Laura F. Steel

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.

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 α 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. (HEPATOLOGY 2005;41:634–642.)

A Proteomic Approach for the Discovery of Early Detection Markers of Hepatocellular Carcinoma

Individuals chronically infected with hepatitis B or C virus (HBV, HCV) are at high risk for the development of hepatocellular carcinoma (HCC), with disease progression occurring relentlessly over many years. The diagnosis of HCC usually occurs at late stages in the disease when there are few effective treatment options and the prognosis for patients with HCC is very poor. The long latency period, together with clearly identified at risk populations, provide opportunities for earlier detection that will allow more timely and effective treatment of this devastating cancer. We are using a proteomic approach to test the hypothesis that changes in the amount of certain serum polypeptides, or changes in their post-translational modifications, can be used to predict the onset of HCC. Advances in the standardization of two dimensional gel electrophoresis (2DE) coupled with computerized image analysis now permit the reproducible resolution of thousands of polypeptides per run. Serum polypeptides from individuals at different stages in the disease continuum are being resolved by 2DE to identify those that change with disease progression. Polypeptides found by this method can be further characterized by mass spectrometry. In addition, the potential for changes in the glycan structure of certain polypeptides to serve as a marker for disease progression can be explored. The proteomic approach is expected to liberate us from the need to “cherry pick” or guess the best biomarkers and let the data tell us which are the best indicators of disease. Information may also be gleaned about the pathobiology of the disease process.

The Cellular TAR RNA Binding Protein, TRBP, Promotes HIV-1 Replication Primarily by Inhibiting the Activation of Double-Stranded RNA-Dependent Kinase PKR

ABSTRACT 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.

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.

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 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.

SEQUENCE AND DEVELOPMENTAL REGULATION OF THE GENE THAT ENCODES THE DICTYOSTELIUM DISCOIDEUM L3 RIBOSOMAL PROTEIN

We have isolated and characterized genomic and cDNA recombinant plasmids that encode the Dictyostelium discoideum (Dd) ribosomal protein L3 (rpL3). Genomic plasmids were identified using a probe derived from the Saccharomyces cerevisiae TCM1 gene, that encodes the yeast rpL3. The DdL3 gene contains two introns and encodes a protein 398 amino acids in length that shows a high degree of homology to the conserved rpL3 protein of both lower and higher eukaryotes. During development, both the pattern of accumulation of DdL3 mRNA and changes in its translational activity are identical to those observed for other r-protein mRNAs.

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.—Sanghvi, V. R., Steel, L. F. RNA silencing as a cellular defense against HIV‐1 infection: progress and issues. FASEB J. 26, 3937–3945 (2012). www.fasebj.org