The continual emergence of HIV strains that are resistant to currently approved anti-HIV drugs is an increasing threat to the effective treatment of HIV infection and control of the HIV/AIDS epidemic. Therefore, the discovery and development of new anti-HIV drugs with novel antiviral mechanisms and targets are urgently needed. The long-term objective of this application is to develop a prototype of a new class of anti-HIV drug that targets a virus-host cell protein-protein interaction that is crucial for viral infectivity. One example of this kind of interaction occurs between the HIV-1 Vif protein and the host cell cytidine deaminase, APOBEC3G. In an HIV-1 infected cell, Vif binds directly to APOBEC3G and prevents its incorporation into newly assembled virions. In the absence of Vif, APOBEC3G is packaged into HIV-1 virions and prevents the productive infection of newly infected cells due to extensive mutation of viral minus strand DNA during reverse transcription.
The specific aims of the proposed research project are to identify Vif- and/or AOBEC3G-derived peptides that will block the Vif-APOBEC3G interaction in vitro and then test these peptides for their ability to inhibit HIV-1 infectivity in non-permissive T cell lines and primary CD4+ T cells. Inhibitory peptides will be identified using an in vitro binding assay in which a library of 15-mer overlapping synthetic peptides, derived from the Vif and ABOBEC3G sequences, will be tested for their ability to block the Vif-APOBEC3G interaction. Inhibition of protein-protein interaction will be detected by SDS-PAGE followed by Western blotting. Peptides found to be capable of blocking the Vif-APOBEC3G interaction in vitro will be tested for their ability to inhibit HIV-1 infectivity in cell culture. H9/IIIB T cells, which are chronically infected with Vif-competent HIV-1, will be treated with inhibitory peptides attached to a cell-penetrating peptide (CPP) sequence. After incubation for 24 hours, the titer of infectious virus in CPP-peptide treated culture supernatants will be determined and compared to the infectious viral titer in supernatants of an untreated control culture. CPP-peptides that show anti-Vif activity in this transient assay will be tested for their ability to block HIV-1 growth in continuous replication assays in H9 and primary CD4+ T cell cultures. Demonstration that viral infectivity can be inhibited by cell-penetrating peptides that block HIV-1 Vif binding to APOBEC3G is the initial step in the process of developing a new class of potent peptide and/or peptidomimetic anti-HIV therapeutics. Lay summary. The HIV-1 Vif protein is absolutely required for the production of infectious HIV-1 virus. Drugs that are able to inhibit the normal function of Vif may have potent antiviral activity.
The specific aims of this proposal are to identify peptide inhibitors of Vif function and test their ability to interfere with the production of infectious virus in CD4+ lymphocytes, the primary target of HIV infection. This research is relevant to public health because it may lead to the development of new therapeutic agents for the treatment of HIV/AIDS. ? ? ? ? ? ?
| Donahue, John P; Vetter, Michael L; Mukhtar, Nizar A et al. (2008) The HIV-1 Vif PPLP motif is necessary for human APOBEC3G binding and degradation. Virology 377:49-53 |
