Who else can stop HIV-1 reverse transcriptase?
Counteracting the HIV-1 epidemic requires more than one approach, this much has become clear over recent decades. To develop new treatment ideas we can often learn from the human body, which already has decent defence systems in place – though these are sometimes outsmarted by the virus.
Darja Pollpeter & Michael Malim
The paper in Nature Microbiology is here: http://go.nature.com/2hMIMcn
Part of our defences are the restriction factors, a collection of cell-encoded proteins that potently inhibit virus infections, act at the single-cell level, and contribute to innate immunity. The APOBEC3 proteins (initially APOBEC3G, A3G) were the first restriction factors found to be active against HIV-11. They were discovered through addressing how the HIV-1 Vif protein regulates infection, which it does by triggering proteasome-mediated destruction of APOBEC3 proteins.
A3G inhibits HIV-1 through: 1) DNA editing (namely cytidine deamination) that causes guanosine-to-adenosine hypermutation and catastrophic genetic damage to the virus, and 2) suppressed viral DNA synthesis, a process called reverse transcription. The former effect is well understood whereas the latter, though recognised since the 1990s, has remained enigmatic.
This paper was plinthed upon two issues we saw as central for illuminating how A3G suppresses the reverse transcriptase (RT) enzyme of HIV-1: first, the development of a new deep sequencing method for studying reverse transcription at single nucleotide resolution in virus infected cells, and, second, the careful affirmation of A3G’s previously reported interaction with HIV-1 RT2 using three distinct techniques.
Our main conclusions are that: 1) nascent deaminated HIV-1 DNA is recognised and processed by cellular DNA repair enzymes; 2) features of HIV-1 reverse transcription in infected cells are quite different in comparison to reconstituted in vitro reactions; 3) A3G binds to RT, rather than viral genomic RNA, to suppress RT’s biosynthetic function in a site- and sequence-independent manner; and 4) RT function can be regulated via direct interactions with cellular proteins (in this instance, A3G).
Going forwards, we will apply this method of analysing reverse transcription to probe the mechanisms of action of other restriction factors (e.g., TRIM5 or MX2), as well as different classes of RT inhibitors. We will also seek to define the interaction interfaces between RT and A3G, which may expose unrecognised vulnerabilities of the RT enzyme.
Finally, it is entertaining for us to recall some of the high and low points of this project:
- We learned the hard way just how biased ssDNA ligation can be, but ultimately succeeded in achieving the prerequisite unbiased linker ligation for the deep sequencing method. Thank you to Chun Kit Kwok and colleagues for their previous work3
- Developing a new method can take a long long time! Thanks to Jernej Ule for his cheerleading; assuring us that it is OK, and we are “almost there”
- Learning about FRET-FLIM. We were deeply impressed by the concordance of data from these assays with many years of assorted protein-protein interaction assays, and with the capability of intra-virion FRET-FLIM (Image 1). Thanks to Maddy
- Our gratitude to the insightful reviewer who suggested the A3G double mutant experiment (Fig 6d in the main article): this demonstrated segregation of the editing and RT suppression phenotypes, and, therefore, illustrates the contributions of both activities to full A3G anti-viral function
- Working with many terrific colleagues, including three outstanding intercalated BSc students – Jamil, Rupert and Sashika
- Scratching around late at night in the final stages of publication searching through years old electronic archives for uncropped immunoblot scans
The link to the full article ‘Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted anti-viral functions of APOBEC3G’ in Nature Microbiology is here: Pollpeter et al.
1. Sheehy, A.M., Gaddis, N.C., Choi, J.D. & Malim, M.H. Nature 418, 646-50 (2002).
2. Wang, X. et al. J Virol (2012).
3. Kwok, C.K., Ding, Y., Sherlock, M.E., Assmann, S.M. & Bevilacqua, P.C. Anal Biochem 435, 181-6 (2013).