Hepatitis B virus (HBV) is a major etiological factor of hepatocellular carcinoma (HCC). However, the precise pathogenetic mechanisms linking HBV infection and HCC remain uncertain. It has been reported that decreased antioxidant enzyme activities are associated with severe liver injury and hepatocarcinogenesis in mouse models. It is unclear if HBV can interfere with the activities of antioxidant enzymes. We established a HBV transgenic mouse line, which spontaneously developed HCC at 2 years of age. We studied the activities of the antioxidant enzymes in the liver of the HBV transgenic mice. Our results showed that the antioxidant enzymes glutathione peroxidase and superoxide dismutase 2 were down-regulated in HBV transgenic mice and correlated with JNK activation. HBV enhanced the Fas-mediated activation of caspase 6, caspase 8 and JNK without enhancing the activation of caspase 3 and hepatocellular apoptosis. As a proper redox balance is important for maintaining cellular homeostasis, these effects of HBV on the host antioxidant system and Fas-signaling may play an important role in HBV-induced hepatocarcinogenesis.

Hepatitis B virus (HBV) is a small DNA virus that requires cellular transcription factors for the expression of its genes. To understand the molecular mechanisms that regulate HBV gene expression, we conducted a yeast one-hybrid screen to identify novel cellular transcription factors that may control HBV gene expression. Here, we demonstrate that Kruppel-like factor 15 (KLF15), a liver-enriched transcription factor, can robustly activate HBV surface and core promoters. Mutations in the putative KLF15 binding site in the HBV core promoter abolished the ability of KLF15 to activate the core promoter in luciferase assays. Furthermore, the overexpression of KLF15 stimulated the expression of HBV surface antigen (HBsAg) and the core protein and enhanced viral replication. Conversely, small interfering RNA knockdown of the endogenous KLF15 in Huh7 cells resulted in a reduction in HBV surface- and core-promoter activities. In electrophoretic mobility shift and chromatin immunoprecipitation assays, KLF15 binds to DNA probes derived from the core promoter and the surface promoter. Introduction of an expression vector for KLF15 short hairpin RNA, together with the HBV genome into the mouse liver using hydrodynamic injection, resulted in a significant reduction in viral gene expression and DNA replication. Additionally, mutations in the KLF15 response element in the HBV core promoter significantly reduced viral DNA levels in the mouse serum. Conclusion: KLF15 is a novel transcriptional activator for HBV core and surface promoters. It is possible that KLF15 may serve as a potential therapeutic target to reduce HBV gene expression and viral replication. (HEPATOLOGY 2011;54:109-121)

Hepatocellular carcinoma (HCC), the third leading cause of cancer deaths worldwide, is most commonly caused by chronic hepatitis B virus (HBV) infection. However, whether HBV plays any direct role in carcinogenesis, other than indirectly causing chronic liver injury by inciting the host immune response, remains unclear. We have established two independent transgenic mouse lines expressing the complete genome of a mutant HBV ("preS2 mutant") that is found at much higher frequencies in people with HCC than those without. The transgenic mice show evidence of stress in the endoplasmic reticulum (ER) and overexpression of cyclin D1 in hepatocytes. These mice do not show any evidence of chronic liver injury, but by 2 years of age a majority of the male mice develop hepatocellular neoplasms, including HCC. Unexpectedly, we also found a significant increase in hepatocarcinogenesis independent of necroinflammation in a transgenic line expressing the entire wildtype HBV. As in the mutant HBV mice, HCC was found only in aged—2-year-old—mice of the wildtype HBV line. The karyotype in all the three transgenic lines appears normal and none of the integration sites of the HBV transgene in the mice is near an oncogene or tumor suppressor gene. The significant increase of HCC incidence in all the three transgenic lines—expressing either mutant or wildtype HBV—therefore argues strongly that in absence of chronic necroinflammation, HBV can contribute directly to the development of HCC.

The molecular and cellular mechanisms underlying nephropathic cystinosis, which exhibits generalized proximal tubular dysfunction and progressive renal failure, remain largely unknown. Renal biopsies from patients with this disorder can reveal abnormally large mitochondria, but the relevance of this and other ultrastructural abnormalities is unclear. We studied the ultrastructure of fibroblasts and renal proximal tubular epithelial cells from patients with three clinical variants of cystinosis: Nephropathic, intermediate, and ocular. Electron microscopy revealed the presence of morphologically abnormal mitochondria and abnormal patterns of mitochondrial autophagy (mitophagy) with a high number of autophagic vacuoles and fewer mitochondria (P < 0.02) in nephropathic cystinosis. In addition, we observed increased apoptosis in renal proximal tubular epithelial cells, greater expression of LC3-II/LC3-I (microtubule-associated protein 1 light chain 3), and significantly more autophagosomes in the nephropathic variant. The autophagy inhibitor 3-methyl adenine rescued cell death in cystinotic cells. Cystinotic cells had increased levels of beclin-1 and aberrant mitochondrial function with a significant decrease in ATP generation and an increase in reactive oxygen species. This study provides ultrastructural and functional evidence of abnormal mitophagy in nephropathic cystinosis, which may contribute to the renal Fanconi syndrome and progressive renal injury.

Nature 457, 906–909 (2009) In this Letter, the name of T. S. Benedict Yen was incorrectly listed as Benjamin Yen. This author is affiliated with the Pathology Service, VA Medical Center, and the Department of Pathology, University of California, and not with the Department of Medicine, University ofCalifornia, as originally listed.

The hepatitis B virus X protein (HBX) plays key regulatory roles in viral replication and the development of hepatocellular carcinoma. HBX is an unstable protein; its instability is attributed to rapid degradation through the ubiquitin-proteasome pathway. Here, we show that the middle and carboxyl-terminal domains of HBX, independently fused to GFP, render the recombinant proteins susceptible to proteasomal degradation, while the amino-terminal domain has little effect on the ubiquitination or stability of HBX. Mutation of any single or combination of up to five of six lysine residues, all located in the middle and carboxyl-terminal domain, did not prevent HBX from being ubiquitinated, ruling out any specific lysine as the sole site of ubiquitination. Surprisingly, HBX in which all six lysines were mutated and showed no evidence of ubiquitination, was still susceptible to proteasomal degradation. These results suggest that both ubiquitin-dependent and -independent proteasomal degradation processes are operative in HBX turnover.

The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins, which, collectively, represent a large fraction of the total protein output of a mammalian cell. Therefore, the protein flux through the ER must be carefully monitored for abnormalities, including the buildup of misfolded proteins. Mammalian cells have evolved an intricate set of signaling pathways from the ER to the cytosol and nucleus, to allow the cell to respond to the presence of misfolded proteins within the ER. These pathways, known collectively as the unfolded protein response, are important for normal cellular homeostasis and organismal development and may also play key roles in the pathogenesis of many diseases. This review provides background information on the unfolded protein response and discusses a selection of diseases whose pathogenesis involves ER stress.

UNLABELLED:Autophagy is important for cellular homeostasis and can serve as innate immunity to remove intracellular pathogens. Here, we demonstrate by a battery of morphological and biochemical assays that hepatitis C virus (HCV) induces the accumulation of autophagosomes in cells without enhancing autophagic protein degradation. This induction of autophagosomes depended on the unfolded protein response (UPR), as the suppression of UPR signaling pathways suppressed HCV-induced lipidation of the microtubule-associated protein light chain 3 (LC3) protein, a necessary step for the formation of autophagosomes. The suppression of UPR or the suppression of expression of LC3 or Atg7, a protein that mediates LC3 lipidation, suppressed HCV replication, indicating a positive role of UPR and the incomplete autophagic response in HCV replication. CONCLUSION:Our studies delineate the molecular pathway by which HCV induces autophagic vacuoles and also demonstrate the perturbation of the autophagic response by HCV. These unexpected effects of HCV on the host cell likely play an important role in HCV pathogenesis.

A 65‐year‐old Latino man presented to his dermatologist for the removal of two melanocytic nevi from the back. The first nevus was removed from the right scapula and contained melanocytes with prominent eosinophilic nuclear inclusion bodies. The second nevus was removed from the paravertebral region, without evidence of inclusion bodies. Ultrastructurally, the inclusions in the first nevus contained dispersed finely granular, homogenous bodies without a limiting membrane. Immunohistochemistry characterized them as ubiquitin‐positive material. Reverse transcriptase in situ polymerase chain reaction analysis was positive for molluscum‐specific primers, suggesting that the inclusions encountered in the first nevus were secondary to a remote, local molluscum viral infection of melanocytes.

HBV is a major risk factor for hepatocellular carcinoma (HCC). However, whether HBV can directly cause HCC or only indirectly via the induction of chronic liver inflammation has been controversial. By using transgenic mice carrying the entire HBV genome as a model, we now demonstrate that HBV by itself is an inefficient carcinogen. However, it can efficiently promote hepatocarcinogenesis initiated by the carcinogen diethylnitrosamine (DEN). This effect of HBV does not involve chronic liver inflammation, is apparently due to enhanced hepatocellular apoptosis and compensatory regeneration following DEN treatment, and does not require the HBV X protein. Conclusion: Our results demonstrate a direct role of HBV in a hepatocarcinogenesis pathway that involves the interaction between this virus and a dietary carcinogen. (HEPATOLOGY 2007;45:16–21.)

OBJECTIVE:To compare the effects of combined administration of cyclophosphamide (CYC) and CTLA-4Ig with the effects of these agents alone on the immunopathology and progression of renal damage in (NZB x NZW)F(1) (B/W) lupus-prone mice, and to explore the clinical implications of this combination by evaluating the ability of CTLA-4Ig to sustain the benefit of CYC in patients with lupus nephritis.METHODS:We carried out a detailed, prospective pathologic and immunohistochemical analysis of the effects of CYC and CTLA-4Ig, alone and in combination, in kidney tissue from B/W mice. The acute effects of these agents on immune cells in the kidney were evaluated by fluorescence-activated cell sorting. We also compared the effect of brief CYC plus sustained CTLA-4Ig administration with the effect of sustained administration of both agents on the progression of renal disease in B/W mice.RESULTS:As a single agent, CTLA-4Ig was generally as effective, and in some cases more effective, than CYC in slowing progression of renal disease. Combined therapy with these two agents very effectively arrested the progression of renal damage and, in some respects, reversed renal pathology. Induction therapy with both CTLA-4Ig and CYC precluded the need for continuous administration of CYC.CONCLUSION:Our results indicate that the combination of CTLA-4Ig and CYC very effectively arrests the progression of murine lupus nephritis. These findings have direct implications for the treatment of lupus nephritis.

ABSTRACT The hepatitis C virus (HCV) core protein represents the first 191 amino acids of the viral precursor polyprotein and is cotranslationally inserted into the membrane of the endoplasmic reticulum (ER). Processing at position 179 by a recently identified intramembrane signal peptide peptidase leads to the generation and potential cytosolic release of a 179-amino-acid matured form of the core protein. Using confocal microscopy, we observed that a fraction of the mature core protein colocalized with mitochondrial markers in core-expressing HeLa cells and in Huh-7 cells containing the full-length HCV replicon. Subcellular fractionation confirmed this observation and showed that the core protein associates with purified mitochondrial fractions devoid of ER contaminants. The core protein also fractionated with mitochondrion-associated membranes, a site of physical contact between the ER and mitochondria. Using immunoelectron microscopy and in vitro mitochondrial import assays, we showed that the core protein is located on the mitochondrial outer membrane. A stretch of 10 amino acids within the hydrophobic C terminus of the processed core protein conferred mitochondrial localization when it was fused to green fluorescent protein. The location of the core protein in the outer mitochondrial membrane suggests that it could modulate apoptosis or lipid transfer, both of which are associated with this subcellular compartment, during HCV infection.

ABSTRACT The persistence of human immunodeficiency virus (HIV) in optimally treated infected individuals poses a major therapeutic problem. In latently infected cells, one of the observed phenotypes is absent elongation of viral transcription. Thus, the positive elongation factor b (P-TEFb), which is usually recruited by NF-κB or Tat, is not present on the HIV long terminal repeat (LTR). Although most attempts to activate these proviruses centered on NF-κB, we investigated effects of Tat. To this end, we generated transgenic mice, which secreted a chimera between Tat and the green fluorescent protein from β cells of the pancreas. This extracellular Tat distributed widely, entered nuclei of resting cells, and specifically transactivated the HIV LTR. No deleterious side effects of Tat were found. Next, we determined that Tat can activate latent proviruses in optimally treated infected individuals. In their cells, T-cell activation or exogenous Tat could induce viral replication equivalently. Thus, P-TEFb could activate the majority of the latent HIV, in this case by Tat.

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Studies of HCV replication and pathogenesis have so far been hampered by the lack of an efficient tissue culture system for propagating HCV in vitro. Although HCV is primarily a hepatotropic virus, an increasing body of evidence suggests that HCV also replicates in extrahepatic tissues in natural infection. In this study, we established a B-cell line (SB) from an HCV-infected non-Hodgkin's B-cell lymphoma. HCV RNA and proteins were detectable by RNase protection assay and immunoblotting. The cell line continuously produces infectious HCV virions in culture. The virus particles produced from the culture had a buoyant density of 1.13 to 1.15 g/ml in sucrose and could infect primary human hepatocytes, peripheral blood mononuclear cells (PBMCs), and an established B-cell line (Raji cells) in vitro. The virus from SB cells belongs to genotype 2b. Single-stranded conformational polymorphism and sequence analysis of the viral RNA quasispecies indicated that the virus present in SB cells most likely originated from the patient's spleen and had an HCV RNA quasispecies pattern distinct from that in the serum. The virus production from the infected primary hepatocytes showed cyclic variations. In addition, we have succeeded in establishing several Epstein-Barr virus-immortalized B-cell lines from PBMCs of HCV-positive patients. Two of these cell lines are positive for HCV RNA as detected by reverse transcriptase PCR and for the nonstructural protein NS3 by immunofluorescence staining. These observations unequivocally establish that HCV infects B cells in vivo and in vitro. HCV-infected cell lines show significantly enhanced apoptosis. These B-cell lines provide a reproducible cell culture system for studying the complete replication cycle and biology of HCV infections.