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Comprehensive identification of host factors involved in the early steps of HIV infection.

Periodic Reporting for period 1 - hRBP-virion (Comprehensive identification of host factors involved in the early steps of HIV infection.)

Periodo di rendicontazione: 2017-05-03 al 2019-05-02

RNA viruses represent a major health threat to humans and other living organisms. In particular, human immunodeficiency virus (HIV) is the second cause of death worldwide due to a single pathogen and currently 37 million people is infected. The high mutation rate of viral RNA genomes usually lead to rapid evolution, making difficult the development of effective antiviral treatments, vaccines and a final cure. Understanding how viruses interact with the host cell remain essential for the eventual discovery of complementary therapeutic strategies.
RNA has a central role in virus biology, yet viral genomes encode only a few proteins able to interact with RNA. Hence, viruses exploit host RNA-binding proteins (RBPs) to accomplish their biological cycle. Although poorly explored until now, many of these RBPs are promising targets for host-based antiviral intervention.
The participation of cellular RBPs in virus infection has been investigated for decades, mostly on a case-by-case basis. However, the universe of proteins enabled with RNA-binding activity has dramatically expanded in recent years. For these reasons, the complement of host RBPs involved in virus infection has remained largely unknown. To fill this gap, we aimed at (i) curating all the human RBPs that had previous links to infection using a tailored computational pipeline; and (ii) creating a novel system-wide method to study experimentally changes in the cellular RNA-binding proteome in response to infection, using a tractable and safe RNA virus model, called sindbis.
Once established in sindbis virus, our methods were applied to study HIV infection. After HIV entry into the host cell, the RNA genome is reversed transcribed into DNA and imported into the nucleus to be integrated into the chromosome. Recent evidence showed that reverse transcription occurs inside the viral capsid shell. This implies that all proteins required for early viral RNA metabolism must be already contained within the capsid and are taken up in the producer cell (summary figure A). The main goal of this project was to define the scope of host RBPs packed within HIV capsids and their biological significance on the early steps of infection.
Two major conclusions arise from this project: (i) hundreds of cellular RBPs are involved in virus infection and affect the infection outcome; and (ii) specific host RBPs are incorporated into HIV capsids and have essential roles in virus spread. Cellular RBPs are thus master regulators of viral replication and represent promising targets for host-based antiviral therapies.
To identify the repertoire of host RBPs involved in virus infection, we first assessed how many of the 1392 experimentally-determined human RBPs are linked to virus infection or immunity. We established the first virus-linked compendium of RBPs, which is comprised by 472 proteins. Many of these bind RNA through poorly understood unconventional mechanisms that remain to be discovered.
We then developed a novel proteome-wide method called comparative RNA-interactome capture to determine the repertoire of cellular RBPs that functionally respond to virus infection. This approach is based on freezing protein-RNA interactions in living cells by UV light irradiation. RNAs are specifically purified with oligo(dT) and the associated proteins are identified by quantitative mass spectrometry. We discovered that the activity of near 250 RBPs is altered upon sindbis virus infection and that these responses are critical for the virus to replicate or for the cell to counteract infection.
Once our RBP characterisation methods were established in a tractable safe pathogen, we extended them to HIV. To study the complement of RBPs assembled with the viral RNA into HIV particles, we developed in virio RNA-interactome capture. This technique builds on RNA-interactome capture and was applied to purified HIV particles produced in human CD4+ T cells (summary figure B). Over 90% of the RNA isolated from viral particles was HIV RNA, and we detected 73 cellular and 8 viral interacting proteins. While many of these RBPs were already linked to HIV, dozens were yet unknown to play a role in HIV infection. To test their function, we first ablated specific proteins by CRISPR/Cas9 gene editing and then measured virus infectivity in cell-to-cell spreading and virus-free infection assays. We observed that several of the HIV-incorporated RBPs are critical for infection, since knocking them out robustly reduce infectivity.
We are currently deciphering the molecular mechanisms of these RBPs in HIV infection. We have established a microscopy-based method to track fluorescently labelled HIV capsids inside a newly infected cell as a proxy for protein function. To unravel the mode of inclusion of host RBPs into viral particles, we compared protein abundance in the virion-incorporated RNA-binding proteome versus the cell proteome. We identified several proteins potentially loaded by active or passive mechanisms, and we are now validating specific cases by immunoprecipitation combined with proteomics and RNA sequencing.
These results have led so far to two peer-reviewed publications in open access, and an additional high-profile publication about the RBPs loaded into HIV particles is expected in the short-term:
Garcia-Moreno M et al. WIREs RNA, 2018. doi: 10.1002/wrna.1498
Garcia-Moreno M et al. Mol Cell, 2019. doi: 10.1016/j.molcel.2019.01.017
A free copy of the accepted manuscripts can be found in Pubmed central and Oxford University Research Archive.
These articles include novel protocols and large datasets with useful information in the fields of virus infection, immunity and RNA biology. The original high-throughput files have been deposited in public repositories (PRIDE: PXD009789 and GEO: GSE125182) and can be reused/reanalysed by the scientific community for specific research purposes.
This project combined next generation proteomics and data analysis with RNA, molecular biology and virology methods to study globally the participation of cellular RBPs in virus infection. These unprecedented interdisciplinary, systematic and comprehensive approaches offer alternative tools to find potential host-based antiviral targets.
Although pioneered in sindbis and HIV, these methods can be extended to other viruses with a considerable health and socioeconomic impact. Notably, we show that many of the host RBPs identified here as pro- or anti-viral can be targeted with drugs available commercially to inhibit virus infection. In addition, knock-out of specific RBPs incorporated into HIV virions strongly reduce virus spread. We anticipate that these cellular proteins have a therapeutic potential. We will exploit this possibility in follow-up collaborations with antiviral drug development laboratories. In the long-term, this could lead to patents and commercial exploitation that will benefit patients infected with HIV.
This project also benefits the scientific community by (i) implementing state-of-the-art methods in virology and RNA biology; (ii) providing new insights into viral replication and cellular antiviral defences; (iii) broadening our knowledge in specific host RBPs; and (iv) opening new avenues of research in HIV and virology fields. The project included an effective exchange of knowledge within a collaborative intra-European network and contributed to European excellence and competitiveness by enhancing world-class basic research.