scholarly journals Evaluation and Prediction of the HIV-1 Central Polypurine Tract Influence on Foamy Viral Vectors to Transduce Dividing and Growth-Arrested Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Sergey Shityakov ◽  
Carola Förster ◽  
Axel Rethwilm ◽  
Thomas Dandekar
Keyword(s):  
Nuclear Import ◽  
Viral Vectors ◽  
Viral Vector ◽  
A549 Cells ◽  
Transfer Task ◽  
Retroviral Vectors ◽  
Polypurine Tract ◽  
A Cell ◽  
Central Polypurine Tract ◽  
Hiv 1

Retroviral vectors are potent tools for gene delivery and various biomedical applications. To accomplish a gene transfer task successfully, retroviral vectors must effectively transduce diverse cell cultures at different phases of a cell cycle. However, very promising retroviral vectors based on the foamy viral (FV) backbone lack the capacity to efficiently transduce quiescent cells. It is hypothesized that this phenomenon might be explained as the inability of foamy viruses to form a pre-integration complex (PIC) with nuclear import activity in growth-arrested cells, which is the characteristic for lentiviruses (HIV-1). In this process, the HIV-1 central polypurine tract (cPPT) serves as a primer for plus-strand synthesis to produce a “flap” element and is believed to be crucial for the subsequent double-stranded cDNA formation of all retroviral RNA genomes. In this study, the effects of the lentiviral cPPT element on the FV transduction potential in dividing and growth-arrested (G1/S phase) adenocarcinomic human alveolar basal epithelial (A549) cells are investigated by experimental and theoretical methods. The results indicated that the HIV-1 cPPT element in a foamy viral vector background will lead to a significant reduction of the FV transduction and viral titre in growth-arrested cells due to the absence of PICs with nuclear import activity.

scholarly journals Reassessment of the Roles of Integrase and the Central DNA Flap in Human Immunodeficiency Virus Type 1 Nuclear Import

2002 ◽  
Vol 76 (23) ◽  
pp. 12087-12096 ◽  
Author(s):  
Jeffrey D. Dvorin ◽  
Peter Bell ◽  
Gerd G. Maul ◽  
Masahiro Yamashita ◽  
Michael Emerman ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
In Situ Hybridization ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Import ◽  
Immunodeficiency Virus ◽  
Central Polypurine Tract ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) can infect nondividing cells productively because the nuclear import of viral nucleic acids occurs in the absence of cell division. A number of viral factors that are present in HIV-1 preintegration complexes (PICs) have been assigned functions in nuclear import, including an essential valine at position 165 in integrase (IN-V165) and the central polypurine tract (cPPT). In this article, we report a comparison of the replication and infection characteristics of viruses with disruptions in the cPPT and IN-V165. We found that viruses with cPPT mutations still replicated productively in both dividing and nondividing cells, while viruses with a mutation at IN-V165 did not. Direct observation of the subcellular localization of HIV-1 cDNAs by fluorescence in situ hybridization revealed that cDNAs synthesized by both mutant viruses were readily detected in the nucleus. Thus, neither the cPPT nor the valine residue at position 165 of integrase is essential for the nuclear import of HIV-1 PICs.

scholarly journals The HIV-1 Central Polypurine Tract Functions as a Second Line of Defense against APOBEC3G/F

2010 ◽  
Vol 84 (22) ◽  
pp. 11981-11993 ◽  
Author(s):  
Chunling Hu ◽  
Dyana T. Saenz ◽  
Hind J. Fadel ◽  
William Walker ◽  
Mary Peretz ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Target Cells ◽  
Second Line ◽  
Wild Type ◽  
Single Cycle ◽  
Polypurine Tract ◽  
Producer Cell ◽  
Central Polypurine Tract ◽  
Hiv 1

ABSTRACT HIV-1 and certain other retroviruses initiate plus-strand synthesis in the center of the genome as well as at the standard retroviral 3′ polypurine tract. This peculiarity of reverse transcription results in a central DNA “flap” structure that has been of controversial functional significance. We mutated both HIV-1 flap-generating elements, the central polypurine tract (cPPT) and the central termination sequence (CTS). To avoid an ambiguity of previous studies, we did so without affecting integrase coding. DNA flap formation was disrupted but single-cycle infection was unaffected in all target cells tested, regardless of cell cycle status. Spreading HIV-1 infection was also normal in most T cell lines, and flap mutant viruses replicated equivalently to the wild type in nondividing cells, including macrophages. However, spreading infection of flap mutant HIV-1 was impaired in non-vif-permissive cells (HuT78, H9, and primary human peripheral blood mononuclear cells [PBMCs]), suggesting APOBEC3G (A3G) restriction. Single-cycle infections confirmed that vif-intact flap mutant HIV-1 is restricted by producer cell A3G/F. Combining the Δvif and cPPT-CTS mutations increased A3G restriction synergistically. Moreover, RNA interference knockdown of A3G in HuT78 cells released the block to flap mutant HIV-1 replication. Flap mutant HIV-1 also accrued markedly increased A3G-mediated G→A hypermutation compared to that of wild-type HIV-1 (a full log10 in the 0.36 kb downstream of the mutant cPPT). We suggest that the triple-stranded DNA structure, the flap, is not the consequential outcome. The salient functional feature is central plus-strand initiation, which functions as a second line of defense against single-stranded DNA editing by A3 proteins that survive producer cell degradation by Vif.

scholarly journals Compensatory Role of Human Immunodeficiency Virus Central Polypurine Tract Sequence in Kinetically Disrupted Reverse Transcription

2008 ◽  
Vol 82 (15) ◽  
pp. 7716-7720 ◽  
Author(s):  
Mark Skasko ◽  
Baek Kim
Keyword(s):  
Human Immunodeficiency Virus ◽  
Dna Synthesis ◽  
Lung Fibroblasts ◽  
Polypurine Tract ◽  
Immunodeficiency Virus ◽  
Deoxynucleoside Triphosphate ◽  
Central Polypurine Tract ◽  
Hiv 1 ◽  
Vector Transduction

ABSTRACT We tested whether the additional positive-strand DNA synthesis initiation of human immunodeficiency virus type 1 (HIV-1) from the central polypurine tract (cPPT) facilitates efficient completion of kinetically disturbed proviral DNA synthesis induced by dysfunctional reverse transcriptase (RT) mutants or limited cellular deoxynucleoside triphosphate (dNTP) pools. Indeed, the cPPT enabled the HIV-1 vectors harboring RT mutants with reduced dNTP binding affinity to transduce human lung fibroblasts (HLFs), without which these mutant vectors normally fail to transduce. The cPPT showed little effect on wild-type HIV-1 vector transduction in HLF, whereas it significantly enhanced vector transduction in HLFs engineered to contain reduced dNTP pools, suggesting a novel compensatory role for cPPT in viruses harboring kinetically impaired RT.

Mutations in the central polypurine tract of HIV-1 result in delayed replication

Virology ◽  
1992 ◽  
Vol 190 (1) ◽  
pp. 440-442 ◽  
Author(s):  
Olav Hungnesi ◽  
Enok Tjøtta ◽  
Bjørn Grinde
Keyword(s):  
Polypurine Tract ◽  
Central Polypurine Tract ◽  
Hiv 1

scholarly journals Impact of the Central Polypurine Tract on the Kinetics of Human Immunodeficiency Virus Type 1 Vector Transduction

2003 ◽  
Vol 77 (8) ◽  
pp. 4685-4694 ◽  
Author(s):  
Bénédicte Van Maele ◽  
Jan De Rijck ◽  
Erik De Clercq ◽  
Zeger Debyser
Keyword(s):  
Nuclear Import ◽  
Transcriptase Inhibitor ◽  
Immunodeficiency Virus ◽  
Central Polypurine Tract ◽  
Reverse Transcriptase Inhibitor ◽  
The Impact ◽  
Kinetics Of ◽  
Hiv 1 ◽  
Vector Transduction

ABSTRACT Lentiviral vectors derived from human immunodeficiency virus type 1 (HIV-1) show great promise as gene carriers for future gene therapy. Insertion of a fragment containing the central polypurine tract (cPPT) in HIV-1 vector constructs is known to enhance transduction efficiency drastically, reportedly by facilitating the nuclear import of HIV-1 cDNA through a central DNA flap. We have studied the impact of the cPPT on the kinetics of HIV-1 vector transduction by real-time PCR. The kinetics of total HIV-1 DNA, two-long-terminal-repeat (2-LTR) circles, and, by an Alu-PCR, integrated proviral DNA were monitored. About 6 to 12 h after transduction, the total HIV-1 DNA reached a maximum level, followed by a steep decrease. The 2-LTR circles peaked after 24 to 48 h and were diluted upon cell division. Integration of HIV-1 DNA was first detected at 12 h postinfection. When HIV-1 vectors that contained the cPPT were used, DNA synthesis was similar but a threefold higher amount of 2-LTR circles was detected, confirming the impact on nuclear import. Moreover, a 10-fold increase in the amount of integrated DNA was observed in the presence of the cPPT. Only in the absence of the cPPT was a saturation in 2-LTR circle formation seen at a high multiplicity of infection, suggesting a role for the cPPT in overcoming a barrier to the nuclear import of HIV-1 DNA. A major effect of the central DNA flap on the juxtaposition of both LTRs is unlikely, since transduction with HIV-1 vectors containing ectopic cPPT fragments resulted in increased amounts of 2-LTR circles as well as integrated DNA. Inhibitors of transduction by cPPT-containing HIV vectors were also studied by real-time PCR. The reverse transcriptase inhibitor azidothymidine (AZT) and the nonnucleoside reverse transcriptase inhibitor α-APA clearly inhibited viral DNA synthesis, whereas integrase inhibitors such as the diketo acid L-708,906 and the pyranodipyrimidine V-165 specifically inhibited integration.

scholarly journals Strand Displacement Synthesis in the Central Polypurine Tract Region of HIV-1 Promotes DNA to DNA Strand Transfer Recombination

1996 ◽  
Vol 271 (47) ◽  
pp. 29605-29611 ◽  
Author(s):  
Gloria M. Fuentes ◽  
Chockalingam Palaniappan ◽  
Philip J. Fay ◽  
Robert A. Bambara
Keyword(s):  
Strand Transfer ◽  
Strand Displacement ◽  
Polypurine Tract ◽  
Central Polypurine Tract ◽  
Dna Strand ◽  
Hiv 1
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