Modification of human immunodeficiency virus type 1 reverse transcriptase to target cells with elevated cellular dNTP concentrations

Retroviruses and DNA viruses utilize cellular dNTPs as substrates for their DNA polymerases during viral replication in infected cells. However, because of S phase-dependent dNTP biosynthesis, the availability of cellular dNTPs significantly varies among cell types (e.g. dividing versus nondividing cells and normal versus tumor cells). Here we tested whether alterations in the dNTP utilization efficiency and dNTP binding affinity of viral DNA polymerases can switch viral infection specificity to cell types with different dNTP concentrations. We employed an HIV-1 reverse transcriptase (RT) mutant (Q151N), which is catalytically active only at high dNTP concentrations because of its reduced dNTP binding affinity. Indeed, the modified HIV-1 vector harboring the Q151N mutant RT preferentially transduced tumor cells containing higher cellular dNTP concentrations than primary cells (e.g. human lung fibroblasts (HLFs) and human keratinocytes). Although the wild type HIV-1 vector transduced both HLFs and tumor cells, the Q151N vector failed to transduce HLFs and keratinocytes but efficiently transduced tumor cells. Pretreatment of HLFs with deoxynucleosides, which increase cellular dNTP pools, enabled the mutant vector to transduce HLFs, suggesting that the transduction failure of the RT mutant vector to primary cells is because of inefficient reverse transcription in low cellular dNTP environments. We also observed that the Q151N vector expressing herpes simplex virus-thymidine kinase renders tumor cells sensitive to gancyclovir. This study validates a novel strategy in which modifications of viral DNA polymerases in various vector systems allow the delivery of target genes exclusively to tumor cells exploiting elevated cellular dNTP concentration as a tumor cell-specific host factor. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc

Potential pitfalls in microRNA profiling: Potential pitfalls in microRNA profiling

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally influence a wide range of cellular processes such as the host response to viral infection, innate immunity, cell cycle progression, migration and apoptosis through the inhibition of target mRNA translation. Due to the growing number of microRNAs and identification of their functional roles, miRNA profiling of many different sample types has become more expansive, especially with relevance to disease signatures. Here, we address some of the advantages and potential pitfalls of the currently available methods for miRNA expression profiling. Some of the topics discussed include isomiRNAs, comparison of different profiling platforms, normalization strategies and issues with regard to sample preparation and experimental analyses

Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy

<p>Abstract</p> <p>Background</p> <p>Unlike CD4+ T cells, HIV-1 infected macrophages exhibit extended life span even upon stress, consistent with their <it>in vivo </it>role as long-lived HIV-1 reservoirs.</p> <p>Results</p> <p>Here, we demonstrate that PI3K/Akt inhibitors, including clinically available Miltefosine, dramatically reduced HIV-1 production from long-living virus-infected macrophages. These PI3K/Akt inhibitors hyper-sensitize infected macrophages to extracellular stresses that they are normally exposed to, and eventually lead to cell death of infected macrophages without harming uninfected cells. Based on the data from these Akt inhibitors, we were able to further investigate how HIV-1 infection utilizes the PI3K/Akt pathway to establish the cytoprotective effect of HIV-1 infection, which extends the lifespan of infected macrophages, a key viral reservoir. First, we found that HIV-1 infection activates the well characterized pro-survival PI3K/Akt pathway in primary human macrophages, as reflected by decreased PTEN protein expression and increased Akt kinase activity. Interestingly, the expression of HIV-1 or SIV Tat is sufficient to mediate this cytoprotective effect, which is dependent on the basic domain of Tat – a region that has previously been shown to bind p53. Next, we observed that this interaction appears to contribute to the downregulation of PTEN expression, since HIV-1 Tat was found to compete with PTEN for p53 binding; this is known to result in p53 destabilization, with a consequent reduction in PTEN protein production.</p> <p>Conclusion</p> <p>Since HIV-1 infected macrophages display highly elevated Akt activity, our results collectively show that PI3K/Akt inhibitors may be a novel therapy for interfering with the establishment of long-living HIV-1 infected reservoirs.</p

Pre-Micro RNA Signatures Delineate Stages of Endothelial Cell Transformation in Kaposi Sarcoma

MicroRNAs (miRNA) have emerged as key regulators of cell lineage differentiation and cancer. We used precursor miRNA profiling by a novel real-time QPCR method (i) to define progressive stages of endothelial cell transformation cumulating in Kaposi sarcoma (KS) and (ii) to identify specific miRNAs that serve as biomarkers for tumor progression. We were able to compare primary patient biopsies to well-established culture and mouse tumor models. Loss of mir-221 and gain of mir-15 expression demarked the transition from merely immortalized to fully tumorigenic endothelial cells. Mir-140 and Kaposi sarcoma–associated herpesvirus viral miRNAs increased linearly with the degree of transformation. Mir-24 emerged as a biomarker specific for KS

Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation

Although oxidative tissue injury often accompanies viral infection, there is little understanding of how it influences virus replication. We show that multiple hepatitis C virus (HCV) genotypes are exquisitely sensitive to oxidative membrane damage, a property distinguishing them from other pathogenic RNA viruses. Lipid peroxidation, regulated in part through sphingosine kinase 2, severely restricts HCV replication in Huh-7 cells and primary human hepatoblasts. Endogenous oxidative membrane damage lowers the 50% effective concentration of direct-acting antivirals, suggesting critical regulation of the conformation of the NS3/4A protease and NS5B polymerase, membrane-bound HCV replicase components. Resistance to lipid peroxidation maps genetically to trans-membrane and membrane-proximal residues within these proteins, and is essential for robust replication in cell culture, as exemplified by the atypical JFH1 strain. Thus, the typical, wild-type HCV replicase is uniquely regulated by lipid peroxidation, providing a novel mechanism for attenuating replication in stressed tissue and possibly facilitating long-term viral persistence

Pre-Micro RNA Signatures Delineate Stages of Endothelial Cell Transformation in Kaposi Sarcoma

MicroRNAs (miRNA) have emerged as key regulators of cell lineage differentiation and cancer. We used precursor miRNA profiling by a novel real-time QPCR method (i) to define progressive stages of endothelial cell transformation cumulating in Kaposi sarcoma (KS) and (ii) to identify specific miRNAs that serve as biomarkers for tumor progression. We were able to compare primary patient biopsies to well-established culture and mouse tumor models. Loss of mir-221 and gain of mir-15 expression demarked the transition from merely immortalized to fully tumorigenic endothelial cells. Mir-140 and Kaposi sarcoma–associated herpesvirus viral miRNAs increased linearly with the degree of transformation. Mir-24 emerged as a biomarker specific for KS

Poisson factor models with applications to non-normalized microRNA profiling.

Next-generation (NextGen) sequencing is becoming increasingly popular as an alternative for transcriptional profiling, as is the case for micro RNAs (miRNA) profiling and classification. miRNAs are a new class of molecules that are regulated in response to differentiation, tumorigenesis or infection. Our primary motivating application is to identify different viral infections based on the induced change in the host miRNA profile. Statistical challenges are encountered because of special features of NextGen sequencing data: the data are read counts that are extremely skewed and non-negative; the total number of reads varies dramatically across samples that require appropriate normalization. Statistical tools developed for microarray expression data, such as principal component analysis, are sub-optimal for analyzing NextGen sequencing data. We propose a family of Poisson factor models that explicitly takes into account the count nature of sequencing data and automatically incorporates sample normalization through the use of offsets. We develop an efficient algorithm for estimating the Poisson factor model, entitled Poisson Singular Value Decomposition with Offset (PSVDOS). The method is shown to outperform several other normalization and dimension reduction methods in a simulation study. Through analysis of an miRNA profiling experiment, we further illustrate that our model achieves insightful dimension reduction of the miRNA profiles of 18 samples: the extracted factors lead to more accurate and meaningful clustering of the cell lines. The PSVDOS software is available on request.Link_to_subscribed_fulltex

Toll-Like Receptor-3 Is Dispensable for the Innate MicroRNA Response to West Nile Virus (WNV)

<div><p>The innate immune response to West Nile virus (WNV) infection involves recognition through toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), leading to establishment of an antiviral state. MiRNAs (miRNAs) have been shown to be reliable biomarkers of TLR activation. Here, we sought to evaluate the contribution of TLR3 and miRNAs to the host response to WNV infection. We first analyzed HEK293-NULL and HEK293-TLR3 cells for changes in the innate immune response to infection. The presence of TLR3 did not seem to affect WNV load, infectivity or phosphorylation of IRF3. Analysis of experimentally validated NFκB-responsive genes revealed a WNV-induced signature largely independent of TLR3. Since miRNAs are involved in viral pathogenesis and the innate response to infection, we sought to identify changes in miRNA expression upon infection in the presence or absence of TLR3. MiRNA profiling revealed 70 miRNAs induced following WNV infection in a TLR3-independent manner. Further analysis of predicted gene targets of WNV signature miRNAs revealed genes highly associated with pathways regulating cell death, viral pathogenesis and immune cell trafficking.</p></div

Venn diagram of miRNAs that were induced in HEK293 Null and HEK293 TLR3 cells.

<p>Venn diagram representation of miRNAs induced following WNV infection of HEK293-Null and HEK293-TLR3 cells or polyI:C stimulation of HEK293-TLR3 cells. The number of shared, induced miRNAs are shown for each sample subset. A detailed list of the miRNAs induced for each category represented in the Venn diagram (HEK293-NULL +WNV, HEK293-TLR3 + WNV and HEK293-TLR3 + polyI:C treatment) is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104770#pone.0104770.s008" target="_blank">Table S6</a>.</p