HIV-1 DNA-capture-seq is a useful tool for the comprehensive characterization of HIV-1 provirus

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.

Phylogenetic and Demographic Characterization of Directed HIV-1 Transmission Using Deep Sequences from High-Risk and General Population Cohorts/Groups in Uganda

“…Our sample comprised study participants who were ART naïve or on therapy with unsuppressed virus, and it is possible that transmissions from individuals who reached viral suppression soon after infection are under-represented in our data. Generation of additional HIV-1 deep sequence data based on sequencing protocols that amplify proviral HIV-1 DNA or sequence virus from low viraemic specimens [54,55] is a consideration for our future analyses. Fifth, transmissions including individuals with co-infections may have been excluded by the definition of source-recipient pairs.…”

Sequencing ultra-rare targets with compound nucleic acid cytometry

“…The final enrichment is the product of each round, allowing a ~6 million-fold enrichment over two rounds. This is ~200-fold higher than enrichments with the next best technology 8,15,23 . To demonstrate the approach, we use it to isolate and sequence single HIV genomes from infected individuals.…”

“…PCR methods recover only the amplified portion and miss information beyond primers 13,14 . Hybridization capture recovers information extending beyond probes, but can require hundreds of probes 15,16 ; this necessitates considerable prior information for probe design, which is often unavailable, especially when little information is known about the region of interest, such as in novel microbe or genetic lesion sequencing 17,18 .…”

HTLV-1 contains a high CG dinucleotide content and is susceptible to the host antiviral protein ZAP

“…Approximately 10 ng of each cDNA was used for linker ligation and library preparation. CAGE libraries were quantified by qPCR and size distribution was evaluated by TapeStation (Agilent Technologies) before sequencing in a NextSeq device (Illumina) as described previously [52]. NET-CAGE was performed as described previously [31].…”