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HIV RNA splicing

Steven A Yukl, Philipp Kaiser, Peggy Kim, Sushama Telwatte, Sunil K Joshi, Mai Vu, Harry Lampiris, Joseph K Wong
Latently infected CD4+ T cells are the main barrier to complete clearance of HIV infection, but it is unclear what mechanisms govern latent HIV infection in vivo. To address this question, we developed a new panel of reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assays specific for different HIV transcripts that define distinct blocks to transcription. We applied this panel of assays to CD4+ T cells freshly isolated from HIV-infected patients on suppressive antiretroviral therapy (ART) to quantify the degree to which different mechanisms inhibit HIV transcription...
February 28, 2018: Science Translational Medicine
Chen Huan, Zhaolong Li, Shanshan Ning, Hong Wang, Xiao-Fang Yu, Wenyan Zhang
The HIV-1 reservoir is a major obstacle to complete eradication of the virus. Although many proteins and RNAs have been characterized as regulators in HIV-1/AIDS pathogenesis and latency, only a few long non-coding RNAs (lncRNAs) have been shown to be closely associated with HIV-1 replication and latency. Here, we demonstrated that lncRNA uc002yug.2 plays a key role in HIV-1 viral replication and latency. uc002yug.2 potentially enhances HIV-1 viral replication, LTR activity as well as the activation of latent HIV-1 in both cell lines and CD4+ T cells from patients...
February 28, 2018: Journal of Virology
Yu-Mi Jung, Kyung-Lee Yu, Seong-Hyun Park, Seong-Deok Lee, Min-Jeong Kim, Ji-Chang You
Y-box binding protein 1 (YB-1) is a member of the cold-shock domain (CSD) protein superfamily. It participates in a wide variety of cellular events, including transcription, RNA splicing, translation, DNA repair, drug resistance, and stress responses. We investigated putative functions of YB-1 in HIV-1 replication. Functional studies using overexpression or knockdown of YB-1 in conjunction with transfection of proviral DNA showed that YB-1 enhances virus production. We found YB-1 regulates HIV-1 production by stimulating viral transcription using HIV-1 LTR sequence U3RU5 with Luciferase assay...
February 12, 2018: BMB Reports
Helene Sertznig, Frank Hillebrand, Steffen Erkelenz, Heiner Schaal, Marek Widera
Alternative splicing plays a key role in the HIV-1 life cycle and is essential to maintain an equilibrium of mRNAs that encode viral proteins and polyprotein-isoforms. In particular, since all early HIV-1 proteins are expressed from spliced intronless and late enzymatic and structural proteins from intron containing, i.e. splicing repressed viral mRNAs, cellular splicing factors and splicing regulatory proteins are crucial for the replication capacity. In this review, we will describe the complex network of cis-acting splicing regulatory elements (SREs), which are mainly localized in the neighbourhoods of all HIV-1 splice sites and warrant the proper ratio of individual transcript isoforms...
January 29, 2018: Virology
Matthew Takata, Steven J Soll, Ann Emery, Daniel Blanco-Melo, Ronald Swanstrom, Paul D Bieniasz
The ~9.5 kilobase HIV-1 genome contains RNA sequences and structures that control many aspects of viral replication, including transcription, splicing, nuclear export, translation, packaging and reverse transcription. Nonetheless, chemical probing and other approaches suggest that the HIV-1 genome may contain many more RNA secondary structures of unknown importance and function. To determine whether there are additional, undiscovered cis-acting RNA elements in the HIV-1 genome that are important for viral replication, we undertook a global silent mutagenesis experiment...
January 29, 2018: PLoS Pathogens
Raymond W Wong, Clifford A Lingwood, Mario A Ostrowski, Tyler Cabral, Alan Cochrane
The capacity of HIV-1 to develop resistance to current drugs calls for innovative strategies to control this infection. We aimed at developing novel inhibitors of HIV-1 replication by targeting viral RNA processing-a stage dependent on conserved host processes. We previously reported that digoxin is a potent inhibitor of this stage. Herein, we identify 12 other cardiac glycoside/aglycones or cardiotonic steroids (CSs) that impede HIV growth in HIV-infected T cells from clinical patients at IC50s (1.1-1.3 nM) that are 2-26 times below concentrations used in patients with heart conditions...
January 16, 2018: Scientific Reports
Rachel A Knoener, Jordan T Becker, Mark Scalf, Nathan M Sherer, Lloyd M Smith
HIV-1 replication requires myriad interactions between cellular proteins and the viral unspliced RNA. These interactions are important in archetypal RNA processes such as transcription and translation as well as for more specialized functions including alternative splicing and packaging of unspliced genomic RNA into virions. We present here a hybridization capture strategy for purification of unspliced full-length HIV RNA-protein complexes preserved in vivo by formaldehyde crosslinking, and coupled with mass spectrometry to identify HIV RNA-protein interactors in HIV-1 infected cells...
December 5, 2017: Scientific Reports
Maritza Puray-Chavez, Philip R Tedbury, Andrew D Huber, Obiaara B Ukah, Vincent Yapo, Dandan Liu, Juan Ji, Jennifer J Wolf, Alan N Engelman, Stefan G Sarafianos
Technical limitations in simultaneous microscopic visualization of RNA, DNA, and proteins of HIV have curtailed progress in this field. To address this need we develop a microscopy approach, multiplex immunofluorescent cell-based detection of DNA, RNA and Protein (MICDDRP), which is based on branched DNA in situ hybridization technology. MICDDRP enables simultaneous single-cell visualization of HIV (a) spliced and unspliced RNA, (b) cytoplasmic and nuclear DNA, and (c) Gag. We use MICDDRP to visualize incoming capsid cores containing RNA and/or nascent DNA and follow reverse transcription kinetics...
December 1, 2017: Nature Communications
Lian-Ju Li, Yin-Guang Fan, Rui-Xue Leng, Hai-Feng Pan, Dong-Qing Ye
The field of m6 A modification and epitranscriptomics has recently attracted much attention. More methods allowing for precise m6 A site profiling and location are developed and crucial players of m6 A modification machinery are increasingly identified. Although some challenges remain, m6 A modification is found to modulate almost all aspects of RNA metabolism, such as splicing, stability, structure, translation, and export. Thus, m6 A modification adds a new layer of post-transcriptional gene expression regulation, and it is implicated in T cell response to HIV infection, type I interferon production, and T cell differentiation and homeostasis...
January 2018: Molecular Immunology
Hannah M Pezzi, Scott M Berry, David J Beebe, Rob Striker
Quantification of the HIV viral reservoir is critical to understanding HIV latency, advancing patient care and ultimately achieving a cure. To quantify the reservoir, a new metric was recently introduced, which quantified cells carrying multiply spliced HIV RNA. The developed assay, Tat/rev Induced Limiting Dilution Assay (TILDA), enables quantification of cells containing multiply-spliced HIV RNA events as an indicator of reservoir size. Due to TILDA's reliance on a limiting dilution format paired with the rarity of target events, numerous individual reactions are required to obtain a single endpoint...
November 13, 2017: Integrative Biology: Quantitative Biosciences From Nano to Macro
George Semango, Bas Heinhuis, Theo S Plantinga, Willeke A M Blokx, Gibson Kibiki, Tolbert Sonda, Daudi Mavura, Elisante John Masenga, Mramba Nyindo, Andre J A M van der Ven, Leo A B Joosten
The intracellular pro-inflammatory mediator interleukin (IL)-32 is associated with tumor progression; however, the mechanisms remain unknown. We studied the IL-32 mRNA expression as well as other pro-inflammatory cytokines and mediators including IL-1α, IL-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, the pro-angiogenic and anti-apoptotic enzyme COX-2, the IL-8 receptor CXCR1, and the intracellular kinase FAK-1. The interaction of IL-32 expression with expression of IL-6, TNFα, IL-8, and COX-2 was also investigated...
October 13, 2017: American Journal of Pathology
Chijioke N Umunnakwe, Karin S Dorman, Drena Dobbs, Susan Carpenter
BACKGROUND: Rev-like proteins are post-transcriptional regulatory proteins found in several retrovirus genera, including lentiviruses, betaretroviruses, and deltaretroviruses. These essential proteins mediate the nuclear export of incompletely spliced viral RNA, and act by tethering viral pre-mRNA to the host CRM1 nuclear export machinery. Although all Rev-like proteins are functionally homologous, they share less than 30% sequence identity. In the present study, we computationally assessed the extent of structural homology among retroviral Rev-like proteins within a phylogenetic framework...
August 22, 2017: Retrovirology
Ina P O'Carroll, Yashna Thappeta, Lixin Fan, Edric A Ramirez-Valdez, Sean Smith, Yun-Xing Wang, Alan Rein
The HIV-1 Rev response element (RRE) is a 351-base element in unspliced and partially spliced viral RNA; binding of the RRE by the viral Rev protein induces nuclear export of RRE-containing RNAs, as required for virus replication. It contains one long, imperfect double helix (domain I), one branched domain (domain II) containing a high-affinity Rev-binding site, and two or three additional domains. We previously reported that the RRE assumes an "A" shape in solution and suggested that the location of the Rev binding sites in domains I and II, opposite each other on the two legs of the A, is optimal for Rev binding and explains Rev's specificity for RRE-containing RNAs...
August 16, 2017: Journal of Virology
John A Hammond, Li Zhou, Rajan Lamichhane, Hui-Yi Chu, David P Millar, Larry Gerace, James R Williamson
HIV-1 requires a specialized nuclear export pathway to transport unspliced and partially spliced viral transcripts to the cytoplasm. Central to this pathway is the viral protein Rev, which binds to the Rev response element in stem IIB located on unspliced viral transcripts and subsequently oligomerizes in a cooperative manner. Previous work identified a number of cellular DEAD-box helicases as in vivo binding partners of Rev, and siRNA experiments indicated a functional role for many in the HIV replication cycle...
July 10, 2017: Journal of Molecular Biology
Diane L Bolton, Kathleen McGinnis, Greg Finak, Pratip Chattopadhyay, Raphael Gottardo, Mario Roederer
CD4 T cells harboring HIV-1/SIV represent a formidable hurdle to eradicating infection, and yet their detailed phenotype remains unknown. Here we integrate two single-cell technologies, flow cytometry and highly multiplexed quantitative RT-PCR, to characterize SIV-infected CD4 T cells directly ex vivo. Within individual cells, we correlate the cellular phenotype, in terms of host protein and RNA expression, with stages of the viral life cycle defined by combinatorial expression of viral RNAs. Spliced RNA+ infected cells display multiple memory and activation phenotypes, indicating virus production by diverse CD4 T cell subsets...
June 2017: PLoS Pathogens
Carin K Ingemarsdotter, Sushmita Poddar, Sarah Mercier, Volker Patzel, Andrew M L Lever
Antiviral strategies targeting hijacked cellular processes are less easily evaded by the virus than viral targets. If selective for viral functions, they can have a high therapeutic index. We used RNA trans-splicing to deliver the herpes simplex virus thymidine kinase-ganciclovir (HSV-tk/GCV) cell suicide system into HIV-producing cells. Using an extensive in silico bioinformatics and RNA structural analysis approach, ten HIV RNA trans-splicing constructs were designed targeting eight different HIV splice donor or acceptor sites and were tested in cells expressing HIV...
June 16, 2017: Molecular Therapy. Nucleic Acids
John D Lapek, Mary K Lewinski, Jacob M Wozniak, John Guatelli, David J Gonzalez
The mechanisms by which human immunodeficiency virus (HIV) circumvents and coopts cellular machinery to replicate and persist in cells are not fully understood. HIV accessory proteins play key roles in the HIV life cycle by altering host pathways that are often dependent on post-translational modifications (PTMs). Thus, the identification of HIV accessory protein host targets and their PTM status is critical to fully understand how HIV invades, avoids detection and replicates to spread infection. To date, a comprehensive characterization of HIV accessory protein host targets and modulation of their PTM status does not exist...
August 2017: Molecular & Cellular Proteomics: MCP
Ahalya Balachandran, Alan Cochrane
The control of RNA processing plays an important role in the nature and quantity of protein generated from mammalian genes. Consequently, efforts to manipulate RNA processing have the capacity to significantly impact gene function. Although multiple strategies have been developed to alter splice site selection using oligonucleotide occlusion of splice sites or splicing regulatory elements, systemic delivery of such agents remains problematic. Outlined in this chapter is a protocol to screen for small molecule inhibitors of HIV-1 Gag expression that have been subsequently determined to modulate viral RNA processing...
June 17, 2017: Methods: a Companion to Methods in Enzymology
Conrad A Vink, John R Counsell, Dany P Perocheau, Rajvinder Karda, Suzanne M K Buckley, Martijn H Brugman, Melanie Galla, Axel Schambach, Tristan R McKay, Simon N Waddington, Steven J Howe
Lentiviral vector genomic RNA requires sequences that partially overlap wild-type HIV-1 gag and env genes for packaging into vector particles. These HIV-1 packaging sequences constitute 19.6% of the wild-type HIV-1 genome and contain functional cis elements that potentially compromise clinical safety. Here, we describe the development of a novel lentiviral vector (LTR1) with a unique genomic structure designed to prevent transfer of HIV-1 packaging sequences to patient cells, thus reducing the total HIV-1 content to just 4...
August 2, 2017: Molecular Therapy: the Journal of the American Society of Gene Therapy
Robert W Yucha, Kristen S Hobbs, Emily Hanhauser, Louise E Hogan, Wildaliz Nieves, Mehmet O Ozen, Fatih Inci, Vanessa York, Erica A Gibson, Cassandra Thanh, Hadi Shafiee, Rami El Assal, Maja Kiselinova, Yvonne P Robles, Helen Bae, Kaitlyn S Leadabrand, ShuQi Wang, Steven G Deeks, Daniel R Kuritzkes, Utkan Demirci, Timothy J Henrich
Reactivation of latent viral reservoirs is on the forefront of HIV-1 eradication research. However, it is unknown if latency reversing agents (LRAs) increase the level of viral transcription from cells producing HIV RNA or harboring transcriptionally-inactive (latent) infection. We therefore developed a microfluidic single-cell-in-droplet (scd)PCR assay to directly measure the number of CD4(+) T cells that produce unspliced (us)RNA and multiply spliced (ms)RNA following ex vivo latency reversal with either an histone deacetylase inhibitor (romidepsin) or T cell receptor (TCR) stimulation...
June 2017: EBioMedicine
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