Molecular Biology International The latest articles from Hindawi Publishing Corporation © 2014 , Hindawi Publishing Corporation . All rights reserved. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase Sun, 17 Aug 2014 12:13:49 +0000 As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu, Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition. Peter McInerney, Paul Adams, and Masood Z. Hadi Copyright © 2014 Peter McInerney et al. All rights reserved. The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons Thu, 10 Jul 2014 00:00:00 +0000 In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5 sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis. Ingo C. Starke, Wilfried Vahjen, Robert Pieper, and Jürgen Zentek Copyright © 2014 Ingo C. Starke et al. All rights reserved. Expression of TPM1, a Novel Sarcomeric Isoform of the TPM1 Gene, in Mouse Heart and Skeletal Muscle Thu, 24 Apr 2014 13:56:25 +0000 We have investigated the expression of TPM1α and TPM1κ in mouse striated muscles. TPM1α and TMP1κ were amplified from the cDNA of mouse heart by using conventional RT-PCR. We have cloned the PCR amplified DNA and determined the nucleotide sequences. Deduced amino acid sequences show that there are three amino acid changes in mouse exon 2a when compared with the human TPM1κ. However, the deduced amino acid sequences of human TPM1α and mouse TPM1α are identical. Conventional RT-PCR data as well as qRT-PCR data, calculating both absolute copy number and relative expression, revealed that the expression of TPM1κ is significantly lower compared to TPM1α in both mouse heart and skeletal muscle. It was also found that the expression level of TPM1κ transcripts in mouse heart is higher than it is in skeletal muscle. To the best of our knowledge, this is the first report of the expression of TPM1κ in mammalian skeletal muscle. Syamalima Dube, Lauren Panebianco, Amr A. Matoq, Henry N. Chionuma, Christopher R. Denz, Bernard J. Poiesz, and Dipak K. Dube Copyright © 2014 Syamalima Dube et al. All rights reserved. Primer Based Approach for PCR Amplification of High GC Content Gene: Mycobacterium Gene as a Model Mon, 24 Mar 2014 16:58:57 +0000 The genome of Mycobacterium is rich in GC content and poses problem in amplification of some genes, especially those rich in the GC content in terminal regions, by standard/routine PCR procedures. Attempts have been made to amplify three GC rich genes of Mycobacterium sp. (Rv0519c and Rv0774c from M. tuberculosis and ML0314c from M. leprae). Out of these three genes, Rv0774c gene was amplified with normal primers under standard PCR conditions, while no amplification was observed in case of Rv0519c and ML0314c genes. In the present investigation a modified primer based approach was successfully used for amplification of GC rich sequence of Rv0519c through codon optimization without changing the native amino acid sequence. The strategy was successfully confirmed by redesigning the standard primers with similar modifications followed by amplification of ML0314c gene. Arbind Kumar and Jagdeep Kaur Copyright © 2014 Arbind Kumar and Jagdeep Kaur. All rights reserved. The Role of Suppressors of Cytokine Signalling in Human Neoplasms Sun, 16 Mar 2014 13:46:47 +0000 Suppressors of cytokine signalling 1–7 (SOCS1–7) and cytokine-inducible SH2-containing protein (CIS) are a group of intracellular proteins that are well known as JAK-STAT and several other signalling pathways negative feedback regulators. More recently several members have been identified as tumour suppressors and dysregulation of their biological roles in controlling cytokine and growth factor signalling may contribute to the development of many solid organ and haematological malignancies. This review explores their biological functions and their possible tumour suppressing role in human neoplasms. Walid Sasi, Anup K. Sharma, and Kefah Mokbel Copyright © 2014 Walid Sasi et al. All rights reserved. A Synthetic Interaction between CDC20 and RAD4 in Saccharomyces cerevisiae upon UV Irradiation Sun, 23 Feb 2014 09:50:56 +0000 Regulation of DNA repair can be achieved through ubiquitin-mediated degradation of transiently induced proteins. In Saccharomyces cerevisiae, Rad4 is involved in damage recognition during nucleotide excision repair (NER) and, in conjunction with Rad23, recruits other proteins to the site of damage. We identified a synthetic interaction upon UV exposure between Rad4 and Cdc20, a protein that modulates the activity of the anaphase promoting complex (APC/C), a multisubunit E3 ubiquitin ligase complex. The moderately UV sensitive Δrad4 strain became highly sensitive when cdc20-1 was present, and was rescued by overexpression of CDC20. The double mutant is also deficient in elicting RNR3-lacZ transcription upon exposure to UV irradiation or 4-NQO compared with the Δrad4 single mutant. We demonstrate that the Δrad4/cdc20-1 double mutant is defective in double strand break repair by way of a plasmid end-joining assay, indicating that Rad4 acts to ensure that damaged DNA is repaired via a Cdc20-mediated mechanism. This study is the first to present evidence that Cdc20 may play a role in the degradation of proteins involved in nucleotide excision repair. Bernadette Connors, Lauren Rochelle, Asela Roberts, and Graham Howard Copyright © 2014 Bernadette Connors et al. All rights reserved. Cancer Stem Cells Accountability in Progression of Head and Neck Squamous Cell Carcinoma: The Most Recent Trends! Wed, 19 Feb 2014 12:07:22 +0000 Cancer stem cells (CSCs) play a major role in local recurrence and metastatic spread in head and neck squamous cell carcinomas (HNSCC). Evidence suggests that cancer stem cells are resistant to conventional therapy. So the emerging concepts of the role of cancer stem cells in the pathobiology of HNSCC should be understood carefully to be able to create new paradigms in treatment plans. Samapika Routray and Neeta Mohanty Copyright © 2014 Samapika Routray and Neeta Mohanty. All rights reserved. Sequence Characterization of Mitochondrial 12S rRNA Gene in Mouse Deer (Moschiola indica) for PCR-RFLP Based Species Identification Mon, 23 Dec 2013 10:04:21 +0000 Mitochondrial 12S rRNA has proven to be a useful molecular marker for better conservation and management of the endangered species. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the mitochondrial 12S rRNA gene has proven to be a reliable and efficient tool for the identification of different Indian deer species of family cervidae. In the present study, mitochondrial 12S rRNA gene sequence of mouse deer (Moschiola indica) belonging to the family Tragulidae was characterized and analysed in silico for its use in species identification. Genomic DNA was isolated from the hair follicles and mitochondrial 12S rRNA gene was amplified using universal primers. PCR product was cloned and sequenced for the first time. The sequence of mouse deer showed 90.04, 90.08, 90.04, 91.2, 90.04, and 90.08% identities with sika deer, sambar, hog deer, musk deer, chital, and barking deer, respectively. Restriction mapping in Lasergene (DNAstar Inc., Madison, WI, USA) revealed that mouse deer mitochondrial 12S rRNA gene sequence can be differentiated from the other deer species in PCR-RFLP using RsaI, DdeI, BsrI, and BstSFI. With the help of predicted pattern, mouse deer can be identified using genomic DNA from a variety of biomaterials, thereby providing molecular aid in wildlife forensics and conservation of the species. Chandra Mohan Siddappa, Mohini Saini, Asit Das, Ramesh Doreswamy, Anil K. Sharma, and Praveen K. Gupta Copyright © 2013 Chandra Mohan Siddappa et al. All rights reserved. Investigation of the Association between Genetic Polymorphism of Microsomal Epoxide Hydrolase and Primary Brain Tumor Incidence Mon, 16 Dec 2013 09:18:07 +0000 mEH is a critical biotransformation enzyme that catalyzes the conversion of xenobiotic epoxide substrates into more polar diol metabolites: it is also capable of inactivating a large number of structurally different molecules. Two polymorphisms affecting enzyme activity have been described in the exon 3 and 4 of the mEH gene. The hypothesis of this study is that inherent genetic susceptibility to a primary brain tumor is associated with mEH gene polymorphisms. The polymorphisms of the mEH gene were determined with PCR-RFLP techniques and 255 Turkish individuals. Our results indicate that the frequency of the mEH exon 4 polymorphism (in controls) is significantly higher than that of primary brain tumor patients (OR = 1.8, 95% CI = 1.0–3.4). This report, however, failed to demonstrate a significant association between mEH exon 3 polymorphism and primary brain tumor susceptibility in this population. Analysis of patients by both histological types of primary brain tumor and gene variants showed no association, although analysis of family history of cancer between cases and controls showed a statistically significant association (, ). Our results marginally support the hypothesis that genetic susceptibility to brain tumors may be associated with mEPHX gene polymorphisms. Ali Aydin, Hatice Pinarbasi, and Mustafa Gurelik Copyright © 2013 Ali Aydin et al. All rights reserved. The RASSF1 Gene and the Opposing Effects of the RASSF1A and RASSF1C Isoforms on Cell Proliferation and Apoptosis Tue, 12 Nov 2013 15:11:27 +0000 RASSF1A has been demonstrated to be a tumor suppressor, while RASSF1C is now emerging as a growth promoting protein in breast and lung cancer cells. To further highlight the dual functionality of the RASSF1 gene, we have compared the effects of RASSF1A and RASSF1C on cell proliferation and apoptosis in the presence of TNF-α. Overexpression of RASSF1C in breast and lung cancer cells reduced the effects of TNF-α on cell proliferation, apoptosis, and MST1/2 phosphorylation, while overexpression of RASSF1A had the opposite effect. We also assessed the expression of RASSF1A and RASSF1C in breast and lung tumor and matched normal tissues. We found that RASSF1A mRNA levels are significantly higher than RASSF1C mRNA levels in all normal breast and lung tissues examined. In addition, RASSF1A expression is significantly downregulated in 92% of breast tumors and in 53% of lung tumors. Conversely, RASSF1C was upregulated in 62% of breast tumors and in 47% of lung tumors. Together, these findings suggest that RASSF1C, unlike RASSF1A, is not a tumor suppressor but instead may play a role in stimulating survival in breast and lung cancer cells. Mark E. Reeves, Matthew Firek, Shin-Tai Chen, and Yousef Amaar Copyright © 2013 Mark E. Reeves et al. All rights reserved. Communication and the Emergence of Collective Behavior in Living Organisms: A Quantum Approach Wed, 30 Oct 2013 13:47:07 +0000 Intermolecular interactions within living organisms have been found to occur not as individual independent events but as a part of a collective array of interconnected events. The problem of the emergence of this collective dynamics and of the correlated biocommunication therefore arises. In the present paper we review the proposals given within the paradigm of modern molecular biology and those given by some holistic approaches to biology. In recent times, the collective behavior of ensembles of microscopic units (atoms/molecules) has been addressed in the conceptual framework of Quantum Field Theory. The possibility of producing physical states where all the components of the ensemble move in unison has been recognized. In such cases, electromagnetic fields trapped within the ensemble appear. In the present paper we present a scheme based on Quantum Field Theory where molecules are able to move in phase-correlated unison among them and with a self-produced electromagnetic field. Experimental corroboration of this scheme is presented. Some consequences for future biological developments are discussed. Marco Bischof and Emilio Del Giudice Copyright © 2013 Marco Bischof and Emilio Del Giudice. All rights reserved. The Transcriptomics of Secondary Growth and Wood Formation in Conifers Tue, 29 Oct 2013 13:27:55 +0000 In the last years, forestry scientists have adapted genomics and next-generation sequencing (NGS) technologies to the search for candidate genes related to the transcriptomics of secondary growth and wood formation in several tree species. Gymnosperms, in particular, the conifers, are ecologically and economically important, namely, for the production of wood and other forestry end products. Until very recently, no whole genome sequencing of a conifer genome was available. Due to the gradual improvement of the NGS technologies and inherent bioinformatics tools, two draft assemblies of the whole genomes sequence of Picea abies and Picea glauca arose in the current year. These draft genome assemblies will bring new insights about the structure, content, and evolution of the conifer genomes. Furthermore, new directions in the forestry, breeding and research of conifers will be discussed in the following. The identification of genes associated with the xylem transcriptome and the knowledge of their regulatory mechanisms will provide less time-consuming breeding cycles and a high accuracy for the selection of traits related to wood production and quality. Ana Carvalho, Jorge Paiva, José Louzada, and José Lima-Brito Copyright © 2013 Ana Carvalho et al. All rights reserved. GAPDH Pseudogenes and the Quantification of Feline Genomic DNA Equivalents Sun, 28 Apr 2013 17:44:01 +0000 Quantitative real-time PCR (qPCR) is broadly used to detect and quantify nucleic acid targets. In order to determine cell copy number and genome equivalents, a suitable reference gene that is present in a defined number in the genome is needed, preferably as a single copy gene. For most organisms, a variable number of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) pseudogenes have been reported. However, it has been suggested that a single-copy of the GAPDH pseudogene is present in the feline genome and that a GAPDH assay can therefore be used to quantify feline genomic DNA (gDNA). The aim of this study was to determine whether one or more GAPDH pseudogenes are present in the feline genome and to provide a suitable alternative qPCR system for the quantification of feline cell copy number and genome equivalents. Bioinformatics and sequencing results revealed that not just one but several closely related GAPDH-like sequences were present in the cat genome. We thus identified, developed, optimized, and validated an alternative reference gene assay using feline albumin (fALB). Our data emphasize the need for an alternative reference gene, apart from the GAPDH pseudogene, for the normalization of gDNA levels. We recommend using the fALB qPCR assay for future studies. A. Katrin Helfer-Hungerbuehler, Stefan Widmer, and Regina Hofmann-Lehmann Copyright © 2013 A. Katrin Helfer-Hungerbuehler et al. All rights reserved. Prevention of Lysosomal Storage Diseases and Derivation of Mutant Stem Cell Lines by Preimplantation Genetic Diagnosis Wed, 26 Dec 2012 07:53:51 +0000 Preimplantation genetic diagnosis (PGD) allows birth of unaffected children for couples at risk for a genetic disorder. We present the strategy and outcome of PGD for four lysosomal storage disorders (LSD): Tay-Sachs disease (TSD), Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (HS), and subsequent development of stem cell lines. For each disease, we developed a family-specific fluorescent multiplex single-cell PCR protocol that included the familial mutation and informative markers surrounding the mutation. Embryo biopsy and PGD analysis were performed on either oocytes (polar bodies one and two) or on single blastomeres from a six-cell embryo. We treated twenty families carrying mutations in these lysosomal storage disorders, including 3 couples requiring simultaneous analysis for two disorders (TSD/GD, TSD/balanced Robertsonian translocation 45XYder(21;14), and HS/oculocutaneus albinism). These analyses led to an overall pregnancy rate/embryo transfer of 38% and the birth of 20 unaffected children from 17 families. We have found that PGD for lysosomal disorders is a safe and effective method to prevent birth of affected children. In addition, by using mutant embryos for the derivation of stem cell lines, we have successfully established GD and HS hESC lines for use as valuable models in LSD research. Gheona Altarescu, Rachel Beeri, Rachel Eiges, Silvina Epsztejn-Litman, Talia Eldar-Geva, Deborah Elstein, Ari Zimran, Ehud J. Margalioth, Ephrat Levy-Lahad, and Paul Renbaum Copyright © 2012 Gheona Altarescu et al. All rights reserved. RASSF Family Proteins Mon, 10 Dec 2012 11:23:00 +0000 Geoffrey J. Clark, Shairaz Baksh, Farida Latif, and Dae-Sik Lim Copyright © 2012 Geoffrey J. Clark et al. All rights reserved. Three-Dimensional Molecular Modeling of a Diverse Range of SC Clan Serine Proteases Mon, 19 Nov 2012 11:15:29 +0000 Serine proteases are involved in a variety of biological processes and are classified into clans sharing structural homology. Although various three-dimensional structures of SC clan proteases have been experimentally determined, they are mostly bacterial and animal proteases, with some from archaea, plants, and fungi, and as yet no structures have been determined for protozoa. To bridge this gap, we have used molecular modeling techniques to investigate the structural properties of different SC clan serine proteases from a diverse range of taxa. Either SWISS-MODEL was used for homology-based structure prediction or the LOOPP server was used for threading-based structure prediction. The predicted models were refined using Insight II and SCRWL and validated against experimental structures. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. The structural geometry of the catalytic core shows clear deviations between taxa, but the relative positions of the catalytic triad residues were conserved. Evolutionary divergence was also exhibited by large variation in secondary structure features outside the core, differences in overall amino acid distribution, and unique surface electrostatic potential patterns between species. Encompassing a wide range of taxa, our structural analysis provides an evolutionary perspective on SC clan serine proteases. Aparna Laskar, Aniruddha Chatterjee, Somnath Chatterjee, and Euan J. Rodger Copyright © 2012 Aparna Laskar et al. All rights reserved. Host-Pathogen Interactions of Retroviruses Wed, 24 Oct 2012 08:36:39 +0000 Abdul A. Waheed, Abraham L. Brass, Suryaram Gummuluru, and Gilda Tachedjian Copyright © 2012 Abdul A. Waheed et al. All rights reserved. A Prevalence of Imprinted Genes within the Total Transcriptomes of Human Tissues and Cells Tue, 11 Sep 2012 13:07:26 +0000 Genomic imprinting is an epigenetic phenomenon that causes a differential expression of paternally and maternally inherited alleles of a subset of genes (the so-called imprinted genes). Imprinted genes are distributed throughout the genome and it is predicted that about 1% of the human genes may be imprinted. It is recognized that the allelic expression of imprinted genes varies between tissues and developmental stages. The current study represents the first attempt to estimate a prevalence of imprinted genes within the total human transcriptome. In silico analysis of the normalized expression profiles of a comprehensive panel of 173 established and candidate human imprinted genes was performed, in 492 publicly available SAGE libraries. The latter represent human cell and tissue samples in a variety of physiological and pathological conditions. Variations in the prevalence of imprinted genes within the total transcriptomes (ranging from 0.08% to 4.36%) and expression profiles of the individual imprinted genes are assessed. This paper thus provides a useful reference on the size of the imprinted transcriptome and expression of the individual imprinted genes. Sergey V. Anisimov Copyright © 2012 Sergey V. Anisimov. All rights reserved. Virtual Interactomics of Proteins from Biochemical Standpoint Wed, 08 Aug 2012 13:22:54 +0000 Virtual interactomics represents a rapidly developing scientific area on the boundary line of bioinformatics and interactomics. Protein-related virtual interactomics then comprises instrumental tools for prediction, simulation, and networking of the majority of interactions important for structural and individual reproduction, differentiation, recognition, signaling, regulation, and metabolic pathways of cells and organisms. Here, we describe the main areas of virtual protein interactomics, that is, structurally based comparative analysis and prediction of functionally important interacting sites, mimotope-assisted and combined epitope prediction, molecular (protein) docking studies, and investigation of protein interaction networks. Detailed information about some interesting methodological approaches and online accessible programs or databases is displayed in our tables. Considerable part of the text deals with the searches for common conserved or functionally convergent protein regions and subgraphs of conserved interaction networks, new outstanding trends and clinically interesting results. In agreement with the presented data and relationships, virtual interactomic tools improve our scientific knowledge, help us to formulate working hypotheses, and they frequently also mediate variously important in silico simulations. Jaroslav Kubrycht, Karel Sigler, and Pavel Souček Copyright © 2012 Jaroslav Kubrycht et al. All rights reserved. Cellular Cofactors of Lentiviral Integrase: From Target Validation to Drug Discovery Tue, 07 Aug 2012 07:37:30 +0000 To accomplish their life cycle, lentiviruses make use of host proteins, the so-called cellular cofactors. Interactions between host cell and viral proteins during early stages of lentiviral infection provide attractive new antiviral targets. The insertion of lentiviral cDNA in a host cell chromosome is a step of no return in the replication cycle, after which the host cell becomes a permanent carrier of the viral genome and a producer of lentiviral progeny. Integration is carried out by integrase (IN), an enzyme playing also an important role during nuclear import. Plenty of cellular cofactors of HIV-1 IN have been proposed. To date, the lens epithelium-derived growth factor (LEDGF/p75) is the best studied cofactor of HIV-1 IN. Moreover, small molecules that block the LEDGF/p75-IN interaction have recently been developed for the treatment of HIV infection. The nuclear import factor transportin-SR2 (TRN-SR2) has been proposed as another interactor of HIV IN-mediating nuclear import of the virus. Using both proteins as examples, we will describe approaches to be taken to identify and validate novel cofactors as new antiviral targets. Finally, we will highlight recent advances in the design and the development of small-molecule inhibitors binding to the LEDGF/p75-binding pocket in IN (LEDGINs). Oliver Taltynov, Belete A. Desimmie, Jonas Demeulemeester, Frauke Christ, and Zeger Debyser Copyright © 2012 Oliver Taltynov et al. All rights reserved. ASGR1 and ASGR2, the Genes that Encode the Asialoglycoprotein Receptor (Ashwell Receptor), Are Expressed in Peripheral Blood Monocytes and Show Interindividual Differences in Transcript Profile Thu, 02 Aug 2012 12:56:45 +0000 Background. The asialoglycoprotein receptor (ASGPR) is a hepatic receptor that mediates removal of potentially hazardous glycoconjugates from blood in health and disease. The receptor comprises two proteins, asialoglycoprotein receptor 1 and 2 (ASGR1 and ASGR2), encoded by the genes ASGR1 and ASGR2. Design and Methods. Using reverse transcription amplification (RT-PCR), expression of ASGR1 and ASGR2 was investigated in human peripheral blood monocytes. Results. Monocytes were found to express ASGR1 and ASGR2 transcripts. Correctly spliced transcript variants encoding different isoforms of ASGR1 and ASGR2 were present in monocytes. The profile of transcript variants from both ASGR1 and ASGR2 differed among individuals. Transcript expression levels were compared with the hepatocyte cell line HepG2 which produces high levels of ASGPR. Monocyte transcripts were 4 to 6 orders of magnitude less than in HepG2 but nonetheless readily detectable using standard RT-PCR. The monocyte cell line THP1 gave similar results to monocytes harvested from peripheral blood, indicating it may provide a suitable model system for studying ASGPR function in this cell type. Conclusions. Monocytes transcribe and correctly process transcripts encoding the constituent proteins of the ASGPR. Monocytes may therefore represent a mobile pool of the receptor, capable of reaching sites remote from the liver. Rebecca Louise Harris, Carmen Wilma van den Berg, and Derrick John Bowen Copyright © 2012 Rebecca Louise Harris et al. All rights reserved. Protease-Mediated Maturation of HIV: Inhibitors of Protease and the Maturation Process Wed, 25 Jul 2012 09:07:57 +0000 Protease-mediated maturation of HIV-1 virus particles is essential for virus infectivity. Maturation occurs concomitant with immature virus particle release and is mediated by the viral protease (PR), which sequentially cleaves the Gag and Gag-Pol polyproteins into mature protein domains. Maturation triggers a second assembly event that generates a condensed conical capsid core. The capsid core organizes the viral RNA genome and viral proteins to facilitate viral replication in the next round of infection. The fundamental role of proteolytic maturation in the generation of mature infectious particles has made it an attractive target for therapeutic intervention. Development of small molecules that target the PR active site has been highly successful and nine protease inhibitors (PIs) have been approved for clinical use. This paper provides an overview of their development and clinical use together with a discussion of problems associated with drug resistance. The second-half of the paper discusses a novel class of antiretroviral drug termed maturation inhibitors, which target cleavage sites in Gag not PR itself. The paper focuses on bevirimat (BVM) the first-in-class maturation inhibitor: its mechanism of action and the implications of naturally occurring polymorphisms that confer reduced susceptibility to BVM in phase II clinical trials. Catherine S. Adamson Copyright © 2012 Catherine S. Adamson. All rights reserved. The Role of TNPO3 in HIV-1 Replication Thu, 19 Jul 2012 13:57:10 +0000 TNPO3, transportin-SR2 or Tnp3, a member of the karyopherin β superfamily of proteins, is important for the ability of human immunodeficiency virus (HIV-1) to achieve productive infection, as TNPO3 depletion in human cells leads to a dramatic reduction of infection. Here we describe and discuss recent findings suggesting that TNPO3 assists HIV-1 replication in the nucleus and in fact that TNPO3 may assist PIC maturation in the nucleus. In addition, the viral determinant for the requirement of TNPO3 in HIV-1 infection is discussed. This paper summarizes the most significant recent discoveries about this important host factor and its role in HIV-1 replication. Felipe Diaz-Griffero Copyright © 2012 Felipe Diaz-Griffero. All rights reserved. Factors Important to the Prioritization and Development of Successful Topical Microbicides for HIV-1 Thu, 12 Jul 2012 13:14:33 +0000 Significant advancements in topical microbicide development have occurred since the prevention strategy was first described as a means to inhibit the sexual transmission of HIV-1. The lack of clinical efficacy of the first generation microbicide products has focused development attention on specific antiretroviral agents, and these agents have proven partially successful in human clinical trials. With greater understanding of vaginal and rectal virus infection, replication, and dissemination, better microbicide products and delivery strategies should result in products with enhanced potency. However, a variety of development gaps exist which relate to product dosing, formulation and delivery, and pharmacokinetics and pharmacodynamics which must be better understood in order to prioritize microbicide products for clinical development. In vitro, ex vivo, and in vivo models must be optimized with regard to these development gaps in order to put the right product at the right place, at the right time, and at the right concentration for effective inhibition of virus transmission. As the microbicide field continues to evolve, we must harness the knowledge gained from unsuccessful and successful clinical trials and development programs to continuously enhance our preclinical development algorithms. Karen W. Buckheit and Robert W. Buckheit Jr. Copyright © 2012 Karen W. Buckheit and Robert W. Buckheit Jr. All rights reserved. The Continuing Evolution of HIV-1 Therapy: Identification and Development of Novel Antiretroviral Agents Targeting Viral and Cellular Targets Tue, 10 Jul 2012 12:53:38 +0000 During the past three decades, over thirty-five anti-HIV-1 therapies have been developed for use in humans and the progression from monotherapeutic treatment regimens to today’s highly active combination antiretroviral therapies has had a dramatic impact on disease progression in HIV-1-infected individuals. In spite of the success of AIDS therapies and the existence of inhibitors of HIV-1 reverse transcriptase, protease, entry and fusion, and integrase, HIV-1 therapies still have a variety of problems which require continued development efforts to improve efficacy and reduce toxicity, while making drugs that can be used throughout both the developed and developing world, in pediatric populations, and in pregnant women. Highly active antiretroviral therapies (HAARTs) have significantly delayed the progression to AIDS, and in the developed world HIV-1-infected individuals might be expected to live normal life spans while on lifelong therapies. However, the difficult treatment regimens, the presence of class-specific drug toxicities, and the emergence of drug-resistant virus isolates highlight the fact that improvements in our therapeutic regimens and the identification of new and novel viral and cellular targets for therapy are still necessary. Antiretroviral therapeutic strategies and targets continue to be explored, and the development of increasingly potent molecules within existing classes of drugs and the development of novel strategies are ongoing. Tracy L. Hartman and Robert W. Buckheit Jr. Copyright © 2012 Tracy L. Hartman and Robert W. Buckheit Jr. All rights reserved. Dynamic Association between HIV-1 Gag and Membrane Domains Thu, 05 Jul 2012 10:15:52 +0000 HIV-1 particle assembly is driven by the structural protein Gag. Gag binds to and multimerizes on the inner leaflet of the plasma membrane, eventually resulting in formation of spherical particles. During virus spread among T cells, Gag accumulates to the plasma membrane domain that, together with target cell membrane, forms a cell junction known as the virological synapse. While Gag association with plasma membrane microdomains has been implicated in virus assembly and cell-to-cell transmission, recent studies suggest that, rather than merely accumulating to pre-existing microdomains, Gag plays an active role in reorganizing the microdomains via its multimerization activity. In this paper, we will discuss this emerging view of Gag microdomain interactions. Relationships between Gag multimerization and microdomain association will be further discussed in the context of Gag localization to T-cell uropods and virological synapses. Ian B. Hogue, G. Nicholas Llewellyn, and Akira Ono Copyright © 2012 Ian B. Hogue et al. All rights reserved. Hippo and rassf1a Pathways: A Growing Affair Thu, 05 Jul 2012 09:46:32 +0000 First discovered in Drosophila, the Hippo pathway regulates the size and shape of organ development. Its discovery and study have helped to address longstanding questions in developmental biology. Central to this pathway is a kinase cascade leading from the tumor suppressor Hippo (Mst1 and Mst2 in mammals) to the Yki protein (YAP and TAZ in mammals), a transcriptional coactivator of target genes involved in cell proliferation, survival, and apoptosis. A dysfunction of the Hippo pathway activity is frequently detected in human cancers. Recent studies have highlighted that the Hippo pathway may play an important role in tissue homoeostasis through the regulation of stem cells, cell differentiation, and tissue regeneration. Recently, the impact of RASSF proteins on Hippo signaling potentiating its proapoptotic activity has been addressed, thus, providing further evidence for Hippo's key role in mammalian tumorigenesis as well as other important diseases. Francesca Fausti, Silvia Di Agostino, Andrea Sacconi, Sabrina Strano, and Giovanni Blandino Copyright © 2012 Francesca Fausti et al. All rights reserved. Retroviral Env Glycoprotein Trafficking and Incorporation into Virions Mon, 02 Jul 2012 10:04:13 +0000 Together with the Gag protein, the Env glycoprotein is a major retroviral structural protein and is essential for forming infectious virus particles. Env is synthesized, processed, and transported to certain microdomains at the plasma membrane and takes advantage of the same host machinery for its trafficking as that used by cellular glycoproteins. Incorporation of Env into progeny virions is probably mediated by the interaction between Env and Gag, in some cases with the additional involvement of certain host factors. Although several general models have been proposed to explain the incorporation of retroviral Env glycoproteins into virions, the actual mechanism for this process is still unclear, partly because structural data on the Env protein cytoplasmic tail is lacking. This paper presents the current understanding of the synthesis, trafficking, and virion incorporation of retroviral Env proteins. Tsutomu Murakami Copyright © 2012 Tsutomu Murakami. All rights reserved. Restriction of Retroviral Replication by Tetherin/BST-2 Mon, 02 Jul 2012 08:14:25 +0000 Tetherin/BST-2 is an important host restriction factor that limits the replication of HIV and other enveloped viruses. Tetherin is a type II membrane glycoprotein with a very unusual domain structure that allows it to engage budding virions and retain them on the plasma membrane of infected cells. Following the initial report identifying tetherin as the host cell factor targeted by the HIV-1 Vpu gene, knowledge of the molecular, structural, and cellular biology of tetherin has rapidly advanced. This paper summarizes the discovery and impact of tetherin biology on the HIV field, with a focus on recent advances in understanding its structure and function. The relevance of tetherin to replication and spread of other retroviruses is also reviewed. Tetherin is a unique host restriction factor that is likely to continue to provide new insights into host-virus interactions and illustrates well the varied ways by which host organisms defend against viral pathogens. Jason Hammonds, Jaang-Jiun Wang, and Paul Spearman Copyright © 2012 Jason Hammonds et al. All rights reserved. The Impact of Macrophage Nucleotide Pools on HIV-1 Reverse Transcription, Viral Replication, and the Development of Novel Antiviral Agents Sun, 01 Jul 2012 10:39:22 +0000 Macrophages are ubiquitous and represent a significant viral reservoir for HIV-1. Macrophages are nondividing, terminally differentiated cells, which have a unique cellular microenvironment relative to actively dividing T lymphocytes, all of which can impact HIV-1 infection/replication, design of inhibitors targeting viral replication in these cells, emergence of mutations within the HIV-1 genome, and disease progression. Scarce dNTPs drive rNTP incorporation into the proviral DNA in macrophages but not lymphocytes. Furthermore, the efficacy of a ribose-based inhibitor that potently inhibits HIV-1 replication in macrophages, has prompted a reconsideration of the previously accepted dogma that 2′-deoxy-based inhibitors demonstrate effective inhibition of HIV-1 replication. Additionally, higher levels of dUTP and rNTP incorporation in macrophages, and lack of repair mechanisms relative to lymphocytes, provide a further mechanistic understanding required to develop targeted inhibition of viral replication in macrophages. Together, the concentrations of dNTPs and rNTPs within macrophages comprise a distinctive cellular environment that directly impacts HIV-1 replication in macrophages and provides unique insight into novel therapeutic mechanisms that could be exploited to eliminate virus from these cells. Christina Gavegnano, Edward M. Kennedy, Baek Kim, and Raymond F. Schinazi Copyright © 2012 Christina Gavegnano et al. All rights reserved.