Exploiting species-specific defects in virion assembly to identify cellular co-factors for HIV-1 replication

As adult, paediatric and adolescent HIV-1 infections continue to spread worldwide, the development of further anti-retroviral treatments remains of the utmost importance; for instance, there is no effective HIV-1 vaccine, and current front-line therapies targeting the viral reverse transcriptase and protease enzymes are compromised by compliance, toxicity and viral resistance concerns. Pursuing other HIV-1 (or human) proteins as anti-viral targets is therefore critical, but their rational selection requires a fundamental understanding of underlying biological principles. Work from many laboratories has contributed to the current understanding of virus assembly ? the process where nascent infectious particles are formed in infected cells in readiness for release and subsequent rounds of viral spread. One experimental approach for addressing key steps in the HIV-1 life cycle is to study them in contexts where they are non-functional, e.g., in cells derived from non-human species. Indeed, it has been clearly established that HIV-1 assembly is markedly inefficient in mouse cells. Here, we will exploit this species-specific defect together with complementing genetic and proteomic methodologies to identify novel cellular co-factors important for the assembly of infectious HIV-1 particles. The underlying basis for function will be addressed using contemporary biochemical, molecular genetic and imaging experiments, with the expectation, for instance, that we will gain novel insight into how the movement of RNA molecules within cells can impact protein function, and how these processes may be impeded to achieve viral inhibition. Lastly, the co-factors that are identified will be applied to cultured mouse cell systems that aim to recapitulate spreading HIV-1 infection, a feat not yet accomplished in the field: achieving this is particularly important as it would pave the way for establishing a living mouse model for HIV-1 infection, which would have tremendous utility for, inter alia, studies of HIV/AIDS pathogenesis, vaccines and therapeutics. The Public Relations Dept of KCL is primarily responsible for communicating our work to the public; and a ?Directory of Experts? is maintained that enables members of the public to identify academics willing to discuss specialist areas. For instance, MHM has given numerous interviews to the press, has appeared on television and has advised the BBC on the content of AIDS-related programming. He is also a Section Editor of a public access journal, PLoS Pathogens, has given overview seminars on the value of scientific research at Community Liaison Meetings (e.g., Cafe Scientifique), and helps organise Conferences attended by non-scientific members of the HIV/AIDS community.

The assembly of infectious HIV-1 particles requires the co-ordinated activities of viral gene products, particularly Gag and RNA, and cellular co-factors and pathways. Unlike human cells, rodent cells are notably deficient in supporting effective HIV-1 core assembly. By understanding the principles for such species-specific replication barriers, investigators have been able to learn a great deal about the interactions of HIV-1 with infected host cells. Indeed, previous work has established that the murine cell assembly deficiency is rescued by human co-factors provided in trans, by manipulating the membrane binding capabilities of the viral Gag protein, or by changing the nuclear export pathway used by viral RNA. Here, we outline an inter-disciplinary and collaborative project that aims to identify human co-factors important for HIV-1 assembly, and to define the mechanistic basis for their activities. First, we will conduct gain-of-function ?genetic? screens in murine cells to identify human cDNAs that can promote HIV-1 assembly; in parallel, and in collaboration with the Frankel and Sundquist groups, we will perform comparative proteomic screens for factors that interact with HIV-1 Gag, Rev or RNA in a manner that correlates with effective assembly. Second, subsequent cell biological, molecular genetic and biochemical assays in both murine- and human-based assay systems will address the molecular mechanism(s) that underlie the rescue of assembly, focusing on question such as interactions with viral and cellular RNAs and proteins, sub-cellular localisation, intra-cellular trafficking and viral infectivity. Importantly, our preliminary data validate the utility of the genetic strategy in that the human orthologues of the SR proteins, SRp40 and SRp55, as well as the nuclear export receptor, CRM1, can each stimulate assembly in murine cells; the mechanisms of action differ, however, in that SR proteins increase Gag expression whereas CRM1 has a dramatic effect on the efficiency of assembly itself. Finally, validated assembly co-factors will be co-expressed with established human co-factors (e.g., CD4, CYCT1) in attempts to create murine cells capable of supporting spreading HIV-1 replication. Though beyond the scope of this work, developing a living mouse model for HIV/AIDS infection has the potential to be highly instructive for work on pathogenesis, immune responses, transmission, vaccines, etc. In sum, we aim to identify novel interactions between HIV-1 and infected host cells that regulate virus assembly, with the goal of better understanding general principles of virus replication and human cell biology, as well as defining rational targets for future therapeutic exploitation.