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Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites
Published on: March 22, 2016
Analysis of human immunodeficiency virus type 1 promoter insertion in vivo
Insights
This study found HIV-1 promoter insertion events in AIDS patients' lung cells. While in vitro studies predicted more, only 1.8% of integrated HIV-1 transcribed adjacent genes in vivo.
Area of Science:
- Molecular Biology
- Virology
- Immunology
Background:
- Human Immunodeficiency Virus type 1 (HIV-1) integration into the host genome is a critical step in viral replication.
- Insertional activation, where viral integration affects host gene expression, is a known phenomenon but its in vivo occurrence and frequency are not well understood.
Purpose of the Study:
- To investigate the occurrence and frequency of HIV-1 insertional activation events in vivo.
- To determine the ratio of provirus integration to promoter insertion events in cells from AIDS patients.
Main Methods:
- Analysis of broncho-alveolar lavage (BAL) cells from AIDS patients using a Polymerase Chain Reaction (PCR) assay.
- Detection of proviral-host sequence transcripts indicating HIV-1 promoter insertion.
- In vitro infection studies to establish the ratio of provirus integration to promoter insertion events, monitoring transcripts with HIV-1 and specific cellular gene sequences (K-ras, CD4).
Main Results:
- HIV-1 promoter insertion events were detected in 9% of analyzed BAL cell aliquots.
- Multiply spliced HIV-1 mRNAs were found in 28% of aliquots.
- In vitro studies indicated that 1.8% of integrated proviruses transcribed adjacent genes, with an expected 25 promoter insertion events in vivo based on proviral load.
Conclusions:
- The study provides evidence for HIV-1 insertional activation in vivo, specifically in lung cells of AIDS patients.
- The observed frequency of promoter insertion events in vivo (five events) was lower than predicted, potentially due to reduced HIV-1 transcriptional activity in vivo compared to cell cultures.
Abstract:
Our study reports the occurrence of HIV-1 insertional activation events in vivo. Using a previously described PCR assay, small aliquots of broncho-alveolar lavage (BAL) cells obtained from AIDS patients were analyzed. Nine percent (5/54) of the aliquots contained proviral-host sequence transcripts indicating HIV-1 promoter insertion, whereas multiply spliced HIV-1 mRNAs were found in 28% (15/54) of the aliquots. In four of five events, insertions affect distinct cellular transcription units expressed in a T-cell line. To establish a ratio between provirus integration and promoter insertion events, an in vitro infection study was performed and transcripts containing HIV-1 and K-ras or CD4 gene sequences, respectively, were monitored. Given the randomness of retrovirus integration, 170 sense-oriented HIV integrations into these gene loci were predicted to occur. Three distinct promoter insertion events were observed, indicating that 1.8% of integrated proviruses transcribed adjacent genes. Based on this result and a mean of 257 proviral copies per 10(6) BAL cells, we would expect to observe 25 promoter insertion events in our in vivo study. That only five events were found may be due to the lower transcriptional activity of HIV-1 in vivo than that in cell cultures.
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