The Role of CKII-COP9 Interactions in HIV-1 Infection
Roland Wolkowicz, San Diego State University, San Diego
Basic Biomedical Sciences
Innovative, Developmental, Exploratory Award (IDEA)
Despite the progress achieved in the development of drugs against HIV, AIDS is still a devastating disease. The virus exploits cellular machinery in order to establish an effective infection. A better understanding of these viral-host interactions is essential in order to develop an effective anti-viral therapy. In this regard, we propose to study a cellular protein complex, the COP9 signalosome (CSN), composed of eight subunits: CSN1 through 8. Though the CSN complex has been shown to be crucial for many important biological events in the cell, very little is known about its involvement in HIV infection.
Our preliminary data based on a biological screen aimed at finding novel anti HIV-1 agents implicate CKII, and possibly CSN3, in the establishment of efficient HIV-1 infection. CKII is a cellular enzyme known to phosphorylate a wide range of proteins. Some studies suggest that CKII phosphorylates CSN2 and CSN7 in red blood cells, and two of the viral proteins - protease and reverse transcriptase, enhancing their activities. Although CKII has been coupled to CSN in previous studies, the effect of CKII on HIV-1 infection through its interaction with the CSN subunits remains elusive.
We hypothesize that CKII plays an important role in efficient HIV-1 infection through its effects on the CSN. This is probably achieved by the modification of one or several CSN subunits, particularly CSN3, through phosphorylation. We thus propose a detailed study on the role of the CSN, particularly CSN3 and its phosphorylation by CKII, in HIV-1 infection and pathogenesis.
The specific aims of this proposal are: a) Discern the phosphorylation potential of the CSN, particularly the CSN3 subunit, by CKII in T-cells, b) Elucidate the effects of the peptide rescued from our biological screen, which shows anti-HIV-1 effects, on the phosphorylation of the CSN, particularly CSN3, in the presence or absence of HIV infection and c) Determine how CSN3 influences biological processes known to be implicated in the HIV-1 life cycle in the presence or absence of infection and/or peptide.
For that purpose, we will determine the physical interactions between HIV proteins, CKII, the CSN or CSN individual subunits through biochemical pull-down experiments. Additional assays will be performed to characterize the ability of CKII to phosphorylate CSN subunits, particularly CSN3, purified from T-cells. Lastly, we will use specific antibodies to characterize the mechanism of action of the rescued peptide by analyzing intracellular molecules shown to be crucial for the establishment of HIV infection, which may be affected by the CSN, CKII and our peptide.
This proposal will reveal the intriguing roles of CKII in connection to the CSN and its phosphorylation for the establishment of productive HIV-1 infection, consequently, shedding light on the roles of the CSN subunits in the biological processes required for HIV-1 infection. CSN subunits, such as CSN3 and its specific sites phosphorylated by CKII, could become novel targets for AIDS therapy. This proposal clearly falls within the Basic Biomedical Sciences, particularly Molecular Virology, aimed at understanding HIV-1 exploitation of the cellular machinery for the establishment of effective infection.