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Abstract
Regardless of recent advances in the development of anti-retroviral drugs, it is still extremely difficult to eradicate HIV-1 from infected individuals. The characterization of the HIV-1 provirus, a type of viral reservoir, with a high resolution is key to HIV-1 cure research. Here, we demonstrate that DNA-capture-seq is a powerful tool to obtain comprehensive information on the HIV-1 provirus. We use biotinylated DNA probes targeting the entire HIV-1 sequence to capture fragments containing HIV-1 sequences from DNA-seq libraries prepared for high throughput sequencing. We demonstrate that the protocol provided the entire proviral sequence from the beginning of the 5′ LTR to the end of the 3′ LTR. Since HIV-1 DNA-probes can hybridize not only viral fragments but also virus-host chimeric ones, the viral integration site information can also be obtained. We verify the efficiency of the protocol by using latently infected cell lines, such as ACH-2 and J1.1, and newly generated ones. The results reveal that the 2 new clones that we analyse harbour one copy of replication-competent provirus, suggesting that latency is not caused by genetic mutations or deletions of the provirus. In conclusion, HIV-1 DNA-capture-seq is a powerful tool to characterize the HIV-1 provirus at a single nucleotide resolution and therefore might be useful for various experiments aiming for an HIV-1 cure.
Highlights
Human immunodeficiency virus type 1 (HIV-1) is an exogenous retrovirus that causes acquired immunodeficiency syndrome (AIDS)
We previously reported that DNA-capture sequencing for retroviruses, HIV-1 and HTLV-1 was useful for detecting retroviral sequences with high sensitivity[31]
We obtained more than 28,000 reads aligning with the provirus from a total of over 560,000 mapped reads, demonstrating that HIV-1 DNA-capture markedly increased the sensitivity of viral sequence detection by over 25,000 times (Fig. 1B)
Summary
Human immunodeficiency virus type 1 (HIV-1) is an exogenous retrovirus that causes acquired immunodeficiency syndrome (AIDS). The presence of HIV-1 as an integrated provirus in the infected cells allows the virus to avoid the effects of the anti-retroviral drugs and the host immune surveillance, making it extremely difficult to eradicate the virus completely. The HIV-1 reservoir in vivo is shaped by multiple factors, including tissue reservoirs, antiviral host immunity, heterogenous infected cell clones and viral www.nature.com/scientificreports/. To understand the mechanism of HIV-1 latency and latency-reversal in these cell models, it is essential to characterize the provirus in these cell lines, including the sequence from the beginning of the 5′LTR to the end of the 3′LTR, its structure and the integration site within the human genome. We have used this novel method to demonstrate that it can be applied for commonly used HIV-1-infected cell lines, in addition to newly established ones, for their characterization and reliable use in this research field
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