with Type-I Diabetes

The ubiquitous form of the sodium–hydrogen exchanger, NHE1, is devoted to the regulation of intracellular pH and cell volume. In addition, NHE1 activity is stimulated by growth factors and increased NHE rates are found in both circulating and immortalized cells during diabetes or diabetic nephropathy. In this context, we searched for polymorphisms of the 5′-flanking regulatory region of NHE1 gene in subjects with type-I diabetes. We identified a C/T transition 696 bases upstream the translation initiation start site which disrupts a repeated palindromic GC sequence. The TT genotype was significantly more frequent in type-1 diabetics and may have functional importance. Genetic linkage between NHE1 and diabetes has been previously described in NOD mice strains with consequences on NHE rates. Hence, the polymorphism described hereby may act as a predisposition factor to type-I diabetes or to diabetic complications, and may be useful to investigate the genetic involvement of NHE1 in human pathophysiology.     

Activation of the Connective Tissue Growth Factor (CTGF)-Transforming Growth Factor β 1 (TGF-β 1) Axis in Hepatitis C Virus-Expressing Hepatocytes

Background

The pro-fibrogenic cytokine connective tissue growth factor (CTGF) plays an important role in the development and progression of fibrosis in many organ systems, including liver. However, its role in the pathogenesis of hepatitis C virus (HCV)-induced liver fibrosis remains unclear.

Methods

In the present study, we assessed CTGF expression in HCV-infected hepatocytes using replicon cells containing full-length HCV genotype 1 and the infectious HCV clone JFH1 (HCV genotype 2) by real-time PCR, Western blot analysis and confocal microscopy. We evaluated transforming growth factor β1 (TGF-β1) as a key upstream mediator of CTGF production using neutralizing antibodies and shRNAs. We also determined the signaling molecules involved in CTGF production using various immunological techniques.

Results

We demonstrated an enhanced expression of CTGF in two independent models of HCV infection. We also demonstrated that HCV induced CTGF expression in a TGF-β1-dependent manner. Further dissection of the molecular mechanisms revealed that CTGF production was mediated through sequential activation of MAPkinase and Smad-dependent pathways. Finally, to determine whether CTGF regulates fibrosis, we showed that shRNA-mediated knock-down of CTGF resulted in reduced expression of fibrotic markers in HCV replicon cells.

Conclusion

Our studies demonstrate a central role for CTGF expression in HCV-induced liver fibrosis and highlight the potential value of developing CTGF-based anti-fibrotic therapies to counter HCV-induced liver damage.     

Tra2-Mediated Recognition of HIV-1 5′ Splice Site D3 as a Key Factor in the Processing of vpr mRNA

Small noncoding HIV-1 leader exon 3 is defined by its splice sites A2 and D3. While 3′ splice site (3′ss) A2 needs to be activated for vpr mRNA formation, the location of the vpr start codon within downstream intron 3 requires silencing of splicing at 5′ss D3. Here we show that the inclusion of both HIV-1 exon 3 and vpr mRNA processing is promoted by an exonic splicing enhancer (ESE_vpr) localized between exonic splicing silencer ESSV and 5′ss D3. The ESE_vpr sequence was found to be bound by members of the Transformer 2 (Tra2) protein family. Coexpression of these proteins in provirus-transfected cells led to an increase in the levels of exon 3 inclusion, confirming that they act through ESE_vpr. Further analyses revealed that ESE_vpr supports the binding of U1 snRNA at 5′ss D3, allowing bridging interactions across the upstream exon with 3′ss A2. In line with this, an increase or decrease in the complementarity of 5′ss D3 to the 5′ end of U1 snRNA was accompanied by a higher or lower vpr expression level. Activation of 3′ss A2 through the proposed bridging interactions, however, was not dependent on the splicing competence of 5′ss D3 because rendering it splicing defective but still competent for efficient U1 snRNA binding maintained the enhancing function of D3. Therefore, we propose that splicing at 3′ss A2 occurs temporally between the binding of U1 snRNA and splicing at D3.     

Processing of the psbA 5′ Untranslated Region in Chlamydomonas reinhardtii Depends upon Factors Mediating Ribosome Association

The 5′ untranslated region of the chloroplast psbA mRNA, encoding the D1 protein, is processed in Chlamydomonas reinhardtii. Processing occurs just upstream of a consensus Shine-Dalgarno sequence and results in the removal of 54 nucleotides from the 5′ terminus, including a stem-loop element identified previously as an important structure for D1 expression. Examination of this processing event in C. reinhardtii strains containing mutations within the chloroplast or nuclear genomes that block psbA translation reveals a correlation between processing and ribosome association. Mutations within the 5′ untranslated region of the psbA mRNA that disrupt the Shine-Dalgarno sequence, acting as a ribosome binding site, preclude translation and prevent mRNA processing. Similarly, nuclear mutations that specifically affect synthesis of the D1 protein specifically affect processing of the psbA mRNA. In vitro, loss of the stem-loop element does not prohibit the binding of a message-specific protein complex required for translational activation of psbA upon illumination. These results are consistent with a hierarchical maturation pathway for chloroplast messages, mediated by nuclear-encoded factors, that integrates mRNA processing, message stability, ribosome association, and translation.