The APOBEC3 deoxycytidine deaminase family functions as host restriction factors 
that can block replication of Vif (virus infectivity factor) deficient HIV-1 
virions to differing degrees by deaminating cytosines to uracils in 
single-stranded (-)HIV-1 DNA. Upon replication of the (-)DNA to (+)DNA, the 
HIV-1 reverse transcriptase incorporates adenines opposite the uracils, thereby 
inducing C/G→T/A mutations that can functionally inactivate HIV-1. Although both 
APOBEC3F and APOBEC3G are expressed in cell types HIV-1 infects and are 
suppressed by Vif, there has been no prior biochemical analysis of APOBEC3F, in 
contrast to APOBEC3G. Using synthetic DNA substrates, we characterized APOBEC3F 
and found that similar to APOBEC3G; it is a processive enzyme and can deaminate 
at least two cytosines in a single enzyme-substrate encounter. However, APOBEC3F 
scanning movement is distinct from APOBEC3G, and relies on jumping rather than 
both jumping and sliding. APOBEC3F jumping movements were also different from 
APOBEC3G. The lack of sliding movement from APOBEC3F is due to an ¹⁹⁰NPM¹⁹² 
motif, since insertion of this motif into APOBEC3G decreases its sliding 
movements. The APOBEC3G NPM mutant induced significantly less mutations in 
comparison to wild-type APOBEC3G in an in vitro model HIV-1 replication assay 
and single-cycle infectivity assay, indicating that differences in DNA scanning 
were relevant to restriction of HIV-1. Conversely, mutation of the APOBEC3F 
¹⁹¹Pro to ¹⁹¹Gly enables APOBEC3F sliding movements to occur. Although APOBEC3F 
¹⁹⁰NGM¹⁹² could slide, the enzyme did not induce more mutagenesis than wild-type 
APOBEC3F, demonstrating that the unique jumping mechanism of APOBEC3F abrogates 
the influence of sliding on mutagenesis. Overall, we demonstrate key differences 
in the impact of APOBEC3F- and APOBEC3G-induced mutagenesis on HIV-1 that 
supports a model in which both the processive DNA scanning mechanism and 
preferred deamination motif (APOBEC3F, 5'TTC; APOBEC3G 5'CCC) influences the 
mutagenic and gene inactivation potential of an APOBEC3 enzyme.