Method for identifying agents capable of inhibiting APOBEC3C activity in HIV-infected cells
The present invention provides methods of identifying agents that reduce a level of active APOBEC3C in a cell. The present invention provides agents that reduce a level of active APOBEC3C in a cell; and compositions comprising the agents. The present invention further provides methods of reducing the mutation rate of a lentivirus in a cell; and methods of reducing the emergence of drug-resistant strains of lentivirus. The present invention further provides methods for treating lentivirus infections.
1. An in vitro method of identifying an agent that reduces the level of active APOBEC3C in a cell, the method comprising:
a) contacting a cell that produces an APOBEC3C protein with a test agent, wherein the cell comprises a replication-competent Vif + human immunodeficiency virus (HIV) proviral construct, said virus being susceptible to the effects of APOBEC3C; and
b) determining i) the number of C→U mutations induced in the minus strand of virus expressed from the proviral construct; ii) the number of G→A mutations in a reverse transcript of the expressed virus; or iii) the number of C→T mutations in the provirus.
2. The method of claim 1 , wherein the APOBEC3C protein is a fusion protein comprising APOBEC3C and a fusion partner that provides a detectable signal.
3. The method of claim 2 , wherein the fusion partner is selected from a fluorescent protein, an enzyme, and an immunological tag.
4. The method of claim 1 , wherein said determining step comprises determining the number of C→U mutations induced in the minus strand of the HIV construct.
5. The method of claim 1 , wherein the APOBEC3C protein is encoded by an expression vector.
6. The method of claim 1 , wherein the cell is a mammalian cell.
7. The method of claim 1 , wherein said determining step comprises determining the number of G→A mutations in a reverse transcript of the expressed virus.
8. The method of claim 1 , wherein said determining step comprises determining the number of C→T mutations in the provirus.