Assembly Cofactors of HIV-1

Lay summary

HIV-1 is the virus responsible for the worldwide AIDS pandemic. Although progression to AIDS in the majority of HIV-infected patients can be prevented with antiretroviral drugs, allowing people infected with HIV to life a normal life, there is no cure and such drugs must be taken for the rest of the patient's life. Additionally, HIV sometimes develops resistance to commonly used antiretrovirals and the drugs themselves can cause serious co-morbidities, making management of the condition more difficult. Further scientific research into the fundamental biological principles of HIV replication and infection will facilitate the development of new classes of drug, giving clinicians more options to manage HIV. Increasing our basic understanding of the virus lifecycle will also help work that aspires to develop life time cures for HIV infection.

In its replication cycle, HIV infects a cell and inserts a DNA copy of its RNA viral genome into the cellular genome. The cellular molecular machinery is then co-opted to produce all the viral proteins that are needed to form new infectious virus particles. These proteins plus the virus genome then move to the cell membrane where they assemble into new virus particles and bud away from the cell surface; these particles proceed to infect new cells thereby sustaining and spreading the infection. There are several lines of scientific evidence indicating that the process of HIV-1 assembly is dependent on interactions between virus components and various cellular cofactors. However, current knowledge of the identities of these critical cellular cofactors is far from complete. This provides us with an important opportunity to discover novel regulators of HIV replication, whose therapeutic inhibition has the potential to block virus growth.

Our proposed research will: i) use state-of-the-art technologies to identify novel cofactors of HIV assembly, and ii) define the molecular mechanisms that underlie their interactions with virus components and determine how these promote assembly of the virus. As these cofactors are important for the virus to replicate, their identification will provide the evidence-based platform from which to explore the development of new antiretroviral drugs.

1) Identifying the RNA(s) that bind HIV-1 Matrix (MA) in living cells.

We will use transfected cells to identify RNA(s) and define binding sites at nucleotide-resolution, using a modified iCLIP protocol coupled with next generation sequencing on the MiSeq platform. After demultiplexing and removal of PCR duplicates, sequences will be aligned to the human and HIV-1 genomes. Partitioning of identified RNA(s) to the cell membrane will be investigated using biochemical techniques such as membrane flotation coupled with RT-qPCR; and microscopy techniques, involving tagging RNAs of interest with MS2 loops for visualisation by fluorescent MS2 coat protein. We will use techniques such as si/shRNA and CRISPR to investigate the effect of knockdown of these RNAs on HIV-1 assembly and infectious virion production, as well as transfection of 5'-O-methylated antisense oligonucleotides to block the RNA-MA interaction.

2) Characterising the HIV-1 gRNA-bound proteome in living cells.

We will use transduced cells and an MS2-based RNA purification protocol, coupled with protein tagging with TMTsixplex labelling reagents and identification by mass spectrometry (Orbitrap Velos Pro) to identify proteins that interact with HIV-1 gRNA. Peptides will be identified and quantified using MaxQuant/Andromeda software. Proteins that specifically interact with gRNA will be knocked down to assess their effects on virus assembly. The mechanism(s) of action of positively-acting cofactors will be elucidated using a range of well-established molecular virology approaches. We will also perform pairwise comparisons of the gRNA-associated proteome in assembly permissive/non-permissive conditions to further enrich for assembly cofactors. Here, we will take advantage of the fact that murine cells are non-permissive for HIV assembly. Where functional differences between human and murine proteins are found, structure-function analyses will be undertaken to map the motifs responsible.

Assembly Cofactors of HIV-1: Impact Summary

This research aims to identify new HIV-1 assembly cofactors, leading to insights into the mechanism of HIV-1 assembly and infectious virion production, and opportunities to subvert this process. As this is an early-stage investigation into identifying novel cell-encoded factors, whilst the scientific impact made during the course of the funding period is likely to be considerable, the economic, health and societal benefits will be realised after the funding period ends.

HIV-1 is typically managed clinically with a lifetime course of highly-active antiretroviral treatment (HAART). This mixture of antiretroviral drugs aims to inhibit viral replication whilst minimising evolution of drug resistance. The latter aim is achieved by designing a regimen that targets multiple viral lifecycle stages. Nevertheless, viruses that have developed resistance to multiple drugs are frequently seen. When effective, this treatment limits replication of the virus but does not eradicate it, as HIV-1 enters a latent phase and cessation of treatment almost always results in a rapid rebound of viral load.

Cellular cofactors necessary for the HIV-1 lifecycle are potential drug targets, and identification of new assembly cofactors will suggest targets for interfering with HIV-1 assembly, which is currently not targeted pharmaceutically. Additionally, a better scientific understanding of assembly, and the ability to interfere in this process, may be one component in developing future novel treatment strategies. A very active avenue of current research is into 'shock and kill' strategies, whereby latent HIV-1 is reactivated in the presence of HAART, killing latently infected cells while preventing reinfection. Developing 'cures' for HIV-1 infection would be highly beneficial to patients, as well as providing significant economic benefit to the NHS where it is estimated that lifetime HAART costs ~£1m.

The potential beneficiaries of this research are pharmaceutical companies, clinicians treating HIV-1/AIDS, HIV-1-infected patients and scientific researchers. Private sector pharmaceutical companies are best placed to design and develop drugs that target identified cofactors, by utilising their extensive R&D expertise. As mentioned above, drugs interfering with assembly may be useful as part of improved HAART regimens, particularly where drug resistance is problematic, and research into assembly processes may be useful to develop reactivation-based therapies for augmenting currently envisioned cure treatments. There is worldwide demand for antiretroviral drugs, so pharmaceutical companies have the opportunity to benefit significantly economically. A wider variety of available antiviral drugs will also help clinicians in treating drug-resistant virus strains, improving the ability of the NHS to care for patients. HIV-1-infected patients have the potential to see the greatest health and quality-of-life benefits from this research. Capitalising on future scientific discoveries to develop protocols for curing HIV-1 infections, while ambitious, would also have massive health and wealth benefits.

The researcher working directly on the project (Dr Sobala) will develop further his laboratory skills in the molecular genetic, biochemical and cell biological analysis of virus replication. This combination of methodologies, with its significant inclusion of cutting-edge next generation sequencing, proteomic and bioinformatic approaches will provide Dr Sobala with the expertise to advance his scientific career. Similarly, greater familiarity with these technologies has the potential to benefit a number of our colleagues working on related projects; the publication of developed methods will benefit researchers worldwide; and dissemination of novel research tools will facilitate lab-based investigations of HIV-1 biology.