Final Report Summary - MDIIHIVIA (Mechanisms of deaminase-independent inhibition of HIV infection by APOBEC3 proteins) The prevention of HIV infection and spread presents one of modern days biggest medical challenges. While anti-HIV drug design and availability has advanced substantially over the last few years, current treatments still do not cure infection, are prone to severe side effects, lead to drug resistance and are expensive. Therefore there is a need for additional interventions and drug targets, which requires a deeper understanding of virus-host interactions. This project is designed to understand a specific aspect of the human cellular innate immune response to HIV-1 infection. More specifically, we are investigating a human protein named APOBEC3G (A3G), which is known to have the ability to potently block HIV infection, but is usually counteracted by a viral protein, named vif. Nevertheless, understanding how A3G can control HIV in the absence of vif, provides ideas and potential targets for antiretroviral therapies and can help predict the effects of such drugs on the virus. It has recently emerged that A3G uses a dual mechanism for its antiviral activities, one that relies on its DNA editing activity and one that is independent of the enzymatic function. Indeed, in the time between proposal submission and the beginning of this fellowship, we provided evidence for editing-independent inhibition by endogenous A3G levels in primary CD4+ T cells, the natural target of HIV infection. These results were published in the Journal of Virology (2012, Gillick, Pollpeter, et al.). However the editing-independent mechanism is not well understood and is the main subject of the presented research. Overall, the project has advanced in line with the proposed work plan. Substantial progress has been made in three areas addressed in the original proposal, which is the 1) The characterization of the interaction between A3G and the HIV-1 reverse transcription enzyme. 2) The assessment of reverse transcription products in the presence and absence of APOBEC3 and 3) The development of a strategy to create a single-stranded viral DNA specific sequencing library.Using multiple different assays, work under this fellowship has provided evidence that the new-found interaction between A3G and HIV RT is a direct protein-protein interaction, which is likely facilitated but not bridged by RNA. This was shown using a) Co-immunoprecipitation assay coupled (or not) to RNAase treatment. b) FRET-FLIM (Fluorescence lifetime imaging microscopy) of proteins expressed in HeLa cells (in collaboration with Dr Maddy Parsons at the Nikon Imaging Center at King’s College London). c) FRET-FLIM on virions packaging A3G. d) By surface plasmon resonance (SPR) using purified proteins (in collaboration with Prof James McDonnell at the Center for Biomolecular Spectroscopy). Of particular interest is the analysis of protein packaged into HIV virions via FRET FLIM, which to our knowledge was not previously described in the literature. The development of this assay system, which combines different known methods, provided evidence that the A3G-RT interaction is relevant in virions, the site of action of A3G mediated inhibition. It can also be applied to other questions about protein –protein contacts within HIV or other virions. Further, detailed mapping studies on the interaction sites on A3G allowed us to create single amino acid point mutation that disrupt interaction with HIV RT. Results of assays evaluating the antiviral activity of such mutants demonstrate that interaction with RT is crucial for A3G’s ability to counteract HIV reverse transcription. The assessment of reverse transcription products in the presence and absence of A3G was first addressed in reconstituted primer extension assays, which have been performed under a variety of conditions as proposed in the fellowship application. Most notably, the results demonstrate that A3G induced reverse transcription pause sites pause sites are dependent on A3G-RNA template interaction. This was demonstrated in three different ways: a) Using A3G RNA binding mutants (A3G R24A), which lost its ability to interfere with reverse transcription in vitro. b) By employing RNA competition assays, in which an A3G RNA substrate, the cellular 7SL RNA, was titrated into the described reconstitution assays. The observation that the presence of excess 7SL RNA can alleviate A3G mediated RT inhibition suggest that A3G-template interaction is required for inhibition. c) Assays in which the order of addition of each component of the reconstituted system is varied also support A3G-RNA binding to be a prerequisite for efficient inhibition. The second major aspect was the evaluation of A3G induced pause sites in reverse transcribed complementary DNA (cDNA) in vivo, starting with reverse transcription in virions and then infected cells. High throughput sequencing libraries of reverse transcription intermediates as well as controls have been constructed and were run on an Illumina MiSeq platform. In depth analysis of the resulting data revealed a bias introduced by the ligation step in the original library creation strategy. This bias was of technical rather than biological origin. In order to eliminate this bias, several rounds of evaluation and optimization were required in this method development. The problem was solved by applying two different ligation strategies. To our knowledge it is the first report on high throughput sequencing of viral single stranded DNA, which allows precise 3’ mapping and our description of findings and necessary modification will provide very valuable information for other scientist seeking to observe single stranded DNA species at a deep sequencing level.In addition to the proposed research, the recruited researcher applied her expertise to studies on another HIV restriction factor, termed SAMHD1. The main results from this research describe the nuclear import of SAMHD1 and its sensitivity to the retroviral accessory protein Vpx. This work was published in Retrovirology (2014, Schaller, Pollpeter et al.). To summarize, our results demonstrate that both A3G’s ability to interact with RNA and its new-found ability to interact with the HIV enzyme reverse transcriptase are underlying the interference of A3G with the HIV life cycle. In addition, we developed two new assay systems, including a strategy to perform deep sequencing of HIV reverse transcription intermediates. The sequencing assay can now be employed to investigate the consequences of other restriction and resistance factors as well as antiretroviral drugs and immune-stimulants, such as cytokines, on reverse transcription. Our results will be lead to a peer-reviewed article in the near future.
