Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes.

Many viruses modulate calcium (Ca2+) signaling to create a cellular environment that is more permissive to viral replication, but for most viruses that regulate Ca2+ signaling, the mechanism underlying this regulation is not well understood. The hepatitis B virus (HBV) HBx protein modulates cytosolic Ca2+ levels to stimulate HBV replication in some liver cell lines. A chronic HBV infection is associated with life-threatening liver diseases, including hepatocellular carcinoma (HCC), and HBx modulation of cytosolic Ca2+ levels could have an important role in HBV pathogenesis. Whether HBx affects cytosolic Ca2+ in a normal hepatocyte, the natural site of an HBV infection, has not been addressed. Here, we report that HBx alters cytosolic Ca2+ signaling in cultured primary hepatocytes. We used single cell Ca2+ imaging of cultured primary rat hepatocytes to demonstrate that HBx elevates the cytosolic Ca2+ level in hepatocytes following an IP3-linked Ca2+ response; HBx effects were similar when expressed alone or in the context of replicating HBV. HBx elevation of the cytosolic Ca2+ level required extracellular Ca2+ influx and store-operated Ca2+ (SOC) entry and stimulated HBV replication in hepatocytes. We used both targeted RT-qPCR and transcriptome-wide RNAseq analyses to compare levels of SOC channel components and other Ca2+ signaling regulators in HBV-expressing and control hepatocytes and show that the transcript levels of these various proteins are not affected by HBV. We also show that HBx regulation of SOC-regulated Ca2+ accumulation is likely the consequence of HBV modulation of a SOC channel regulatory mechanism. In support of this, we link HBx enhancement of SOC-regulated Ca2+ accumulation to Ca2+ uptake by mitochondria and demonstrate that HBx stimulates mitochondrial Ca2+ uptake in primary hepatocytes. The results of our study may provide insights into viral mechanisms that affect Ca2+ signaling to regulate viral replication and virus-associated diseases.

Intrinsic hepatocyte dedifferentiation is accompanied by upregulation of mesenchymal markers, protein sialylation and core alpha 1,6 linked fucosylation.

Alterations in N-linked glycosylation have long been associated with cancer but for the most part, the reasons why have remained poorly understood. Here we show that increased core fucosylation is associated with de-differentiation of primary hepatocytes and with the appearance of markers indicative of a transition of cells from an epithelial to a mesenchymal state. This increase in core fucosylation was associated with increased levels of two enzymes involved in α-1,6 linked fucosylation, GDP-mannose 4, 6-dehydratase (Gmds) and to a lesser extent fucosyltransferase 8 (Fut8). In addition, the activation of cancer-associated cellular signaling pathways in primary rat hepatocytes can increase core fucosylation and induce additional glycoform alterations on hepatocyte proteins. Specifically, we show that increased levels of protein sialylation and α-1,6-linked core fucosylation are observed following activation of the ß-catenin pathway. Activation of the Akt signaling pathway or induction of hypoxia also results in increased levels of fucosylation and sialylation. We believe that this knowledge will help in the better understanding of the genetic factors associated with altered glycosylation and may allow for the development of more clinically relevant biomarkers.

Hepatitis B virus and microRNAs: Complex interactions affecting hepatitis B virus replication and hepatitis B virus-associated diseases.

Chronic infection with the hepatitis B virus (HBV) is the leading risk factor for the development of hepatocellular carcinoma (HCC). With nearly 750000 deaths yearly, hepatocellular carcinoma is the second highest cause of cancer-related death in the world. Unfortunately, the molecular mechanisms that contribute to the development of HBV-associated HCC remain incompletely understood. Recently, microRNAs (miRNAs), a family of small non-coding RNAs that play a role primarily in post-transcriptional gene regulation, have been recognized as important regulators of cellular homeostasis, and altered regulation of miRNA expression has been suggested to play a significant role in virus-associated diseases and the development of many cancers. With this in mind, many groups have begun to investigate the relationship between miRNAs and HBV replication and HBV-associated disease. Multiple findings suggest that some miRNAs, such as miR-122, and miR-125 and miR-199 family members, are playing a role in HBV replication and HBV-associated disease, including the development of HBV-associated HCC. In this review, we discuss the current state of our understanding of the relationship between HBV and miRNAs, including how HBV affects cellular miRNAs, how these miRNAs impact HBV replication, and the relationship between HBV-mediated miRNA regulation and HCC development. We also address the impact of challenges in studying HBV, such as the lack of an effective model system for infectivity and a reliance on transformed cell lines, on our understanding of the relationship between HBV and miRNAs, and propose potential applications of miRNA-related techniques that could enhance our understanding of the role miRNAs play in HBV replication and HBV-associated disease, ultimately leading to new therapeutic options and improved patient outcomes.

RNA silencing as a cellular defense against HIV-1 infection: progress and issues.

MicroRNAs (miRNAs) are known to have a role in gene regulation that is closely integrated into the pathways that control virtually all fundamental cell processes of growth, differentiation, metabolism, and death. Whether silencing RNAs and the cellular pathways that generate them are also used in antiviral defense in higher eukaryotes, as they are in plants and lower eukaryotes, has been the subject of much study. Results to date point to a complex interplay between viruses and vertebrate host cells that can vary considerably among different viruses. Here, we review current knowledge regarding interactions between HIV-1 and host cell RNA silencing mechanisms. Important questions in this field remain unresolved, including whether HIV-1 itself encodes small silencing RNAs that might either promote or repress its replication, whether host cell miRNAs can directly target viral transcripts or can alter the course of infection indirectly through effects on cellular genes necessary for viral replication, and whether HIV-1 produces proteins or RNAs that suppress the host-silencing pathway. We summarize evidence and controversies related to the potential role of RNA silencing pathways as a defense against HIV-1 infection.

Polycistronic expression of interfering RNAs from RNA polymerase III promoters.

In many RNA silencing applications, there is a benefit to expressing multiple interfering RNAs simultaneously. This can be achieved by using a single RNA polymerase II promoter to express multiple micro(mi)RNA-formatted interfering RNAs that are arranged in a polycistronic cluster, mimicking the organization of naturally clustered, endogenous miRNAs. While RNA pol III promoters are often used to express individual short hairpin (sh) RNAs, we have recently shown that pol III promoters can also be used to drive polycistronic expression of miRNA-formatted interfering RNAs. Here, we present methods for the assembly of polycistronic miRNA expression vectors that use pol III promoters. In addition, we present methods for testing the potency and the level of expression of each of the individual miRNAs encoded in the construct.

The cellular TAR RNA binding protein, TRBP, promotes HIV-1 replication primarily by inhibiting the activation of double-stranded RNA-dependent kinase PKR.

The TAR RNA binding protein, TRBP, is a cellular double-stranded RNA (dsRNA) binding protein that can promote the replication of HIV-1 through interactions with the viral TAR element as well as with cellular proteins that affect the efficiency of translation of viral transcripts. The structured TAR element, present on all viral transcripts, can impede efficient translation either by sterically blocking access of translation initiation factors to the 5'-cap or by activating the dsRNA-dependent kinase, PKR. Several mechanisms by which TRBP can facilitate translation of viral transcripts have been proposed, including the binding and unwinding of TAR and the suppression of PKR activation. Further, TRBP has been identified as a cofactor of Dicer in the processing of microRNAs (miRNAs), and sequestration of TRBP by TAR in infected cells has been proposed as a viral countermeasure to potential host cell RNA interference-based antiviral activities. Here, we have addressed the relative importance of these various roles for TRBP in HIV-1 replication. Using Jurkat T cells, primary human CD4(+) T cells, and additional cultured cell lines, we show that depletion of TRBP has no effect on viral replication when PKR activation is otherwise blocked. Moreover, the presence of TAR-containing mRNAs does not affect the efficacy of cellular miRNA silencing pathways. These results establish that TRBP, when expressed at physiological levels, promotes HIV-1 replication mainly by suppressing the PKR-mediated antiviral response, while its contribution to HIV-1 replication through PKR-independent pathways is minimal.

A re-examination of global suppression of RNA interference by HIV-1.

The nature of the interaction between replicating HIV-1 and the cellular RNAi pathway has been controversial, but it is clear that it can be complex and multifaceted. It has been proposed that the interaction is bi-directional, whereby cellular silencing pathways can restrict HIV-1 replication, and in turn, HIV-1 can suppress silencing pathways. Overall suppression of RNAi has been suggested to occur via direct binding and inhibition of Dicer by the HIV-1 Tat protein or through sequestration of TRBP, a Dicer co-factor, by the structured TAR element of HIV-1 transcripts. The role of Tat as an inhibitor of Dicer has been questioned and our results support and extend the conclusion that Tat does not inhibit RNAi that is mediated by either exogenous or endogenous miRNAs. Similarly, we find no suppression of silencing pathways in cells with replicating virus, suggesting that viral products such as the TAR RNA elements also do not reduce the efficacy of cellular RNA silencing. However, knockdown of Dicer does allow increased viral replication and this occurs at a post-transcriptional level. These results support the idea that although individual miRNAs can act to restrict HIV-1 replication, the virus does not counter these effects through a global suppression of RNAi synthesis or processing.

Expression of interfering RNAs from an HIV-1 Tat-inducible chimeric promoter.

The therapeutic value of antiviral interfering RNAs could be improved by technologies that limit their expression to the infected cell population. The HIV-1 Tat-inducible viral LTR and LTR-containing chimeric promoters have previously been used to drive expression of antiviral RNAs and proteins directed against HIV-1. Here, we characterize an alternative promoter, consisting of a chicken ß-actin core promoter fused to the viral TAR element, for the conditional expression of interfering RNAs. This promoter, that we refer to as the CK-TAR promoter, can induce levels of silencing comparable to the viral LTR in response to Tat produced from co-transfected plasmids or during viral replication. While the CK-TAR promoter shows a modest level of basal activity, similar to the viral LTR, it is less responsive to the extracellular stimuli tested including LPS, TNFα, and PMA. The CK-TAR promoter is an alternative Tat-inducible promoter with the potential to minimize the risk of vector mobilization and to drive polycistronic expression of interfering RNAs.

RNA polymerase III can drive polycistronic expression of functional interfering RNAs designed to resemble microRNAs.

In both research and therapeutic applications of RNA interference, it is often advantageous to silence several targets simultaneously. Toward this end, several groups have developed vectors that utilize the model of endogenously encoded micro (mi) RNAs, where a single RNA polymerase II promoter can drive the expression of multiple interfering RNAs. Stronger pol III promoters have been used to drive individual short hairpin (sh) RNAs, but to date, it has been necessary to repeat the promoter in each silencing cassette to achieve multiplexed expression from a single vector. Here, we show that it is possible to drive polycistronic expression from a single pol III promoter when the interfering RNAs are formatted to resemble miRNAs rather than shRNAs. As many as four miRNAs designed to target hepatitis B virus (HBV) transcripts are shown to be processed and functional in reporter assays as well as in the context of replicating virus in cell culture systems. Although it has been observed that high levels of expression of shRNAs can lead to cytotoxicity, we find no significant evidence in transient transfection assays that the HBV-miRNAs produced by our vectors compete for the activity of endogenously produced miR-122 or for processing of an exogenously expressed miR-EGFP.

Vector design for liver-specific expression of multiple interfering RNAs that target hepatitis B virus transcripts.

RNA interference (RNAi) is a process that can target intracellular RNAs for degradation in a highly sequence-specific manner, making it a powerful tool that is being pursued in both research and therapeutic applications. Hepatitis B virus (HBV) is a serious public health problem in need of better treatment options, and aspects of its life cycle make it an excellent target for RNAi-based therapeutics. We have designed a vector that expresses interfering RNAs that target HBV transcripts, including both viral RNA replicative intermediates and mRNAs encoding viral proteins. Our vector design incorporates many features of endogenous microRNA (miRNA) gene organization that are proving useful for the development of reagents for RNAi. In particular, our vector contains an RNA pol II driven gene cassette that leads to tissue-specific expression and efficient processing of multiple interfering RNAs from a single transcript, without the co-expression of any protein product. This vector shows potent silencing of HBV targets in cell culture models of HBV infection. The vector design will be applicable to silencing of additional cellular or disease-related genes.

SELDI-TOF MS profiling of serum for detection of the progression of chronic hepatitis C to hepatocellular carcinoma.

Proteomic profiling of serum is an emerging technique to identify new biomarkers indicative of disease severity and progression. The objective of our study was to assess the use of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify multiple serum protein biomarkers for detection of liver disease progression to hepatocellular carcinoma (HCC). A cohort of 170 serum samples obtained from subjects in the United States with no liver disease (n = 39), liver diseases not associated with cirrhosis (n = 36), cirrhosis (n = 38), or HCC (n = 57) were applied to metal affinity protein chips for protein profiling by SELDI-TOF MS. Across the four test groups, 38 differentially expressed proteins were used to generate multiple decision classification trees to distinguish the known disease states. Analysis of a subset of samples with only hepatitis C virus (HCV)-related disease was emphasized. The serum protein profiles of control patients were readily distinguished from each HCV-associated disease state. Two-way comparisons of chronic hepatitis C, HCV cirrhosis, or HCV-HCC versus healthy had a sensitivity/specificity range of 74% to 95%. For distinguishing chronic HCV from HCV-HCC, a sensitivity of 61% and a specificity of 76% were obtained. However, when the values of known serum markers alpha fetoprotein, des-gamma carboxyprothrombin, and GP73 were combined with the SELDI peak values, the sensitivity and specifity improved to 75% and 92%, respectively. In conclusion, SELDI-TOF MS serum profiling is able to distinguish HCC from liver disease before cirrhosis as well as cirrhosis, especially in patients with HCV infection compared with other etiologies.

Assays for glucosidase inhibitors with potential antiviral activities: secreted alkaline phosphatase as a surrogate marker.

As secretion of the middle (MHBs) glycoprotein of hepatitis B virus is highly dependent upon the action of the host oligosaccharide processing enzymes glucosidase I and II, drugs that inhibit this enzyme have been proposed as potential antiviral agents. To facilitate the identification of new, more effective inhibitors of MHBs secretion, an assay has been developed based on the expression of this glycoprotein alone by transfection of Huh7 hepatoma cells. The data clearly demonstrate that both mono- and di-glycosylated forms of MHBs are produced in this system and both forms are equally dependent upon glucosidase processing for secretion. In addition, inclusion of a co-transfected reporter construct that encodes secreted alkaline phosphatase (SEAP) to permit normalization of transfection revealed that the SEAP gene product was itself sensitive to glucosidase inhibition. This sensitivity also was observed in HepG2 human hepatoma cells. Thus, measuring SEAP secretion may be another method for evaluating glucosidase inhibition. In addition, this finding has important implications for the use of a SEAP reporter in screens of potential antiviral agents.

Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans.

Chronic infection with hepatitis B virus (HBV) is associated with the majority of hepatocellular carcinoma (HCC). The diagnosis of HCC is usually made in the late stages of the disease, when treatment options are limited and prognosis is poor. We therefore have developed a method of glycoproteomic analysis in an attempt to discover serum markers that can assist in the early detection of HBV-induced liver cancer. Briefly, a comparative method for analysis of oligosaccharides released from serum glycoproteins and for recovery and identification of proteins with aberrant glycosylation, as a function of cancer diagnosis, is described. The model we have used is the woodchuck (Marmota monax), which shares similarities in the glycosylation pattern associated with liver proteins in human HCC. In this report, we show that woodchucks diagnosed with HCC have dramatically higher levels of serum-associated core alpha-1,6-linked fucose, as compared with woodchucks without a diagnosis of HCC. The coupling of this methodology with 2D gel proteomics has permitted the identification of several glycoproteins with altered glycosylation as a function of cancer. One such glycoprotein, Golgi Protein 73 (GP73), was found to be elevated and hyperfucosylated in animals with HCC. Further, the study showed GP73 to be elevated in the serum of people with a diagnosis of HCC, providing a validation of our approach. The potential of this technology for biomarker discovery and the implications of increased levels of GP73 in liver cancer are discussed.

Methods of comparative proteomic profiling for disease diagnostics.

The recent development of numerous technologies for proteome analysis holds the promise of new and more precise methods for disease diagnosis. In this review, we provide an overview of some of these technologies including two-dimensional gel electrophoresis (2DE), historically the workhorse of proteomic analysis, as well as some newer approaches such as liquid phase separations combined with mass spectrometry, and protein microarrays. It is evident that each method has its own strengths and weaknesses and no single method will be optimal in all applications. However, the continuing development of innovative strategies for protein separation and analysis is providing a wealth of new tools for multi-dimensional protein profiling that will advance our capabilities in disease diagnostics and our understanding of disease pathology.

Comparative proteomic analysis of de-N-glycosylated serum from hepatitis B carriers reveals polypeptides that correlate with disease status.

Analysis of the polypeptide profile in tissues, cells, and sera by high-resolution two-dimensional (2-D) gel electrophoresis offers promise in the identification of biomarkers that correlate with disease. However, sera contain many polypeptides bearing N-linked glycosylation that can complicate interpretation. Therefore, we tested the possibility that de-N-glycosylation of the polypeptides present in human serum would result in a simplification of serum proteome profiles. Briefly, polypeptides present in human serum were left untreated or subjected to de-N-glycosylation by incubation with PNGase F and resolved by high-resolution 2-D gel electrophoresis. De-N-glycosylation reduced the number of glycoform variants, enhanced the resolution of many polypeptides and allowed other polypeptides to become visible. As an initial test of concept, clinically relevant serum samples from individuals with or without diagnosis of hepatocellular carcinoma were compared. Several polypeptides, apparent only after de-N-glycosylation, were shown to correlate with disease. Although the results are preliminary and the identities of all the putative biomarkers not yet known, the data suggest that de-N-glycosylation offers a method to enhance the resolution of serum polypeptide profiles and has value in comparative proteomic studies.

A strategy for the comparative analysis of serum proteomes for the discovery of biomarkers for hepatocellular carcinoma.

Many of the emerging technologies for the global evaluation of gene expression, at both the RNA and protein level, are being applied to the problem of finding biomarkers for human disease progression. These analyses can be made difficult, however, by variation between samples that arises from both technical and nondisease related physiological or genetic causes. In an effort to identify serum polypeptides whose presence or absence correlates with the clinical status of patients at high risk for hepatocellular carcinoma (HCC), we have developed a strategy that helps to focus the analysis on meaningful changes in protein levels above the background of variation. For the current study we divided the patient population into four clinically defined diagnostic groups that represent a generally increasing risk for HCC. Chronic infection with hepatitis B virus (HBV) is a major risk factor for HCC and our groups included patients with no indication of liver disease (healthy), those with inactive chronic HBV, those with active chronic HBV, and patients with a diagnosis of HCC and history of chronic HBV infection. Serum polypeptides from these patients were first analyzed in two-dimensional gels by combining the serum from patients in each of the four groups to generate composite gel profiles. Analysis of these composite gels allowed us to identify two relatively abundant features that were reduced in the HCC group as compared to the healthy group. Tryptic fragment mass fingerprinting identified the features as a carboxy terminal fragment of complement C3 and an isoform of apolipoprotein A1. These two features were examined by two-dimensional gel electrophoresis of serum from each individual in the four groups in order to verify that the inter-group differences seen in composite gels reported changes in abundance for most members of the group, rather than extreme changes for a small fraction of the group. These preliminary studies suggest that a proteomic methodology can be used for the identification of serum biomarkers for HCC and other liver disease.

Efficient and specific removal of albumin from human serum samples.

Patient serum or plasma is frequently monitored for biochemical markers of disease or physiological status. Many of the rapidly evolving technologies of proteome analysis are being used to find additional clinically informative protein markers. The unusually high abundance of albumin in serum can interfere with the resolution and sensitivity of many proteome profiling techniques. We have used monoclonal antibodies against human serum albumin (HSA) to develop an immunoaffinity resin that is effective in the removal of both full-length HSA and many of the HSA fragments present in serum. This resin shows markedly better performance than dye-based resins in terms of both the efficiency and specificity of albumin removal. Immunoglobulins are another class of highly abundant serum protein. When protein G resin is used together with our immunoaffinity resin, Ig proteins and HSA can be removed in a single step. This strategy could be extended to the removal of any protein for which specific antibodies or affinity reagents are available.