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Featured Projects

Featured Projects for 2007-2008

Featured Projects for 2006-2007


2007-2008

  • Physiological role of APOBEC3 • Yoshiyuki Hakata, Ph.D
    The cytidine deaminase APOBEC3G is encapsidated in D vif HIV-1 during virus assembly and blocks its replication after viral infection. The mouse ortholog is also active against HIV. APOBEC3G family members evolved prior to HIV and recent findings suggest that they may have been a means of blocking the deleterious transposition of endogenous genetic elements in somatic or germ cells. It is also possible that APOBEC3G plays additional roles in the immune system, either related to its role as a DNA mutator or potentially as an RNA editor. The APOBEC3G gene is under tight transcriptional control and characterization of its expression may provide clues as to its function. I propose to study the physiological role of APOBEC3G in Knock-out mice. They will provide important information regarding the role of APOBEC3G, which ultimately will help to develop antiviral therapeutics targeting APOBEC3G. The specific aims of the project are to determine whether APOBEC3G regulates lymphocyte development, whether the knock-out mice have immune response defects, whether retrotransposon transposition is increased in APOBEC3G null mice, and whether the mice are susceptible to retroviral infection.
    Yoshiyuki Hakata and Nathaniel Landau. (2006) Reversed Organization of Mouse and Human apobec3 cytidine deaminase Domains. J. Biol. Chem.281 36624-36631
  • Metabolic shift of mycobacterium tuberculosis during infection and its regulation • Lanbo Shi, PhD
    M. tuberculosis is a very successful air-borne pathogen that infects one third of the world population. Several lines of evidence show that the persisting bacilli in its dormant state are probably in a very slow growth rate or growth arrest. Current anti-TB drugs are not effective against dormant bacilli because they target bacterial growth. There is therefore, an urgent need for anti-TB drugs that target persistent bacilli efficiently. This objective is particularly important for the treatment of HIV positive individuals co-infected with TB since TB is the leading cause of death in HIV positive population. In a murine model of M. persistence, we have shown by transcriptional profiling that the transition to growth arrest is accompanied by a shift in the bacterial physiological/metabolic state. This is revealed by changes in M. tuberculosis gene levels involved in the central metabolic pathway. The current proposal aims to dissect the role and regulatory mechanisms of several central metabolic pathways that are found to be up-regulated during transition to growth arrest. Mutant strains fro these genes will be obtained from the TARGET project at John Hopkins University and will be tested for ability (i) to establish chronic infection in mice; (ii) to survive in models of growth arrest in vitro. It is expected that some mutants will show decreased ability to persist in mice or decreased survival rate after treatment in vitro. The study of metabolic regulation in M. tuberculosis will be centered on the factors known to have a role in the adaptation to the extra cellular environment and stress, such as the alternative sigma factors : SigE and SigH. Mice will be infected with mutant strains for SigE and SigH and potential transcriptional target genes will be studied by real time RT-PCR in the lungs or in cultures treated by NO or hypoxia. Defining the role of central metabolic pathways and the underlying regulatory mechanisms have profound implications for the design of novel anti-TB therapeutics.
  • The Immunoglobulin gene usage for anti-V3 monoclonal antibodies • Mirek Gorny, MD, PhD
    These studies are designed toexamine the relationship between immunoglobulin (Ig) variable region gene usage of heavy chains (VH) of human anti-V3 monoclonal antibodies (mAbs) and their functional activity. The human anti-V3 mAbs generated from HIv-1 infected individuals have the capacity to neutralize primary isolates in in-vitro assays and may play important role in the protection against HIV-1 infection if they are induced by vaccine in the healthy volunteers. These mAbs display diversity in the range of their cross reactivity and neutralization that may be related to their immunogenetic background. The most useful approach to study the genetic background of mAbs is the analysis of the VH germline gene usage for the variable fragment of the heavy chain. Our preliminary results showed significantly increased use of the VH5 gene family in anti-V3 human mAbs (P=0.0048) as compared to the normal repertoire of VH gene families. The VH5 derived anti-V3 mAbs demonstrated broader cross-reactivity with the V3-fusion proteins (V3-FP) from clade A, B, and C than the non-VH5-derived mAbs suggesting that particular germline gene usage correlates with highly functional antibody. The proposed work will examine the relationship between VH gene family usage of 42 human anti-V3 mAbs developed in our laboratory, their cross reactivity, and neutralization of pseudotyped viruses. VH gene usage will be determined by PCR sequencing the variable fragment of the heavy chain of particular mAb and comparing its homology with repertoire of germline genes using the International ImMunoGene Tics information system. Cross reactivity will be analyzed by testing mAb binding to V3-FPs and V3 peptides representing the V3 sequence of primary isolates from clade A, B, and C. Neutralizing activity will be tested against viruses pseudotyped with Env proteins derived from primary isolates of clade A, B, and C. Two specific aims are targeted for this one-year pilot project:
    • Aim 1. To determine the VH gene usage for the anti-V3 mAbs compared to mAbs specific for other epitopes of the envelope proteins of HIV-1.
    • Aim 2: To determine the correlation between VH gene usage and the neutralizing activity and cross-reactivity of anti-V3 mAbs.
    The results of this work will be important for both basic studies of the immune system and for understanding the immunogenetics of the antibody production which would have critical implications for HIV vaccine development.

2006-2007

  • Effect of HIV Protease Inhibitors on the Pre-Erythrocytic Stages of Plasmodium • Charlotte Hobbs, MD
    Sub-Saharan Africa is where the HIV/AIDS epidemic intersects with malaria, one of the leading causes of infection-related death in children. The incidence of malaria is increasing world-wide in part due to drug resistance on the part of the parasite. In the laboratory, protease inhibitors have been shown to kill the erythrocytic stages of Plasmodium and are therefore a potential new class of anti-malarial drugs. Because of the overlap between malaria and HIV in sub-Saharan Africa, we propose to determine if the protease inhibitors (PIs) used to treat HIV infection would also be effective against Plasmodium. If this proves to be the case, this could determine the first-line therapy for HIV in this part of the world.
    Our data demonstrate that there is an anti-Plasmodial effect of certain HIV PIs on the pre-erythrocytic forms of the parasite. The drugs with the most potent effect were saquinavir and lopinavir at levels that approach those achieved in clinical practice. Atazanavir, amprenavir and nelfinavir, at levels that approach those achieved in clinical practice, did not have an effect. These data parallel previously published data evaluating the inhibitory effects of these drugs on the erythrocytic stages and suggest that the same protease inhibitors may have an effect on two different life cycle stages of the parasite. HIV protease inhibitors would be unique as a class of drugs that affects both the pre-erythrocytic and erythrocytic stages of Plasmodium and that already are used in clinical practice. However, further work is needed to elucidate minimum levels at which these effects may be seen in vivo.
  • Consequences of HIV-1 Cellular Co-infection • David N. Levy, Ph.D.
    Historically, our thinking about HIV-1 infection at the cellular level has comprised only one conceptual template: a single integrated provirus within each infected cell. However, recent studies have demonstrated that the typical in vivo infected cell contains >1 integrated provirus. The consequences of prevalent coinfection include rapid genetic recombination and altered virus replication and pathogenesis. In addition, other studies have demonstrated that for every successful integration of a cDNA provirus, there are 10-100 other cDNA proviruses which fail to integrate. Thus HIV-1 infection can be reasonably described as massive coinfection with low probability of successful integration. Recent studies have shown that this unintegrated viral DNA is transcriptionaly active and generates a restricted set of HIV-1 early proteins. In order to fully describe HIV-1 coinfection we will need to account for both the integrated and unintegrated viral DNA.
    This project employs in vitro molecular and virological techniques that are unique to this laboratory in order to study the influence of multiple infections on the death of infected cells, the production of new virus and the switch from early to late gene expression. In the first half of the funding period of this grant, we have developed new tools to study these outcomes and have initiated investigations. At this stage in our research, particular emphasis has been placed on understanding the role of unintegrated DNA (uDNA).
    Our study of the switch from early to late gene expression tentatively shows that the current understanding of some temporal aspects may need modification. Our studies into HIV-1 cellular coinfection have revealed that unintegrated DNA can influence HIV-1 gene expression. Each of the aims in the funded grant proposal will be influenced by these findings. Thus our understanding of the true nature of HIV-1 coinfection continues to expand.
  • Proteasome-Regulated Genes and Mycobacterium tuberculosis Pathogenesis • K. Heran Darwin, PhD
    HIV/AIDS is the leading cause of death of adults by an infectious agent. A large proportion of AIDS-associated deaths are due to tuberculosis following immunosuppression by HIV. Thus, reducing the prevalence of Mtb infections among a growing population of individuals immunocompromised by HIV is essential. An effective vaccine to Mtb has yet to be developed and current chemotherapies are prolonged and often toxic. Targeting the proteasome and genes regulated by the proteasome may be an effective strategy for treating not only tuberculosis, but also diseases caused by other mycobacterial species such as M. avium and M. leprae.
    From our experiments to date, it appears that the Mtb proteasome regulates at least one gene that impacts the progression of disease in mice. This will require further investigation. In addition, we are examining additional genes that are dependent on the proteasome for expression. We will also test mutants of these genes in mice. Finally, preliminary data show that an mpa mutant is attenuated in mice that lack IFN?r. This suggests that Mpa is important for defending against other host factors independent of iNOS, or that the mpa mutant has other (metabolic) defects that affect growth in vivo. Future experiments will test these hypotheses.
    Richard A. Festa, Michael J. Pearce, and K. Heran Darwin - Characterization of the Proteasome Accessory Factor (paf) Operon in Mycobacterium tuberculosis JOURNAL OF BACTERIOLOGY, Apr. 2007, p. 3044–3050 Vol. 189, No. 8
  • Control of Glucocorticoid Receptor Protein Stability by HIV gag through TSG101 • Michael Garabedian, Ph.D.
    We have previously shown that TSG101 binds the glucocorticoid receptor (GR) and stabilizes the receptor by impeding its degradation by the proteasome, which provides a novel means of controlling receptor levels post transcriptionally. Given that that HIV binds TSG101 and utilizes it for viral budding, we would predict that the steady state GR levels would be reduced in HIV infected macrophages or T-cells producing virus, by virtue of HIV sequestering TSG101 away from GR, thereby rendering TSG101 rate-limiting in the cell. In support for this idea, several studies have shown that chronically HIV infected T-cell line contains fewer GRs and are resistant glucocorticoid-induced cell death. This may represent a novel strategy used by the virus to circumvent host defense mechanisms controlled by GR. Whether the reduction in GR levels in HIV-infected cells is dependent upon TSG101 remains to be explored. (Ismaili, N., et al. J Biol Chem, 2005. 280: p.11120)
    We have now demonstrate that overexpression HIV-1 Gag-pol protein in leads to a reduction in GR levels, thus mimicking the effect of TSG101 depletion. Thus, HIV-1 has the capacity to decrease GR levels, most likely through TSG101 appropriation by Gag.
    We have recently shown, in collaboration with Derya Unutmaz’s lab, that human T-cells infected with HIV exhibit a faster migrating GR immunoreactive species. We are now in process of examining whether HIV gag and TSG101 plays a role in promoting this unique receptor species.