The Interactions Of Dengue Virus RNA Dependent RNA Polymerase (NS5) With Other Viral And Host Factors.
Funder
National Health and Medical Research Council
Funding Amount
$170,165.00
Summary
Dengue fever is a mosquito-borne disease that is prevalent in tropical countries. It is estimated that 40% of the global population is at risk of dengue infection. Classical dengue fever is not life threatening. However, the more serious disease, dengue haemorrhagic fever-shock syndrome requires intensive medical attention to prevent fatality. A significant number of deaths are recorded each year especially in the underdeveloped countries. Dengue is periodically also a problem in northern Austra ....Dengue fever is a mosquito-borne disease that is prevalent in tropical countries. It is estimated that 40% of the global population is at risk of dengue infection. Classical dengue fever is not life threatening. However, the more serious disease, dengue haemorrhagic fever-shock syndrome requires intensive medical attention to prevent fatality. A significant number of deaths are recorded each year especially in the underdeveloped countries. Dengue is periodically also a problem in northern Australia. There is no cure for dengue fever. The present research aims to use a knowledge-based approach to develop novel antiviral strategies based on preventing the critical protein interactions required for the normal virus life cycle. Two of the most important proteins involved in dengue virus replication are called the NS3 and NS5 proteins. The protein-protein interaction (contact) that occurs between NS5 and NS3 is crucial for the replication of the virus. Little is known about this interaction at present, and the studies we propose will directly address this issue. We have previously shown that a 37 amino acid in the middle of NS5 contains a nuclear localisation signal that can target the normally cytoplasmic protein to the nucleus of the infected cell. What the function of this protein is in the nucleus is not known. We will use a technique called the yeast two-hybrid test to address the question of dengue virus protein interactions in the common bakers yeast. This method is very sensitive and powerful and will provide important insights that will contribute to the development of a rapid high-throughput test to screen the extensive extract collection from Australia's marine biodiversity, held by the Australian Institute of Marine Sciences, to discover suitable inhibitors of NS3-NS5 interaction.Read moreRead less
Regulation Of Nuclear Import Of Viral Oncoproteins And Transcription Factors By Protein-protein Interactions
Funder
National Health and Medical Research Council
Funding Amount
$650,383.00
Summary
The present application examines the controls that exerted over proteins that localize in the nucleus of eukaryotic cells. This relates relates integrally to cellular processes such as growth, development and oncogenesis. This research area is not represented elsewhere in Australia, and the particular experimental strategies to approach the problem, revolving around the use of special quantitative microscopic techniques are novel internationally. One part of the application seeks to examine tran ....The present application examines the controls that exerted over proteins that localize in the nucleus of eukaryotic cells. This relates relates integrally to cellular processes such as growth, development and oncogenesis. This research area is not represented elsewhere in Australia, and the particular experimental strategies to approach the problem, revolving around the use of special quantitative microscopic techniques are novel internationally. One part of the application seeks to examine transport within the cell of complexes of interacting proteins, rather than single proteins, under as close as possible to physiologically relevant conditions. This will be truly unique, and of great importance to our comprehension of eukaryotic cell function. This application examines particular types of negative control over protein nuclear localization. Since many proteins show such regulation, and in particular important proteins controlling cell growth and division, the results are fundamentally important to our understanding of how cells function in general. Further, this understanding may be applied in disease situations, such as viral-mediated oncogenesis. In the work we propose to do, viral proteins with functions relating to cancer will be examined in detail, as well as a cellular protein which is recognised by them - the tumor suppressor Rb. We intend to examine several viral oncoproteins which target Rb; one is a protein (E7) from the Human Papilloma Virus which has been frequently associated with cervical carcinomas and other cancers. Accordingly, the results may have direct application to viral-induced cancer, and our work may lead to understanding of the regulation of protein transport to the nucleus. This may thus afford a new approach at the pharmacological level to combat transformation.Read moreRead less
Approaches to combat AIDS and its causative agent, the human immunodeficiency virus HIV-1, have thus far proved ineffective. The proposed research program intends to investigate the nuclear import of two HIV-1 proteins which have central roles in HIV infection. We will apply our expertise in the area of the regulation of nuclear import of viral proteins, and build on our observations with respect to these proteins to attempt to establish the mechanistic basis of their nuclear import, and how thi ....Approaches to combat AIDS and its causative agent, the human immunodeficiency virus HIV-1, have thus far proved ineffective. The proposed research program intends to investigate the nuclear import of two HIV-1 proteins which have central roles in HIV infection. We will apply our expertise in the area of the regulation of nuclear import of viral proteins, and build on our observations with respect to these proteins to attempt to establish the mechanistic basis of their nuclear import, and how this differs from the conventional nuclear import pathways used by normal cellular proteins. We already have evidence that nuclear import of HIV-Tat is regulated in novel fashion by cellular factors, and intend, through determining its mechanistic basis, to be able to form the basis of a strategy to block this import pathway specifically, and thereby inhibit HIV replication. This may form the basis in the future of a new pharmaceutical approach to combat HIV-AIDS.Read moreRead less
NUCLEAR AND TRANSGOLGI TARGETING AND MEMBRANE INDUCTION BY DENGUE NS5 RNA-DEPENDENT RNA POLYMERASE INTERDOMAIN REGION
Funder
National Health and Medical Research Council
Funding Amount
$450,750.00
Summary
Dengue virus is the causative agent of a mosquito-borne disease, Dengue fever, relevant to northern Queensland, where antibodies from a previous infection can complex with virus of a different serotype in a subsequent infection, and cause a severe, potentially fatal form of the disease (Dengue haemorrhagic fever-Dengue shock syndrome). The present proposal seeks to further understanding of the role of the dengue RNA-dependent RNA polymerase NS5, which is essential for viral RNA replication, with ....Dengue virus is the causative agent of a mosquito-borne disease, Dengue fever, relevant to northern Queensland, where antibodies from a previous infection can complex with virus of a different serotype in a subsequent infection, and cause a severe, potentially fatal form of the disease (Dengue haemorrhagic fever-Dengue shock syndrome). The present proposal seeks to further understanding of the role of the dengue RNA-dependent RNA polymerase NS5, which is essential for viral RNA replication, within the viral infectious cycle. We intend to examine the subcellular targeting properties of a short central region (the interdomain) of NS5, which appears to play multiple roles in targeting to both the perinuclear Golgi-membranes and to the nucleus, as well as in inducing intracellular membranes derived from the Golgi which are the site of viral replication. We will determine how NS5 localisation-membrane induction may differ in insect and primate cells, and attempt to isolate binding partners of NS5 from the nucleus and Golgi compartment of insect and primate cells using various different approaches. Our studies should assist in understanding NS5's critical role in the Dengue infectious cycle, and contribute towards devising new anti-viral strategies such as vaccination and-or therapies targeted at the NS5 interdomain.Read moreRead less
Engineered Histones As DNA Carriers With Application In Therapeutic Gene Delivery
Funder
National Health and Medical Research Council
Funding Amount
$417,750.00
Summary
We intend to apply our knowledge of protein transport to the nucleus to enhance the delivery of DNA to target cells. This relates to the use of gene therapy to treat genetic defects such as inborn errors of metabolism, where a disease-causing lack-of-function mutation can be overcome by engineering cells within the organism which express, in the necessary quantities and in response to the appropriate regulatory signals, the particular component which is lacking. A limiting factor in gene therapy ....We intend to apply our knowledge of protein transport to the nucleus to enhance the delivery of DNA to target cells. This relates to the use of gene therapy to treat genetic defects such as inborn errors of metabolism, where a disease-causing lack-of-function mutation can be overcome by engineering cells within the organism which express, in the necessary quantities and in response to the appropriate regulatory signals, the particular component which is lacking. A limiting factor in gene therapy approaches is the low efficiency of nuclear uptake of introduced DNA, where it has been estimated that < 1% of the DNA taken up is actually expressed. Our proposal seeks to develop approaches to enhance non-viral-mediated gene delivery, in particular by optimising this critical, limiting step of the delivery of exogenous DNA to the nucleus. We intend to apply knowledge from studies of nuclear targeting and chromatin assembly to improve gene transfer technologies. We will build on our work showing that specific signals for nuclear import - nuclear targeting signals (NTSs) - can be used to enhance nuclear gene delivery and expression. Since DNA in the normal cellular context is in the form of chromatin - a specific complex with proteins such as histones - we intend to use reconstituted chromatin as the transfecting DNA, whereby histones engineered to include NTSs and other modular sequence elements will be used. Chromatin should not only enable NTSs and other sequence modules to be linked to the DNA but also protect against nuclease-mediated degradation prior to nuclear entry, condense the DNA to enable more efficient cellular-nuclear entry, and ensure expression of the transfected reporter gene by presenting it to the cell in a physiological context. Our approaches should contribute to bringing gene therapy closer to reality in the clinic.Read moreRead less