Analysis of early human immunodeficiency virus type 1 DNA synthesis by use of a new sensitive assay for quantifying integrated provirus - PubMed (original) (raw)
Analysis of early human immunodeficiency virus type 1 DNA synthesis by use of a new sensitive assay for quantifying integrated provirus
Audrey Brussel et al. J Virol. 2003 Sep.
Abstract
A novel Alu-long terminal repeat (LTR)-based real-time nested-PCR assay was developed to quantify integrated human immunodeficiency virus type 1 (HIV-1) DNA in infected cells with both accuracy and high sensitivity (six proviruses within 50,000 cell equivalents). Parallel assays for total HIV-1 DNA and two-LTR HIV-1 DNA circles allowed the synthesis and fate of the different HIV-1 DNA species to be monitored upon a single round of viral replication.
Figures
FIG. 1.
Real-time PCR strategies and location of primers and probes for the quantification of integrated HIV-1 DNA (a), total HIV-1 DNA (b), and two-LTR circles (c). Thin arrows, primers; thick arrows, probes.
FIG. 2.
Characteristics of _Alu_-LTR nested PCR. (a) Fluorescence curves generated by two-step amplification of serial dilutions of HeLa R7 Neo cell DNA. The copy numbers for each standard dilution are shown over the corresponding fluorescence curve. (b) Linear regression for quantifying integrated HIV-1 DNA. (c) Specificity of the _Alu_-LTR nested PCR. Fluorescence curves generated by amplification of viral DNA from CEM cells infected with the VSV-G-pseudotyped HIV-1 R7 Neo virus and collected in the presence of Alu primers (pink, 8 h p.i.; green, 48 h p.i.), in the absence of Alu primers (blue, 8 h p.i.; red, 48 h p.i.), or without a preamplification step (purple, 8 h p.i.; orange, 48 h p.i.) are shown.
FIG. 3.
Quantification of total HIV-1 DNA (a), integrated HIV-1 DNA (b), and two-LTR circles (c) during a single round of replication of the VSV-G-pseudotyped HIV-1 R7 Neo virus in CEM cells. Values are means ± standard errors of the means. The depicted results were obtained from a representative experiment.
Similar articles
- Alu-LTR real-time nested PCR assay for quantifying integrated HIV-1 DNA.
Brussel A, Delelis O, Sonigo P. Brussel A, et al. Methods Mol Biol. 2005;304:139-54. doi: 10.1385/1-59259-907-9:139. Methods Mol Biol. 2005. PMID: 16061972 - Analysis of human immunodeficiency virus type 1 integration by using a specific, sensitive and quantitative assay based on real-time polymerase chain reaction.
Yamamoto N, Tanaka C, Wu Y, Chang MO, Inagaki Y, Saito Y, Naito T, Ogasawara H, Sekigawa I, Hayashida Y. Yamamoto N, et al. Virus Genes. 2006 Feb;32(1):105-13. doi: 10.1007/s11262-005-5851-2. Virus Genes. 2006. PMID: 16525741 - Impact of the central polypurine tract on the kinetics of human immunodeficiency virus type 1 vector transduction.
Van Maele B, De Rijck J, De Clercq E, Debyser Z. Van Maele B, et al. J Virol. 2003 Apr;77(8):4685-94. doi: 10.1128/jvi.77.8.4685-4694.2003. J Virol. 2003. PMID: 12663775 Free PMC article. - Human Immunodeficiency Virus Type 1 Two-Long Terminal Repeat Circles: A Subject for Debate.
Olivares I, Pernas M, Casado C, López-Galindez C. Olivares I, et al. AIDS Rev. 2016 Jan-Mar;18(1):23-31. AIDS Rev. 2016. PMID: 26936759 Review. - HIV-1 gene expression: lessons from provirus and non-integrated DNA.
Wu Y. Wu Y. Retrovirology. 2004 Jun 25;1:13. doi: 10.1186/1742-4690-1-13. Retrovirology. 2004. PMID: 15219234 Free PMC article. Review.
Cited by
- Evidence for gene expression by unintegrated human immunodeficiency virus type 1 DNA species.
Brussel A, Sonigo P. Brussel A, et al. J Virol. 2004 Oct;78(20):11263-71. doi: 10.1128/JVI.78.20.11263-11271.2004. J Virol. 2004. PMID: 15452245 Free PMC article. - HCV-coinfection is related to an increased HIV-1 reservoir size in cART-treated HIV patients: a cross-sectional study.
López-Huertas MR, Palladino C, Garrido-Arquero M, Esteban-Cartelle B, Sánchez-Carrillo M, Martínez-Román P, Martín-Carbonero L, Ryan P, Domínguez-Domínguez L, Santos IL, Moral SF, Benito JM, Rallón N, Alcamí J, Resino S, Fernández-Rodríguez A, Coiras M, Briz V; Multidisciplinary Group of viral coinfection HIV/Hepatitis (COVIHEP). López-Huertas MR, et al. Sci Rep. 2019 Apr 3;9(1):5606. doi: 10.1038/s41598-019-41788-9. Sci Rep. 2019. PMID: 30944340 Free PMC article. - Residual HIV-1 DNA Flap-independent nuclear import of cPPT/CTS double mutant viruses does not support spreading infection.
Iglesias C, Ringeard M, Di Nunzio F, Fernandez J, Gaudin R, Souque P, Charneau P, Arhel N. Iglesias C, et al. Retrovirology. 2011 Nov 10;8:92. doi: 10.1186/1742-4690-8-92. Retrovirology. 2011. PMID: 22074589 Free PMC article. - The role of unintegrated DNA in HIV infection.
Sloan RD, Wainberg MA. Sloan RD, et al. Retrovirology. 2011 Jul 1;8:52. doi: 10.1186/1742-4690-8-52. Retrovirology. 2011. PMID: 21722380 Free PMC article. Review. - HuR interacts with human immunodeficiency virus type 1 reverse transcriptase, and modulates reverse transcription in infected cells.
Lemay J, Maidou-Peindara P, Bader T, Ennifar E, Rain JC, Benarous R, Liu LX. Lemay J, et al. Retrovirology. 2008 Jun 10;5:47. doi: 10.1186/1742-4690-5-47. Retrovirology. 2008. PMID: 18544151 Free PMC article.
References
- Barbosa, P., P. Charneau, N. Dumey, and F. Clavel. 1994. Kinetic analysis of HIV-1 early replicative steps in a coculture system. AIDS Res. Hum. Retrovir. 10:53-59. - PubMed
- Brussel, A., D. Mathez, S. Broche-Pierre, R. Lancar, T. Calvez, P. Sonigo, and J. Leibowitch. 2003. Longitudinal monitoring of 2-long terminal repeat circles in peripheral blood mononuclear cells from patients with chronic HIV-1 infection. AIDS 17:645-652. - PubMed
- Butler, S. L., M. S. Hansen, and F. D. Bushman. 2001. A quantitative assay for HIV DNA integration in vivo. Nat. Med. 7:631-634. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources