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Determining 3’-termini and sequences of nascent single stranded viral DNA molecules during HIV-1 reverse transcription in infected cells

Research output: Contribution to journalArticle

Original languageEnglish
JournalJournal of visualized experiments : JoVE
Publication statusSubmitted - 2018

King's Authors

Abstract

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and host cell proteins on viral DNA synthesis. Here we address the lack of a cell based, high coverage and high resolution assay that is capable of defining retroviral reverse transcription intermediates within the physiological context of virus infection. The described method captures the 3’-termini of nascent complementary DNA (cDNA) molecules within HIV-1 infected cells at single nucleotide resolution. The protocol involves harvesting whole cell DNA, targeted enrichment of viral DNA via hybrid capture, adaptor ligation, size separation by gel purification, PCR amplification, deep sequencing and data analysis. A key step is the efficient and unbiased ligation of adaptor molecules to open 3’-DNA termini. Application of the described method determines the abundance of reverse transcripts of each particular length in a given sample. It also provides information about the (internal) sequence variation in reverse transcripts and thereby any potential mutations. In general, the assay is suitable for any questions relating to DNA 3’-extension provided that the template sequence is known.

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