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A defining feature of retroviruses is significant diversity and enormous evolutionary potential based partly on error-prone reverse transcriptase enzymes (approximately one mutation per genome per replication cycle) (Chiu and Greene, 2008). Other sources of viral mutation come from cellular RNA polymerase (during transcription of the genome) and genomic recombination, as reverse transcriptase can utilize both copies of the viral RNA packaged into the viral core (Rambaut, 2004). This rapid evolution is a particular problem in the case of HIV-1. This virus has a particularly high replication rate and maintains a large population size, which together with an error prone polymerase, allow the virus to quickly evolve to evade immune responses and develop resistance to antiviral drugs. This variation also makes creating an effective HIV vaccine more difficult. APOBEC3 is also a source of viral mutagenesis. The degree to which APOBEC3 is responsible for viral evolution is unknown, but it was early noted that G-to- A mutations account for a large percentage of drug-resistance mutations (Berkhout and de Ronde, 2004). Although APOBEC3 can cause lethal mutagenesis based on missense and nonsense codon changes, if APOBEC3 is restricted by Vif-mediated degradation, limited mutations may occur. Thus, APOBEC3 may not only serve as an antiretroviral factor, but also as a potential proviral factor by aiding viral evolution.

Several important experiments have provided evidence that APOBEC3-mediated mutagenesis can in fact aid viral evolution and lead to drug resistance. The majority of these experiments have utilized in vitro transfection and infection systems with human APOBEC3G. Using a wide range of APOBEC3 expression levels in single round infections, Sadler and colleagues found a number of integrated HIV proviruses with only a single APOBEC3 mutation (at least within the viral gene examined). Although APOBEC3G levels affected virus infectivity, low- level, non-lethal mutagenesis occurred both in the presence and absence of Vif (Sadler, 2010). In silico analysis also supports a role for APOBEC3G in HIV-1 evolution, through simulation of multiple rounds of infection using prediction models to map mutations based on calculated APOBEC3G target probabilities. These models also assessed G-to-A nonsynonymous and synonymous mutations in present-day HIV compared to ancestral genomes, and showed a higher proportion of nonsynonymous mutations compared to random controls, implying some selection for APOBEC3G-mediated mutations (Jern, 2009).

In vitro experiments have also directly tested the ability for APOBEC3G to cause drug resistance through viral mutation. The use of the nucleoside analog reverse transcriptase inhibitor 3TC often leads to drug resistance in patients based on a single amino acid substitution in the catalytic site of RT. This mutation is at position 184, changing the normally occurring methionine in the conserved YMDD motif to isoleucine, valine, or threonine (Boucher, 1993). Monotherapy with this drug leads to drug resistance within a few weeks in HIV-infected patients (Sarafianos, 1999). The M184I mutation is specifically caused by mutation from AUG to AUA, which also lies within the preferred editing context of APOBEC3G (5’-GG-3’). Wild-type virus grown in vitro in the presence of IC90 levels of 3TC rapidly developed resistance via the M184I

mutation in cells engineered to express APOBEC3. The development of resistance did not occur in cells lacking APOBEC3 during the timeframe assessed, nor did the mutation become fixed in viral populations not grown with 3TC (Kim, 2010).

Natural variations in the Vif protein may largely affect APOBEC3 activity. A partially active or inactive Vif could profoundly impact viral sequence evolution within virally infected

individuals. Viruses isolated from HIV-infected patient samples show a large diversity in the vif coding region, with variation of up to 8% from the consensus sequence (Simon, 2005). When tested in vitro, these naturally occurring variants had variable activity against APOBEC3, with selected mutants failing to effectively block these enzymes. For example, Vif harboring a K22E mutation fails to restore HIV-1 infectivity in the presence of APOBEC3G (Simon, 2005).

Virus containing this mutant Vif was propagated in PBMCs in vitro for two weeks, and resulting viruses had over thirty times more mutations compared to wild-type viruses undergoing similar propagation. These mutations were characterized by a strong bias toward G-to-A mutations in the APOBEC3G editing context (Mulder, 2008). Over forty percent of recovered viral sequences also harbored the G-to-A mediated M184I 3TC-resistance mutation, compared to none in the wild-type viral population. However, all of the viruses harboring this drug resistance mutation were contained within replication-defective proviruses, based on premature stop codons also induced by APOBEC3. Infection with a viral quasi-species containing these resistance- containing replication-defective viruses together with wild-type sequences eventually led to drug- resistant, replication-competent viruses produced through viral recombination in vitro (Mulder, 2008). These experiments provide essential evidence that APOBEC3 can result in drug resistance, even in the complete absence of selective pressure by existing drug.

These experiments are corroborated by analysis of patient viral samples. A cohort of HIV-1 infected individuals failing antiretroviral therapy was compared to a cohort of treatment naive infected patients. The patients failing therapy typically have a higher rate of drug resistant viruses (Sethi, 2003). Virus was isolated from plasma, and protease, reverse transcriptase and Vif genes were amplified and sequenced. Mutation K22H in Vif was more frequently seen in viruses isolated from the cohort failing treatment. Of viruses harboring this Vif mutation, 72% had at least two drug-resistance-associated mutations in a GA or GG dinucleotide context, compared to 42% of all viruses with a wild-type K22 Vif (Fourati, 2010). Thus, a partially effective Vif is associated with APOBEC3-mediated drug resistance mutations in vivo.