Comparison of G-to-A Mutation Frequencies Induced by APOBEC3 Proteins in H9 Cells and Peripheral Blood Mononuclear Cells in the Context of Impaired Processivities of Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase Variants

Abstract

APOBEC3 proteins can inhibit human immunodeficiency virus type 1 (HIV-1) replication by inducing G-to-A mutations in newly synthesized viral DNA. However, HIV-1 is able to overcome the antiretroviral activity of some of those enzymes by the viral protein Vif. We investigated the impact of different processivities of HIV-1 reverse transcriptases (RT) on the frequencies of G-to-A mutations introduced by APOBEC3 proteins. Wild-type RT or the M184V, M184I, and K65R؉M184V RT variants, which are increasingly impaired in their processivities, were used in the context of a vif-deficient molecular HIV-1 clone to infect H9 cells and peripheral blood mononuclear cells (PBMCs). After two rounds of infection, a part of the HIV-1 env gene was amplified, cloned, and sequenced. The M184V mutation led to G-to-A mutation frequencies that were similar to those of the wild-type RT in H9 cells and PBMCs. The frequencies of G-to-A mutations were increased after infection with the M184I virus variant. This effect was augmented when using the K65R؉M184V virus variant (P < 0.001). Overall, the G-to-A mutation frequencies were lower in PBMCs than in H9 cells. Remarkably, 38% ؎ 18% (mean ؎ standard deviation) of the env clones derived from PBMCs did not harbor any G-to-A mutation. This was rarely observed in H9 cells (3% ؎ 3%). Our data imply that the frequency of G-to-A mutations induced by APOBEC3 proteins can be influenced by the processivities of HIV-1 RT variants. The high number of nonmutated clones derived from PBMCs leads to several hypotheses, including that additional antiretroviral mechanisms of APOBEC3 proteins other than their deamination activity might be involved in the inhibition of vif-deficient viruses.

Vif Substitution Enables Persistent Infection of Pig-Tailed Macaques by Human Immunodeficiency Virus Type 1

“…There is also evidence indicating that catalytically inactive A3G still blocks HIV-1 replication (33,62), although other studies show that deaminase activity is essential (61,77). Finally, A3G editing activity in T cells and G-to-A mutations in viral cDNAs from vif-deleted HIV-1-infected PBMC are lower than expected (49,85), further suggesting hypermutation may have a limited role in inhibiting viral replication.…”

IFN-α Induces APOBEC3G, F, and A in Immature Dendritic Cells and Limits HIV-1 Spread to CD4+ T Cells

“…Primers M13 forward (59-GTAAAACGACG-GCCAG-39) and M13 reverse (59-CAGGAAACAGCTATGAC-39) were used for sequencing. G-to-A mutations were analyzed by using the program Hypermut 2.0 that is freely available at http://www.hiv.lanl.gov/content/ sequence/HYPERMUT/hypermut.html (28).…”

Defining APOBEC3 Expression Patterns in Human Tissues and Hematopoietic Cell Subsets

“…In addition to these editing effects, a growing amount of literature indicates that APOBEC3 proteins also diminish the accumulation of HIV-1 reverse transcripts in infected cells, most likely by interfering with the process of reverse transcription (3,5,8,26,29,31,45,47,49,51). The relative contributions of these functions to viral suppression under differing infection conditions remain to be determined (40). HIV-1 Vif overcomes the activity of APOBEC3 proteins by recruiting them to a cullin 5-elongin B/C-Rbx ubiquitin ligase complex and inducing polyubiquitylation, proteasomal degradation, and exclusion from viral particles (18,44,50,52,70,73,85).…”