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Retrovirology Nov 2010HIV uncoating is defined as the loss of viral capsid that occurs within the cytoplasm of infected cells before entry of the viral genome into the nucleus. It is an... (Review)
Review
HIV uncoating is defined as the loss of viral capsid that occurs within the cytoplasm of infected cells before entry of the viral genome into the nucleus. It is an obligatory step of HIV-1 early infection and accompanies the transition between reverse transcription complexes (RTCs), in which reverse transcription occurs, and pre-integration complexes (PICs), which are competent to integrate into the host genome. The study of the nature and timing of HIV-1 uncoating has been paved with difficulties, particularly as a result of the vulnerability of the capsid assembly to experimental manipulation. Nevertheless, recent studies of capsid structure, retroviral restriction and mechanisms of nuclear import, as well as the recent expansion of technical advances in genome-wide studies and cell imagery approaches, have substantially changed our understanding of HIV uncoating. Although early work suggested that uncoating occurs immediately following viral entry in the cell, thus attributing a trivial role for the capsid in infected cells, recent data suggest that uncoating occurs several hours later and that capsid has an all-important role in the cell that it infects: for transport towards the nucleus, reverse transcription and nuclear import. Knowing that uncoating occurs at a later stage suggests that the viral capsid interacts extensively with the cytoskeleton and other cytoplasmic components during its transport to the nucleus, which leads to a considerable reassessment of our efforts to identify potential therapeutic targets for HIV therapy. This review discusses our current understanding of HIV uncoating, the functional interplay between infectivity and timely uncoating, as well as exposing the appropriate methods to study uncoating and addressing the many questions that remain unanswered.
Topics: Capsid; Capsid Proteins; HIV Infections; HIV-1; Humans; Virulence; Virus Integration; Virus Uncoating
PubMed: 21083892
DOI: 10.1186/1742-4690-7-96 -
Blood Apr 2020In this issue of , Six et al provide evidence for the existence of myeloid- and lymphoid-dominant human hematopoietic stem and progenitor cells (HSPCs) using clonal...
In this issue of , Six et al provide evidence for the existence of myeloid- and lymphoid-dominant human hematopoietic stem and progenitor cells (HSPCs) using clonal tracking in patients treated with gene therapy for Wiskott-Aldrich syndrome (WAS) and β-hemoglobinopathies.
Topics: Genetic Therapy; HIV Integrase; Hematopoietic Stem Cell Transplantation; Humans; Virus Integration
PubMed: 32271907
DOI: 10.1182/blood.2020005115 -
Viruses Mar 2021Chronic hepatitis B infection remains a globally important cause of morbidity and mortality and has recently undergone a renaissance in therapeutic interest with... (Review)
Review
Chronic hepatitis B infection remains a globally important cause of morbidity and mortality and has recently undergone a renaissance in therapeutic interest with increased pre-clinical and clinical testing of new drug classes. One of the first new classes in the clinic was RNA interference agents, which have the potential to impact the entire viral life cycle by reducing all virus-produced mRNA. Early clinical testing with the first of these agents in the clinic, ARC-520, demonstrated that rapid and deep reductions in viral proteins, RNA and DNA could be produced with this approach, but also the surprising insight that HBsAg production from incomplete HBV DNA integrated into the host genome appears to play a heretofore unappreciated and important role in maintaining circulating HBsAg, thought to play a fundamental role in preventing host clearance of the virus. Thus, accounting for viral DNA integration in novel HBV treatment approaches may prove to be essential to achieving successful finite therapies of this difficult to treat chronic infection.
Topics: DNA, Viral; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatitis B, Chronic; Humans; RNA Interference; RNA, Small Interfering; Virus Integration
PubMed: 33808298
DOI: 10.3390/v13040581 -
Virology Sep 2009Integrase (IN) is the enzyme responsible for provirus integration of retroviruses into the host cell genome. We used an Avian Sarcoma and Leukemia Viruses (ASLV)...
Avian sarcoma and leukemia virus (ASLV) integration in vitro: mutation or deletion of integrase (IN) recognition sequences does not prevent but only reduces the efficiency and accuracy of DNA integration.
Integrase (IN) is the enzyme responsible for provirus integration of retroviruses into the host cell genome. We used an Avian Sarcoma and Leukemia Viruses (ASLV) integration assay to investigate the way in which IN integrates substrates mutated or devoid of one or both IN recognition sequences. We found that replacing U5 by non-viral sequences (U5del) or U3 by a mutated sequence (pseudoU3) resulted in two and three fold reduction of two-ended integration (integration of the two ends from a donor DNA) respectively, but had a slight effect on concerted integration (integration of both ends at the same site of target DNA). Further, IN was still able to integrate the viral ends of the double mutant (pseudoU3/U5del) in a two-ended and concerted integration reaction. However, efficiency and accuracy (i.e. fidelity of size duplication and of end cleavage) of integration were reduced.
Topics: Alpharetrovirus; Animals; Base Sequence; Binding Sites; DNA Primers; DNA, Viral; Genes, Viral; In Vitro Techniques; Integrases; Models, Biological; Mutation; Sequence Deletion; Virus Integration
PubMed: 19638332
DOI: 10.1016/j.virol.2009.06.031 -
Journal of Biomolecular Structure &... Feb 2020Communicated by Ramaswamy H. Sarma.
Integrating molecular dynamics simulation and molecular mechanics/generalized Born surface area calculation into pharmacophore modeling: a case study on the proviral integration site for Moloney murine leukemia virus (Pim)-1 kinase inhibitors.
Communicated by Ramaswamy H. Sarma.
Topics: Molecular Dynamics Simulation; Moloney murine leukemia virus; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-pim-1; Proviruses; Thermodynamics; Virus Integration
PubMed: 30678548
DOI: 10.1080/07391102.2019.1571946 -
Retrovirology Jan 2017Assessing the location and frequency of HIV integration sites in latently infected cells can potentially inform our understanding of how HIV persists during combination...
BACKGROUND
Assessing the location and frequency of HIV integration sites in latently infected cells can potentially inform our understanding of how HIV persists during combination antiretroviral therapy. We developed a novel high throughput sequencing method to evaluate HIV integration sites in latently infected cell lines to determine whether there was virus replication or clonal expansion in these cell lines observed as multiple integration events at the same position.
RESULTS
We modified a previously reported method using random DNA shearing and PCR to allow for high throughput robotic processing to identify the site and frequency of HIV integration in latently infected cell lines. Latently infected cell lines infected with intact virus demonstrated multiple distinct HIV integration sites (28 different sites in U1, 110 in ACH-2 and 117 in J1.1 per 150,000 cells). In contrast, cell lines infected with replication-incompetent viruses (J-Lat cells) demonstrated single integration sites. Following in vitro passaging of the ACH-2 cell line, we observed a significant increase in the frequency of unique HIV integration sites and there were multiple mutations and large deletions in the proviral DNA. When the ACH-2 cell line was cultured with the integrase inhibitor raltegravir, there was a significant decrease in the number of unique HIV integration sites and a transient increase in the frequency of 2-LTR circles consistent with virus replication in these cells.
CONCLUSION
Cell lines latently infected with intact HIV demonstrated multiple unique HIV integration sites indicating that these cell lines are not clonal and in the ACH-2 cell line there was evidence of low level virus replication. These findings have implications for the use of latently infected cell lines as models of HIV latency and for the use of these cells as standards.
Topics: Cell Line; HIV; High-Throughput Nucleotide Sequencing; Humans; Virus Integration; Virus Latency; Virus Replication
PubMed: 28086908
DOI: 10.1186/s12977-016-0325-2 -
Orthodontics & Craniofacial Research Feb 2007To provide a comprehensive literature review describing recent developments of the recombinant adeno-associated virus (rAAV) vector and exploring the therapeutic... (Review)
Review
OBJECTIVES
To provide a comprehensive literature review describing recent developments of the recombinant adeno-associated virus (rAAV) vector and exploring the therapeutic application of rAAV for bone defects, cartilage lesions and rheumatoid arthritis.
DESIGN
Narrative review.
RESULT
The review outlines the serotypes and genome of AAV, integration and life cycle of the rAAV vectors, the immune response and regulating system for AAV gene therapy. Furthermore, the advancements of rAAV gene therapy for bone growth together with cartilage repair are summarized.
CONCLUSION
Recombinant adeno-associated virus vector is perceived to be one of the most promising vector systems for bone and cartilage gene therapy approaches and further investigations need to be carried out for craniofacial research.
Topics: Arthritis, Rheumatoid; Bone Diseases; Cartilage Diseases; DNA, Recombinant; Dependovirus; Genetic Therapy; Genetic Vectors; Humans; Virus Integration
PubMed: 17284242
DOI: 10.1111/j.1601-6343.2007.00381.x -
Journal of Virology Jan 1992Deletion mutants of simian immunodeficiency virus (SIVmac) which were unable to integrate into host cells were generated by removing a portion of the integrase (IN)...
Deletion mutants of simian immunodeficiency virus (SIVmac) which were unable to integrate into host cells were generated by removing a portion of the integrase (IN) domain of the pol gene. The resulting plasmid was transfected into HUT-78 and human rhabdomyosarcoma cells. In comparison with the parental plasmid DNA transfected in parallel, the deletion mutant was found to direct efficient production of virus in both cell systems. Viruses derived from wild-type and mutant proviral DNAs were also tested for their relative replicative abilities in HUT-78 and U937 cells, and the kinetics of virus production was found to vary between these two cell systems. Analysis of DNA from infected cell nuclei showed that the deletion mutant lacked the ability to integrate despite being able to produce infectious virus. Using the sensitive polymerase chain reaction technique, we have clearly demonstrated the absence of the IN domain in the deletion mutant after infection and replication in HUT-78 cells. Such mutants might form the basis for the development of an experimental live attenuated vaccine.
Topics: Base Sequence; DNA, Viral; Humans; Kinetics; Molecular Sequence Data; Mutation; Plasmids; Polymerase Chain Reaction; Simian Immunodeficiency Virus; Transfection; Tumor Cells, Cultured; Virus Integration
PubMed: 1727479
DOI: 10.1128/JVI.66.1.167-171.1992 -
Nature Medicine Jul 2013The clinical application of adeno-associated virus vectors (AAVs) is limited because of concerns about AAV integration-mediated tumorigenicity. We performed...
The clinical application of adeno-associated virus vectors (AAVs) is limited because of concerns about AAV integration-mediated tumorigenicity. We performed integration-site analysis after AAV1-LPL(S447X) intramuscular injection in five lipoprotein lipase-deficient subjects, revealing random nuclear integration and hotspots in mitochondria. We conclude that AAV integration is potentially safe and that vector breakage and integration may occur from each position of the vector genome. Future viral integration-site analyses should include the mitochondrial genome.
Topics: Animals; Binding Sites; Dependovirus; Genetic Therapy; Genetic Vectors; Humans; Hyperlipoproteinemia Type I; Injections, Intramuscular; Lipoprotein Lipase; Mice; Mice, Inbred C57BL; Models, Biological; Mutagenesis, Insertional; Transcriptome; Virus Integration
PubMed: 23770691
DOI: 10.1038/nm.3230 -
Gene Therapy Aug 2013Integrating vectors can lead to the dysregulation of nearby chromosomal genes, with important consequences for clinical trials and cellular engineering. This includes...
Integrating vectors can lead to the dysregulation of nearby chromosomal genes, with important consequences for clinical trials and cellular engineering. This includes the retroviral and lentiviral vectors commonly used for deriving induced pluripotent stem cells (iPSCs). We previously used integrating foamy virus (FV) vectors expressing OCT4, SOX2, MYC and KLF4 to reprogram osteogenesis imperfecta mesenchymal stem cells (MSCs). Here, we have studied the effects of 10 FV vector proviruses on neighboring gene expression in four iPSC lines and their corresponding iPSC-derived MSC (iMSCs). Gene expression profiles in these iPSC lines showed that none of the 38 genes within 300 kb up- or downstream of integrated proviruses had a significant difference in mRNA levels, including five genes with proviruses in their transcription units. In the iMSCs derived from these iPSCs, the same type of analysis showed a single dysregulated transcript out of 46 genes found near proviruses. This frequency of dysregulation was similar to that of genes lacking nearby proviruses, so it may have been due to interclonal variation and/or measurement inaccuracies. While the number of integration sites examined in this paper is limited, our results suggest that integrated FV proviruses do not impact the expression of chromosomal genes in pluripotent human stem cells or their differentiated derivatives. This interpretation is consistent with previous reports that FV vectors have minimal genotoxicity, even when integrating near or within genes.
Topics: Cell Differentiation; Cell Line; Gene Expression Regulation, Developmental; Genetic Vectors; Humans; Induced Pluripotent Stem Cells; Kruppel-Like Factor 4; Spumavirus; Virus Integration
PubMed: 23388702
DOI: 10.1038/gt.2013.6