Infectious Diseases

HIV research - mechanisms of resistance to HIV infection at the CD4+ T cell level; immunological marker of disease; gene therapy in HIV infection.

020 7188 2662

The major focus of my research is to understand how the composition and structure of retroviral genomic ribonucleoprotein particles (gRNPs), particularly that of HIV-1, regulates the different steps of their lifecycle.  Currently, we are focusing on how HIV-1 Gag translation is regulated and how this is similar and different to that of cellular transcripts.  Other research interests include how the composition of the HIV-1 gRNP regulates virion assembly and reverse transcription.     

020 7188 8272

The laboratory has combined efforts in virology, stem cell biology and molecular biology to elucidate the mechanism of site-specific integration of adeno-associated virus (AAV). In order to establish latency, this non-pathogenic, non-autonomous parvovirus has evolved to integrate its genome site-specifically into human chromosome 19. It is this unique mechanism that could potentially be exploited in the development of safe gene and cell based therapies. To date, gene therapy studies have mainly employed gamma-retroviral vectors, which establish persistence through integration in a largely random fashion. Inherent to this approach is that the chromosomal context and thus the expression of a transgene will vary between vector-transduced cells. In addition, while in differentiated cells the potential for insertional mutagenesis might be negligible, in cells with high proliferation potential, e.g. stem cells, this aspect becomes important. The mutagenesis potential has been documented by the emergence of leukemia as a result of retrovirally mediated gene therapy of X-linked SCID in an otherwise highly successful clinical trial. At the basis of the development of technologies that employ site-specific integration is a thorough understanding of the site and mechanism of integration. Our findings that the chromosomal signals required for human site-specific integration are conserved in the mouse genome in a region corresponding to the human target site opened up the possibility to study site-specific integration in mouse embryonic stem cells (ES). Stringent in vitro and in vivo assays developed for this cell type allowed for the read-out of potential effects of disruption of the genes that are embedded in the integration site. Detailed molecular analysis of the integration event in mouse ES cells as well as other human cell types demonstrated that site-specific integration of transgenes or wild type virus occurs predominantly in a gene called MBS85, and this without causing a functional disruption of the gene. This was confirmed using the various ES-cell specific in vitro and in vivo assays. In addition, we could demonstrate that transgene expression from this particular site remained robust throughout differentiation of mouse ES cells, a prerequisite for the development of gene transfer technologies in stem cells that incorporate the mechanism of site-specific integration. Future experiments will be designed to unravel the intricacies of the integration mechanism. We will address questions such as which proteins, besides the viral Rep protein, are involved and do genomic structures play a role? In addition, we will continue our efforts to optimize gene targeting strategies in human ES cells (and iPS cells) as preliminary experiments have shown that AAV-mediated transgene integration is feasible in the cell type that holds the promise to be used in future cell therapies. The study of human ES cell differentiation and derivation of mature tissue-specific cells will likely require genetic manipulation and in a setting in which stem cells will be expanded, differentiated and ultimately transplanted, prior knowledge of the site of integration and possible effects of the transgene cassette on the regulation of surrounding genes will be most advantageous. Finally, we are exploring new technologies that would allow for Rep-mediated site-specific transgene integration in hematopoietic stem cells that have proven to be resistant to AAV infection.

020 7188 8276

020 7188 3385

The role of iron in development of immunity, particularly in its role in driving inflammatory processes both in autoimmune diseases such as rheumatoid arthritis as well as infectious diseases.

020 7848 6044

020 7188 8275

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With the successful introduction of effective vaccines against the oncogenic types of human papillomavirruses the focus of my research has been retargetted against human immunodeficiency virus. In particular I am interested in the potential virological and host factors which may be involved in the delay of disease onset. To this end we are currently studying patients who have an extended disease-free period (long term non-progressors).

020 7188 1180

020 7188 1285

The last step in retroviral life cycle is the separation of the nascent viral particle from the infected cell. HIV-1 and other retroviruses encode a so-called Late Budding domain (L-domain) whose mutation induces the accumulation of immature virions that remain tethered to the plasma membrane by a membranous stalk. Work by our group and other laboratories has identified the cellular protein Tsg101 as the cellular partner that facilitates HIV-1 and Ebola virus budding thorough the interaction with a highly conserved aminoacid motif (PTAP) in the L-domain of these pathogens. Subsequently, it has been shown that other viruses that include HIV-2, HTLV and Lassa fever virus also require Tsg101 for particle budding, thus emphasizing the importance of this protein in human disease. Tsg101, the mammalian orthologue of the yeast protein Vps23, is one of the subunits of a 350 kd complex (endosomal sorting complex required for transport-I, ESCRT-I) which also includes Vps28 and Vps37. Tsg101 is a component of the class E pathway and is required both for the budding of viruses that encode PTAP type L-domains and the topologically equivalent process of vesicle budding into Multivesicular Bodies (MVB). Recent work by our laboratory and others has identified 19 human genes of the class E pathway that participate in budding of highly divergent enveloped viruses and, perhaps, MVB biogenesis. Importantly, a greater understanding of how HIV-1 viruses exploit the host cell to facilitate the budding process could provide opportunities for chemotherapeutic intervention using a completely novel class of antiviral compounds that would be active against a number of human pathogens including HIV-1, HIV-2, Ebola virus, HTLV, Lassa fever virus and essentially any virus that exploits the PTAP/Tsg101 interaction during budding. Our work with the ESCRT proteins has recently led to the discovery of and exciting link between retroviral budding and abscission, the last step in cell division. We have described that Tsg101 and ALIX are recruited to the midbody by Cep55 to mediate the separation of the daughter cells through a mechanism that is topologically similar to HIV-1 budding.

020 7188 7137

Our lab is interested in the broad fields of virus-host interactions and HIV/AIDS molecular pathogenesis. To this end, we employ assorted molecular-genetic, cultured cell, biochemical, structural, bioinformatic and cohort-based methods to study the biological principles that underpin HIV infection, replication and disease. Indeed, over the last twenty years, our research has spanned many facets of HIV replication, including viral RNA processing and nuclear export, virus particle assembly, the infection of non-proliferating cells and the role(s) played by the regulatory/accessory proteins of HIV.

Current work in the group addresses: 1) the activity of the cellular viral resistance factor, APOBEC3G, its antagonism by the virus-encoded Vif protein; 2) the impact of cellular RNA-rich microdomains, P-bodies and stress granules, in HIV replication and APOBEC3G biology; 3) the immediate cellular response to HIV infection and the interplay between alpha interferon and HIV infection; and 4) the link between viral RNA trafficking and HIV particle assembly.  

020 7188 0149

020 7188 0147

The study of adeno-associated virus (AAV) has brought our laboratory to the intersection of basic virological, genetic and biochemical studies with translational efforts, both in the gene transfer arena and the newly evolving stem cell discipline. In addition, we have committed considerable efforts to the establishment of an AAV vector core, with particular emphasis on scientific and organizational issues that allow for an efficient and appropriate process in providing recombinant AAV to a number of collaborating laboratories. The biology of AAV and AAV-based vectors Adeno-associated viruses (AAVs) have been studied since the early 1960s. In contrast to other human DNA viruses it has become clear that there is no significant correlation between the widespread infection by AAV throughout the population and any known disease entity. Studies throughout the past several decades have led to an emerging view that AAV might have evolved a possibly optimal relationship with its host through a unique life style that allows the virus to only replicate in cells that are infected by other viruses, which by themselves are deleterious to the host cell. Through this dependency AAV might have overcome an apparent challenge to viral life cycles in general: on one hand viruses depend on their respective hosts for replication, on the other, most viruses hurt the hosts through their replication to various degrees. Through its dependency, AAV will only replicate in cells that are affected by the consequences of helper virus infection. Thus, if our findings from tissue culture studies can be extrapolated to the human host, infection by AAV could indeed be viewed as beneficial to the host in that cells that are infected by adenovirus, herpes viruses and possibly papilloma viruses will die as a result of AAV replication. In light of this aspect it is no surprise that the AAVs appear widespread throughout the vertebrate kingdom. Molecular aspects. Possibly one of the most intriguing aspects of AAV biology is that it is the only known eukaryotic virus with the unique ability to integrate site-specifically into the human genome (Berns and Linden, 1995; Linden and Berns, 2000; Linden et al., 1996, Dutheil and Linden, 2006). On this background our laboratory has been active for a number of years in efforts to elucidate the molecular mechanisms underlying AAV2 site-specific integration and, related to this mechanism, DNA replication (Ward et al., 2003; Ward et al., 2001; Ward and Linden, 2000). We have approached these questions from different angles, including the genetic characterization of the human target locus for site-specific integration (Dutheil et al., 2000; Dutheil et al., 2004), the biochemical characterization of the AAV Rep proteins that are responsible for all aspects of the AAV life cycle, including site-specific integration (Smith et al., 1999; Yoon et al., 2001; Yoon-Robarts et al., 2004; Yoon-Robarts and Linden, 2003) and, more recently, the biophysical/structural basis for Rep action (James et al., 2004; James et al., 2003). These efforts have led us to be among the first to define the structure of SF3 helicases, and, as a result, to conclude that these proteins that are frequently found in viruses such as papilloma and polyoma viruses, in fact belong to the AAA+ proteins, a broad family of ATPases that are associated with a variety of functions ranging from membrane fusion, protein degradation and now also functions that are relating to several viral mechanisms. These include, but are not limited to DNA replication and genome packaging. Based on these findings we are now actively engaged in dissecting the biochemical and structural determinants underlying the molecular mechanisms supported by these viral AAA+ (vAAA+) proteins. Recombinant AAV vector core. During the past years we have spent considerable efforts in establishing a viral vector core that generates and purifies recombinant AAV vectors followed by stringent quality control assessments. At this point these vectors are distributed to a range of collaborators that are actively engaged in gene transfer experiments. Our current gene transfer collaborations include studies on pancreatic islet transplants, liver-mediated gene delivery for a number of monogenic diseases such as lysosomal storage diseases, a program that is aimed at the developmental aspects of kidney disease as well as several additional exploratory projects in neurology, neuroscience and Ophthalmology. The underlying philosophy to our efforts is to provide our strength to programs and projects that are founded on long-term and in-depth experience in the disease and animal models by our preclinical and clinical collaborators. In summary, our ongoing studies on the biology of viruses and AAV in particular has provided us with the opportunity to study unique viral and cellular mechanisms and to become part of the efforts in developing strategies that might ultimately become components of future gene and cell-based therapies.

020 7188 3162

020 7188 3385

Mammals have evolved a variety of innate cellular defences that block the replication of retroviruses. Primate lentiviruses in turn have developed mechanisms to evade these host restrictions, helping them to establish chronic infections that, in the case of Human Immunodeficiency Viruses (HIV), eventually lead to AIDS. My interest is in how the small accessory protein Vpu, encoded by HIV-1, overcomes such a cellular defence. We identified a human IFN-induced plasma membrane protein, Tetherin/CD317 that restricts retroviral particle release but can be overcome by Vpu expression. Tetherin/CD317 has an unusual topology and traffics between multiple cellular compartments. Understanding the cellular and molecular basis for Vpu antagonism of Tetherin-mediated restriction of HIV-1 release should indicate novel targets for antiretroviral drug development.