The candidate's long-term career goal is to maintain a strong research program in an area of importance to the biomedical sciences. To date, the candidate's research has made a substantial contribution to the understanding of retrovirus RNA packaging. The salary support provided by an Independent Scientist Award (K02) would relieve the candidate of a significant amount of his teaching load as well as provide relief from administrative duties. This relief would ensure that 75 to 80% of the candidate's time is free for research. The additional research time made available by this Independent Scientist Award will have an immediate and positive effect on the candidate's research productivity. The present proposal extends research being conducted by the candidate to address the protein determinants involved in deltaretrovirus RNA packaging. The members of the deltaretroviruses, which include human T-cell leukemia viruses type 1 and 2 (HTLV-1 and HTLV-2) and bovine leukemia virus (BLV), are medically important because they cause human cancers that cannot be cured and have no vaccines for prevention. The deltaretroviruses replicate to low titers in their natural hosts and are poorly infectious in cell culture. Cocultivation is typically used to infect permissive host cells. Because of these difficulties, information regarding the molecular details of their life cycles, including viral RNA packaging, is limited. The objectives of this proposal are to map the determinants in the Gag polyprotein that are involved in deltaretrovirus RNA packaging. Two observations have been made with deltaretroviruses (specifically BLV) which indicate that the mechanism of RNA packaging is unique among retroviruses. First, the primary packaging signal is located exclusively in the gag gene and not the 5' untranslated leader region. Second, the matrix protein domain of Gag binds specifically to BLV RNA overlapping the primary packaging signal and mutation of basic amino acid residues in the matrix domain can influence RNA packaging efficiencies. In this proposal, genetic approaches will be employed using a newly developed virus-like particle (VLP) model system in order to identify the amino acid residues and/or protein domains in Gag that are involved in RNA packaging. This proposal has 3 specific aims.
Specific Aim 1 will determine if the BLV matrix (MA) and nucleocapsid (NC) protein domains of Gag act cooperatively in viral RNA packaging.
This aim will test the hypothesis that combination of MA and NC mutations can act in an additive manner to decrease viral RNA packaging efficiencies.
Specific Aim 2 will determine the interplay between RNA packaging defects caused by mutations in the BLV MA or NC and disruptions of the stable RNA stem-loop structures that represent the BLV packaging signal.
This aim will test the hypothesis that combination of MA or NC mutants with mutations that disrupt RNA stem-loop structures of the RNA packaging signal will aid in associating RNA packaging defects caused by MA or NC protein mutants to RNA stem-loop structures.
Specific Aim 3 will determine if both the HTLV MA and NC domains are involved in viral RNA packaging.
This aim will test the hypothesis that mutations in the HTLV MA and NC protein domains will correlate with RNA packaging defects. Completion of the aims in this proposal will provide further insight into HTLV/BLV RNA packaging, a group of retroviruses that package viral RNA by novel interactions with the Gag polyprotein precursor. This information should be useful in developing antiretroviral strategies to block deltaretrovirus replication and to prevent retrovirus-induced cancer.
