ABSTRACT
Human LMNA gene mutations result in laminopathies that include Emery-Dreifuss muscular dystrophy (AD-EDMD) and Hutchinson-Gilford progeria, the premature aging syndrome (HGPS). The Lmna null (Lmna(-/-)) and progeroid LmnaΔ9 mutant mice are models for AD-EDMD and HGPS, respectively. Both animals develop severe tissue pathologies with abbreviated life spans. Like HGPS cells, Lmna(-/-) and LmnaΔ9 fibroblasts have typically misshapen nuclei. Unexpectedly, Lmna(-/-) or LmnaΔ9 mice that are also deficient for the inner nuclear membrane protein Sun1 show markedly reduced tissue pathologies and enhanced longevity. Concordantly, reduction of SUN1 overaccumulation in LMNA mutant fibroblasts and in cells derived from HGPS patients corrected nuclear defects and cellular senescence. Collectively, these findings implicate Sun1 protein accumulation as a common pathogenic event in Lmna(-/-), LmnaΔ9, and HGPS disorders.
Subject(s)
Lamin Type A/metabolism , Membrane Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Muscular Dystrophy, Emery-Dreifuss/metabolism , Muscular Dystrophy, Emery-Dreifuss/pathology , Nuclear Proteins/metabolism , Progeria/metabolism , Animals , Cell Line , Cellular Senescence , Disease Models, Animal , Fibroblasts/metabolism , Humans , Mice , Mice, Knockout , Microtubule-Associated Proteins/genetics , Progeria/pathologyABSTRACT
LINC complexes are evolutionarily conserved nuclear envelope bridges, composed of SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne/homology) domain proteins. They are crucial for nuclear positioning and nuclear shape determination, and also mediate nuclear envelope (NE) attachment of meiotic telomeres, essential for driving homolog synapsis and recombination. In mice, SUN1 and SUN2 are the only SUN domain proteins expressed during meiosis, sharing their localization with meiosis-specific KASH5. Recent studies have shown that loss of SUN1 severely interferes with meiotic processes. Absence of SUN1 provokes defective telomere attachment and causes infertility. Here, we report that meiotic telomere attachment is not entirely lost in mice deficient for SUN1, but numerous telomeres are still attached to the NE through SUN2/KASH5-LINC complexes. In Sun1(-/-) meiocytes attached telomeres retained the capacity to form bouquet-like clusters. Furthermore, we could detect significant numbers of late meiotic recombination events in Sun1(-/-) mice. Together, this indicates that even in the absence of SUN1 telomere attachment and their movement within the nuclear envelope per se can be functional.
Subject(s)
Meiosis/genetics , Membrane Proteins/genetics , Microtubule-Associated Proteins/genetics , RNA, Long Noncoding/genetics , Telomere-Binding Proteins/genetics , Animals , Cell Cycle Proteins/genetics , Cytoskeletal Proteins , Mice , Multiprotein Complexes/genetics , Nuclear Envelope/genetics , Nuclear Proteins/genetics , Telomere/geneticsABSTRACT
A major barrier to the elimination of HIV-1 infection is the presence of a pool of long-lived, latently infected CD4+ memory T-cells. The search for treatments to re-activate latent HIV to aid in clearance is hindered by the incomplete understanding of the mechanisms that lead to transcriptional silencing of viral gene expression in host cells. Here we identify a previously unknown role for RUNX1 in HIV-1 transcriptional latency. The RUNX proteins, in combination with the co-factor CBF-ß, are critical transcriptional regulators in T-cells. RUNX1 strongly modulates CD4 expression and contributes to CD4+ T-cell function. We show that RUNX1 can bind DNA sequences within the HIV-1 LTR and that this binding represses transcription. Using patient samples we show a negative correlation between RUNX1 expression and viral load. Furthermore, we find that pharmacologic inhibition of RUNX1 by a small molecule inhibitor, Ro5-3335, synergizes with the histone deacetylase (HDAC) inhibitor SAHA (Vorinostat) to enhance the activation of latent HIV-1 in both cell lines and PBMCs from patients. Our findings indicate that RUNX1 and CBF-ß cooperate in cells to modulate HIV-1 replication, identifying for the first time RUNX1 as a cellular factor involved in HIV-1 latency. This work highlights the therapeutic potential of inhibitors of RUNX1 to re-activate virus and aid in clearance of HIV-1.
Subject(s)
Core Binding Factor Alpha 2 Subunit/antagonists & inhibitors , HIV Infections/virology , Virus Activation/physiology , Virus Latency/physiology , CCAAT-Binding Factor/metabolism , Chromatin Immunoprecipitation , Drug Synergism , Flow Cytometry , HIV-1/physiology , Histone Deacetylase Inhibitors/pharmacology , Humans , Hydroxamic Acids/pharmacology , Microscopy, Confocal , Mutagenesis, Site-Directed , Reverse Transcriptase Polymerase Chain Reaction , Viral Load , VorinostatABSTRACT
It has been 30 years since a 'new' leukaemia termed adult T-cell leukaemia (ATL) was described in Japan, and more than 25 years since the isolation of the retrovirus, human T-cell leukaemia virus type 1 (HTLV-1), that causes this disease. We discuss HTLV-1 infectivity and how the HTLV-1 Tax oncoprotein initiates transformation by creating a cellular environment favouring aneuploidy and clastogenic DNA damage. We also explore the contribution of a newly discovered protein and RNA on the HTLV-1 minus strand, HTLV-1 basic leucine zipper factor (HBZ), to the maintenance of virus-induced leukaemia.
Subject(s)
Basic-Leucine Zipper Transcription Factors/physiology , Cell Transformation, Neoplastic , Cell Transformation, Viral , Gene Products, tax , Leukemia-Lymphoma, Adult T-Cell/pathology , Aneuploidy , Basic-Leucine Zipper Transcription Factors/genetics , DNA Damage , Human T-lymphotropic virus 1 , Humans , Mitosis , Models, BiologicalABSTRACT
Central to the development of new treatments for human immunodeficiency virus 1 (HIV-1) is a more thorough understanding of the viral life cycle and the cellular cofactors upon which this depends. Targeting cellular proteins and their interaction with HIV-1 has the potential to reduce the problem of emerging viral resistance to drugs as mutational escape is more difficult. We performed a short interfering RNA (siRNA) library screen targeting 59 cellular RNA helicases, assessing the effect on both viral capsid protein production and infectious virion formation. Five RNA helicases were identified which, when knocked down, reproducibly decreased infectious particle production: DDX5, DDX10, DDX17, DDX28 and DDX52. Two of these proteins (DDX5 and DDX17) have known roles in HIV-1 replication. A further helicase (DDX10) was a positive hit from a previous genome-wide siRNA screen; however, DDX28 and DDX52 have not previously been implicated as essential cofactors for HIV-1.
Subject(s)
HIV-1/enzymology , HIV-1/physiology , Host-Pathogen Interactions , RNA Helicases/metabolism , Virus Replication , Gene Knockdown Techniques , Genetic Testing , HIV-1/genetics , Humans , RNA Helicases/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolismABSTRACT
Core binding factor (CBF) leukemias, those with translocations or inversions that affect transcription factor genes RUNX1 or CBFB, account for ~24% of adult acute myeloid leukemia (AML) and 25% of pediatric acute lymphocytic leukemia (ALL). Current treatments for CBF leukemias are associated with significant morbidity and mortality, with a 5-y survival rate of ~50%. We hypothesize that the interaction between RUNX1 and CBFß is critical for CBF leukemia and can be targeted for drug development. We developed high-throughput AlphaScreen and time-resolved fluorescence resonance energy transfer (TR-FRET) methods to quantify the RUNX1-CBFß interaction and screen a library collection of 243,398 compounds. Ro5-3335, a benzodiazepine identified from the screen, was able to interact with RUNX1 and CBFß directly, repress RUNX1/CBFB-dependent transactivation in reporter assays, and repress runx1-dependent hematopoiesis in zebrafish embryos. Ro5-3335 preferentially killed human CBF leukemia cell lines, rescued preleukemic phenotype in a RUNX1-ETO transgenic zebrafish, and reduced leukemia burden in a mouse CBFB-MYH11 leukemia model. Our data thus confirmed that RUNX1-CBFß interaction can be targeted for leukemia treatment and we have identified a promising lead compound for this purpose.
Subject(s)
Benzodiazepines/pharmacology , Core Binding Factor Alpha 2 Subunit/metabolism , Core Binding Factor beta Subunit/metabolism , High-Throughput Screening Assays/methods , Leukemia, Myeloid, Acute/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Transcriptional Activation/drug effects , Amino Acid Sequence , Animals , Blotting, Western , Core Binding Factor beta Subunit/genetics , Electrophoretic Mobility Shift Assay , Flow Cytometry , Fluorescence Resonance Energy Transfer/methods , Genetic Vectors/genetics , Hematopoiesis/drug effects , Histological Techniques , Humans , Immunoprecipitation , Jurkat Cells , Mice , Molecular Sequence Data , Protein Interaction Mapping/methods , Surface Plasmon Resonance , ZebrafishABSTRACT
Autophagy, a general homeostatic process for degradation of cytosolic proteins or organelles, has been reported to modulate the replication of many viruses. The role of autophagy in human T-cell leukemia virus type 1 (HTLV-1) replication has, however, been uncharacterized. Here, we report that HTLV-1 infection increases the accumulation of autophagosomes and that this accumulation increases HTLV-1 production. We found that the HTLV-1 Tax protein increases cellular autophagosome accumulation by acting to block the fusion of autophagosomes to lysosomes, preventing the degradation of the former by the latter. Interestingly, the inhibition of cellular autophagosome-lysosome fusion using bafilomycin A increased the stability of the Tax protein, suggesting that cellular degradation of Tax occurs in part through autophagy. Our current findings indicate that by interrupting the cell's autophagic process, Tax exerts a positive feedback on its own stability.
Subject(s)
Autophagy , Gene Products, tax/metabolism , Human T-lymphotropic virus 1/physiology , Virus Replication , Cell Line , Cytoplasm/ultrastructure , Humans , Phagosomes/metabolism , Phagosomes/ultrastructureABSTRACT
Human T-cell lymphotropic virus type 1 (HTLV-1) was originally discovered in the early 1980s. It is the first retrovirus to be unambiguously linked causally to a human cancer. HTLV-1 currently infects approximately 20 million people worldwide. In this chapter, we review progress made over the last 30 years in our understanding of HTLV-1 infection, replication, gene expression, and cellular transformation.
Subject(s)
Cell Transformation, Neoplastic , HTLV-I Infections/complications , Human T-lymphotropic virus 1/physiology , Human T-lymphotropic virus 1/pathogenicity , Leukemia-Lymphoma, Adult T-Cell/etiology , Adult , HTLV-I Infections/virology , HumansABSTRACT
MicroRNAs (miRNAs) are 22-nt non-coding RNAs involved in the regulation of cellular gene expression and potential cellular defense against viral infection. Using in silico analyses, we predicted target sites for 22 human miRNAs in the HIV genome. Transfection experiments using synthetic miRNAs showed that five of these miRNAs capably decreased HIV replication. Using one of these five miRNAs, human miR-326 as an example, we demonstrated that the degree of complementarity between the predicted viral sequence and cellular miR-326 correlates, in a Dicer-dependent manner, with the potency of miRNA-mediated restriction of viral replication. Antagomirs to miR-326 that knocked down this cell endogenous miRNA increased HIV-1 replication in cells, suggesting that miR-326 is physiologically functional in moderating HIV-1 replication in human cells.
Subject(s)
HIV-1/genetics , MicroRNAs/metabolism , Virus Replication , Base Pairing , Base Sequence , Cell Line , Cells, Cultured , Computer Simulation , Genome, Viral , HIV-1/physiology , Humans , Jurkat Cells , MicroRNAs/chemistry , RNA, Messenger/chemistry , RNA, Messenger/metabolismABSTRACT
RNAi plays important roles in many biological processes, including cellular defense against viral infection. Components of the RNAi machinery are widely conserved in plants and animals. In mammals, microRNAs (miRNAs) represent an abundant class of cell encoded small noncoding RNAs that participate in RNAi-mediated gene silencing. Here, findings that HIV-1 replication in cells can be regulated by miRNAs and that HIV-1 infection of cells can alter cellular miRNA expression are reviewed. Lessons learned from and questions outstanding about the complex interactions between HIV-1 and cellular miRNAs are discussed.
Subject(s)
Gene Expression Regulation, Viral , HIV-1/genetics , MicroRNAs/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Animals , Argonaute Proteins/metabolism , CD4-Positive T-Lymphocytes/virology , DEAD-box RNA Helicases/metabolism , Gene Silencing , HeLa Cells , Humans , Models, Biological , RNA Processing, Post-Transcriptional , RNA, Viral/metabolism , Ribonuclease III/metabolismABSTRACT
We demonstrate that at least three different promoter variant strains of HIV-1 subtype C have been gradually expanding and replacing the standard subtype C viruses in India, and possibly in South Africa and other global regions, over the past decade. The new viral strains contain an additional NF-κB, NF-κB-like, or RBEIII site in the viral promoter. Although the acquisition of an additional RBEIII site is a property shared by all the HIV-1 subtypes, acquiring an additional NF-κB site remains an exclusive property of subtype C. The acquired κB site is genetically distinct, binds the p50-p65 heterodimer, and strengthens the viral promoter at the levels of transcription initiation and elongation. The 4-κB viruses dominate the 3-κB "isogenic" viral strains in pairwise competition assays in T-cell lines, primary cells, and the ecotropic human immunodeficiency virus mouse model. The dominance of the 4-κB viral strains is also evident in the natural context when the subjects are coinfected with κB-variant viral strains. The mean plasma viral loads, but not CD4 counts, are significantly different in 4-κB infection suggesting that these newly emerging strains are probably more infectious. It is possible that higher plasma viral loads underlie selective transmission of the 4-κB viral strains. Several publications previously reported duplication or deletion of diverse transcription factor-binding sites in the viral promoter. Unlike previous reports, our study provides experimental evidence that the new viral strains gained a potential selective advantage as a consequence of the acquired transcription factor-binding sites and importantly that these strains have been expanding at the population level.
Subject(s)
HIV Infections/metabolism , HIV Infections/virology , HIV Long Terminal Repeat , HIV-1/genetics , NF-kappa B/metabolism , Transcription, Genetic , Adult , Cohort Studies , Female , Gene Expression Regulation, Viral , HIV Infections/genetics , HIV-1/chemistry , HIV-1/classification , HIV-1/physiology , Humans , Male , Molecular Sequence Data , NF-kappa B/genetics , Protein Binding , Virus Replication , Young AdultABSTRACT
Emerging evidence suggests that supernumerary centrosomes drive genome instability and oncogenesis. Human T-cell leukaemia virus type I (HTLV-I) is etiologically associated with adult T-cell leukaemia (ATL). ATL cells are aneuploid, but the causes of aneuploidy are incompletely understood. Here, we show that centrosome amplification is frequent in HTLV-I-transformed cells and that this phenotype is caused by the viral Tax oncoprotein. We also show that the fraction of Tax protein that localizes to centrosomes interacts with TAX1BP2, a novel centrosomal protein composed almost entirely of coiled-coil domains. Overexpression of TAX1BP2 inhibited centrosome duplication, whereas depletion of TAX1BP2 by RNAi resulted in centrosome hyperamplification. Our findings suggest that the HTLV-I Tax oncoprotein targets TAX1BP2 causing genomic instability and aneuploidy.
Subject(s)
Cell Transformation, Neoplastic/metabolism , Centrosome/metabolism , Gene Products, tax/metabolism , Human T-lymphotropic virus 1/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Leukemia-Lymphoma, Adult T-Cell/metabolism , Aneuploidy , Animals , CHO Cells , COS Cells , Cell Transformation, Neoplastic/genetics , Chlorocebus aethiops , Cricetinae , Gene Products, tax/genetics , Genomic Instability/physiology , HeLa Cells , Human T-lymphotropic virus 1/genetics , Humans , Intracellular Signaling Peptides and Proteins/genetics , Jurkat Cells , Leukemia-Lymphoma, Adult T-Cell/genetics , Membrane Proteins , Molecular Sequence Data , RNA Interference , RNA, Small Interfering/physiology , RatsABSTRACT
5'-mRNA capping is an early modification that affects pre-mRNA synthesis/splicing, RNA cytoplasmic transport, and mRNA translation and turnover. In eukaryotes, a 7-methylguanosine (m7G) cap is added to newly transcribed RNA polymerase II (RNAP II) transcripts. A subset of RNAP II-transcribed cellular RNAs, including small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and telomerase RNA, is further hypermethylated at the exocyclic N2 of the guanosine to create a trimethylguanosine (TMG)-capped RNA. Some of these TMG-capped RNAs are transported within the nucleus and from the nucleus to the cytoplasm by the CRM-1 (required for chromosome region maintenance) protein. CRM-1 is also used to export Rev/RRE-dependent unspliced/ partially spliced HIV-1 RNAs. Here we report that like snRNAs and snoRNAs, some Rev/RRE-dependent HIV-1 RNAs are TMG-capped. The methyltransferase responsible for TMG modification of HIV-1 RNAs is the human PIMT (peroxisome proliferator-activated receptor-interacting protein with methyltransferase) protein. TMG capping of unspliced/partially spliced HIV-1 RNAs represents a new regulatory mechanism for selective expression.
Subject(s)
Guanosine/analogs & derivatives , HIV-1/genetics , RNA, Viral/genetics , rev Gene Products, Human Immunodeficiency Virus/genetics , Binding Sites/genetics , Blotting, Western , Gene Expression Regulation, Viral , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Guanosine/metabolism , HIV-1/metabolism , HeLa Cells , Humans , Microscopy, Confocal , Mutation , Protein D-Aspartate-L-Isoaspartate Methyltransferase/genetics , Protein D-Aspartate-L-Isoaspartate Methyltransferase/metabolism , RNA/genetics , RNA/metabolism , RNA Caps/metabolism , RNA Processing, Post-Transcriptional , RNA, Viral/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Telomerase/genetics , Telomerase/metabolism , Transfection , Virus Replication/drug effects , rev Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
Although RNA interference (RNAi) is known to play an important part in defense against viruses of invertebrates, its contribution to mammalian anti-viral defense has been a matter of dispute. This is surprising because all components of the RNAi machinery necessary for robust RNAi-mediated restriction of viruses are conserved in mammals, and the introduction of synthetic small interfering RNAs (siRNAs) into cells efficiently silences the replication of viruses that contain siRNA complementary sequences in those cells. Here, I discuss the reasons for the dispute, and review the evidence that RNAi is a part of the physiological defense of mammalian cells against viral infections.
Subject(s)
Host-Pathogen Interactions , RNA Interference , RNA, Small Interfering/genetics , Virus Diseases/genetics , Virus Diseases/immunology , Virus Physiological Phenomena , Animals , Humans , MicroRNAs/genetics , RNA, Viral/genetics , Retroelements/geneticsABSTRACT
CDK9/cyclin T1, a key enzyme in HIV-1 transcription, is negatively regulated by 7SK RNA and the HEXIM1 protein. Dephosphorylation of CDK9 on Thr(186) by protein phosphatase 1 (PP1) in stress-induced cells or by protein phosphatase M1A in normally growing cells activates CDK9. Our previous studies showed that HIV-1 Tat protein binds to PP1 through the Tat Q(35)VCF(38) sequence, which is similar to the PP1-binding RVXF motif and that this interaction facilitates HIV-1 transcription. In the present study, we analyzed the effect of expression of the central domain of nuclear inhibitor of PP1 (cdNIPP1) in an engineered cell line and also when cdNIPP1 was expressed as part of HIV-1 pNL4-3 in place of nef. Stable expression of cdNIPP1 increased CDK9 phosphorylation on Thr(186) and the association of CDK9 with 7SK RNA. The stable expression of cdNIPP1 disrupted the interaction of Tat and PP1 and inhibited HIV-1 transcription. Expression of cdNIPP1 as a part of the HIV-1 genome inhibited HIV-1 replication. Our study provides a proof-of-concept for the future development of PP1-targeting compounds as inhibitors of HIV-1 replication.
Subject(s)
Cyclin-Dependent Kinase 9/metabolism , Endoribonucleases/physiology , HIV-1/genetics , Phosphoprotein Phosphatases/physiology , RNA-Binding Proteins/physiology , Transcription, Genetic , Animals , Anti-HIV Agents , Cell Line , Endoribonucleases/genetics , Gene Products, tat/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Phosphoprotein Phosphatases/genetics , Phosphorylation , Protein Phosphatase 1/metabolism , RNA-Binding Proteins/genetics , Rabbits , Threonine/metabolism , Virus ReplicationABSTRACT
MicroRNAs (miRNAs) are small non-coding RNAs that control a multitude of critical processes in mammalian cells. Increasing evidence has emerged that host miRNAs serve in animal cells to restrict viral infections. In turn, many viruses encode RNA silencing suppressors (RSS) which are employed to moderate the potency of the cell's miRNA selection against viral replication. Some viruses also encode viral miRNAs. In this review, we summarize findings from human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1) that illustrate examples of host cell miRNAs that target the viruses, of RSS encoded by viruses, and of host cell miRNA profile changes that are seen in infected cells. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.
Subject(s)
MicroRNAs/metabolism , RNA, Viral/metabolism , Retroviridae/genetics , Gene Expression Regulation, Viral , HIV-1/genetics , Human T-lymphotropic virus 1/genetics , Humans , MicroRNAs/genetics , Models, Genetic , RNA Interference , RNA, Viral/genetics , Virus ReplicationABSTRACT
The importance of geographic diversity in publishing is emphasized in this editorial.
Subject(s)
Biodiversity , Publishing/statistics & numerical data , Science , Humans , Population DynamicsABSTRACT
Recent literature highlights at Retrovirology are described. Predictions are made regarding "hot" retrovirology research trends for the coming year based on recent journal access statistics. Changes in Retrovirology editor and the frequency of the Retrovirology Prize are announced.
Subject(s)
Awards and Prizes , Periodicals as Topic/statistics & numerical data , Congresses as Topic , Humans , Peer Review, Research/trends , Periodicals as Topic/trends , Retroviridae/genetics , Virology/trendsABSTRACT
Nine years after its founding, Retrovirology has moved to the forefront of virology journals in Impact Factor.
Subject(s)
Access to Information , Periodicals as Topic/standards , Humans , Internet , Journal Impact Factor , Periodicals as Topic/economics , RetroviridaeABSTRACT
Human T-cell Leukemia Virus type 1 (HTLV-1) and 2 (HTLV-2) are two closely related human retroviruses. HTLV-1 is associated with an aggressive Adult T-cell Leukemia (ATL) while there is no evidence for an association of HTLV-2 with any human malignancies. The two viruses encode transactivator proteins, Tax-1 and Tax-2 respectively. In ATL, Tax-1 is thought to play a central role in the transformation of a normal T-cell into a leukemic cell; however, it has not been entirely clear how post-translational modifications of Tax-1 influence its transforming activity. Here, we discuss three recent papers that report on the ubiquitination and sumoylation of Tax-1 and Tax-2. We comment on their divergent findings implicating the importance (or lack of importance) of these modifications and other events on Tax activation of NF-κB as related to cellular transformation.