Resting CD4⁺T Cells from Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals Carry Integrated HIV-1 Genomes within Actively Transcribed Host Genes

Abstract

Resting CD4؉ T-cell populations from human immunodeficiency virus type 1 (HIV- 1

Establishment of novel cell lines latently infected with human immunodeficiency virus 1

“…PCR conditions for the first inverse PCR were 3 mins of pre-denaturation at 94°C, 30 cycles of 30 secs at 94°C, 1 min at 65°C, 2 mins at 68°C, and 4 minutes final extension at 68°C. Second inverse PCR was carried out with the inner LTR and gag primers reported by Han et al (2004) and PCR conditions were the same as in the first inverse PCR. The PCR products were analyzed on 1% agarose gel electrophoresis, eluted, and then directly sequenced (Fig.…”

“…Genomic DNA was digested with PstI (New England Biolabs) at 37°C for 8 hrs and ligated using T4 DNA ligase (Roche Diagnostics) at 16°C overnight, and inactivated at 70°C for 10 mins and at 4°C for 5 mins. To analyze the junction sequences between the 5ʹ-end of the HIV-1 viral genome and host cell DNA, a circular DNA was used for the first inverse PCR with outer LTR and gag primers reported by Han et al (2004). The circular DNA was amplified with PCR mixture containing EF-Taq PCR buffer (SolGent), dNTP (10 mmol/l), each 10 pmol first inverse PCR primers, and EF-Taq polymerase (SolGent).…”

“…Inverse PCR described by Han et al (2004) was used to warrant the accuracy of HIV-1 integration assay for NCHA cell lines. The optimal initial amount of genomic DNA extracted from cell lines was 50 μg in the inverse PCR.…”

Natural history of adult T-cell leukemia/lymphoma and approaches to therapy

“…These are equivalent to the frequency calculated based on random integration (33.2%) (Doi et al, 2005). By contrast, HIV-1, another human retrovirus, integrates predominantly (91%) into actively transcribed genes of T lymphocytes (Han et al, 2004), and integration sites are evenly distributed within the transcription units. Additionally, the frequency of integration into transcription units is 34.2% for murine leukemia virus (MLV), and these sites are clustered near the transcriptional After a long latent period, ATL develops in about 5% of asymptomatic carriers.…”

Exploring HIV latency using transcription profiling

“…Combined with RT-ddPCR, which enables absolute cDNA quantification 24 , this approach provides a considerable advantage over previous work that mostly focuses on one mechanism of latency at a time and has typically utilised in vitro models of latency, which may not recapitulate what happens in vivo. [18][19][20]22,23,[26][27][28] . Unlike previously employed strategies, specific blocks to transcription and the magnitude of their impact on the prevailing levels of HIV RNA can be simultaneously assessed by determining the expression of these processive transcripts.…”