APOBEC3C tandem domain proteins create super restriction factors against HIV-1

Humans encode proteins, called restriction factors, that inhibit replication of viruses like HIV-1. One family of antiviral proteins, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 ( APOBEC3, shortened to A3) acts by deaminating cytidines to uridines during the reverse transcription reaction of HIV-1. The A3 locus encodes seven genes, named A3A - A3H . These genes either have one or two cytidine deaminase domains and several of these A3s potently restrict HIV-1. A3C, which has only a single cytidine deaminase domain, however, inhibits HIV-1 only very weakly. We tested novel double domain protein combinations by genetically linking two A3C genes to make a synthetic tandem domain protein. This protein created a “super restriction factor” that had more potent antiviral activity than the native A3C protein, which correlated with increased packaging into virions. Furthermore, disabling one of the active sites of the synthetic tandem domain protein results in an even greater increase in the antiviral activity—recapitulating a similar evolution seen in A3F and A3G (double domain A3s that only use a single catalytically active deaminase domain). These A3C tandem domain proteins do not have an increase in mutational activity, but instead inhibit formation of reverse transcription products which correlates with their ability to form large higher order complexes in cells. Finally, the A3C-A3C super restriction factor largely escaped antagonism by the HIV-1 viral protein, Vif. Importance As a part of the innate immune system, humans encode proteins that inhibit viruses like HIV-1. These broadly acting antiviral proteins do not protect humans from viral infections because viruses encode proteins that antagonize the host antiviral proteins to evade the innate immune system. One such example of a host antiviral protein is APOBEC3C (A3C), which weakly inhibits HIV-1. Here, we show that we can improve the antiviral activity of A3C by duplicating the DNA sequence to create a synthetic tandem domain, and furthermore, are relatively resistant to the viral antagonist, Vif. Together, these data give insights about how nature has evolved a defense against viral pathogens like HIV.