APOBEC3G and APOBEC3F Act in Concert To Extinguish HIV-1 Replication

Abstract

The multifunctional HIV-1 accessory protein Vif counters the antiviral activities of APOBEC3G (A3G) and APOBEC3F (A3F), and some Vifs counter stable alleles of APOBEC3H (A3H). Studies in humanized mice have shown that HIV-1 lacking Vif expression is not viable. Here, we look at the relative contributions of the three APOBEC3s to viral extinction. Inoculation of bone marrow/liver/thymus (BLT) mice with CCR5-tropic HIV-1JRCSF(JRCSF) expressing a vif gene inactive for A3G but not A3F degradation activity (JRCSFvifH42/43D) displayed either no or delayed replication. JRCSF expressing a vif gene mutated to inactivate A3F degradation but not A3G degradation (JRCSFvifW79S) always replicated to high viral loads with variable delays. JRCSF with vif mutated to lack both A3G and A3F degradation activities (JRCSFvifH42/43DW79S) failed to replicate, mimicking JRCSF without Vif expression (JRCSFΔvif). JRCSF and JRCSFvifH42/43D, but not JRCSFvifW79S or JRCSFvifH42/43DW79S, degraded APOBEC3D. With one exception, JRCSFs expressing mutant Vifs that replicated acquired enforced vif mutations. These mutations partially restored A3G or A3F degradation activity and fully replaced JRCSFvifH42/43D or JRCSFvifW79S by 10 weeks. Surprisingly, induced mutations temporally lagged behind high levels of virus in blood. In the exceptional case, JRCSFvifH42/43D replicated after a prolonged delay with no mutations in vif but instead a V27I mutation in the RNase H coding sequence. JRCSFvifH42/43D infections exhibited massive GG/AG mutations in pol viral DNA, but in viral RNA, there were no fixed mutations in the Gag or reverse transcriptase coding sequence. A3H did not contribute to viral extinction but, in combination with A3F, could delay JRCSF replication. A3H was also found to hypermutate viral DNA.

Importance: Vif degradation of A3G and A3F enhances viral fitness, as virus with even a partially restored capacity for degradation outgrows JRCSFvifH42/43D and JRCSFvifW79S. Unexpectedly, fixation of mutations that replaced H42/43D or W79S in viral RNA lagged behind the appearance of high viral loads. In one exceptional JRCSFvifH42/43D infection, vif was unchanged but replication proceeded after a long delay. These results suggest that Vif binds and inhibits the non-cytosine deaminase activities of intact A3G and intact A3F, allowing JRCSFvifH42/43D and JRCSFvifW79S to replicate with reduced fitness. Subsequently, enhanced Vif function is acquired by enforced mutations. In infected cells, JRCSFΔvif and JRCSFvifH42/43DW79S are exposed to active A3F and A3G and fail to replicate. JRCSFvifH42/43D Vif degrades A3F and, in some cases, overcomes A3G mutagenic activity to replicate. Vif may have evolved to inhibit A3F and A3G by stoichiometric binding and subsequently acquired the ability to target these proteins to proteasomes.

FIG 1

Mutations in Vif that disrupt degradation of APOBEC3G or APOBEC3F do not affect virus replication in vitro. (A) Viruses were produced by transfection of 293T cell with proviral clones. CCR5-expressing CEM-SS cells were infected with wild-type JRCSF or JRCSF with point mutations in Vif that disrupt degradation of either APOBEC3G, APOBEC3F, or both. The culture supernatant was monitored longitudinally by ELISA for HIV p24^gag protein as a measure of virus replication. (B) HEK-293T cells were cotransfected with the proviral clones in panel A and an expression vector for either V5-tagged APOBEC3G or APOBEC3F. The cell lysates were resolved by SDS-PAGE and probed with anti-V5 to assess APOBEC3 degradation in the presence of the designated Vif mutants. Western blots for Vif and GAPDH were conducted as controls. (C) Same as panel B except that HA-tagged APOBEC3D degradation was probed with anti-HA.

FIG 2

Absolute restriction of HIV in vivo requires both APOBEC3G and APOBEC3F. (A) (Top) The plasma of BLT humanized mice infected with JRCSFvifH42/43D (blue lines) was monitored longitudinally for the presence of HIV RNA. Wild-type JRCSF RNA is presented in aggregate (n = 7) (black diamonds). (Bottom) Nested PCR to detect HIV DNA was performed on genomic-DNA extracts from each tissue listed from the infected humanized mice. +, tissues positive for HIV DNA; −, tissues where HIV DNA was not detected. (B) (Top) Viral RNA was present in the plasma of 4/4 mice infected with JRCSFvifW79S (red lines) at levels comparable to those of wild-type JRCSF (n = 7) (black diamonds). (Bottom) Nested PCR to detect HIV DNA was performed on genomic DNA extracted from each tissue listed from the infected BLT humanized mice. +, tissues positive for HIV DNA; −, tissues where HIV DNA was not detected. (C) (Top) Plasma viral RNA was not observed in 4/4 mice infected with JRCSFvifH42/43DW79S (green lines) for up to 8 weeks postexposure, in contrast to infection with wild-type JRCSF (black diamonds). (Bottom) Viral DNA was not detected in any tissue of JRCSFvifH42/43DW79S-inoculated BLT humanized mice (−). (D) Sequencing of viral DNA amplified from the tissues of mice infected with JRCSFvifH42/43D (G3 and G4) revealed heavy G-to-A mutation at GG sites (blue bar). Few G-to-A mutations were present at GA sites in viral DNA from mice infected with JRCSFvifH42/43D or JRCSFvifW79S, and no mutations were observed in WT1 JRCSF DNA. The data are presented as means ± SEM.

FIG 3

In vivo enforced mutation of vif in JRCSFvifH42/43D and JRCSFvifW79S. (A) HIV vif sequences amplified from plasma virions at terminal time points of JRCSFvifH42/43D infection show mutations changing the aspartate codons in mouse G3 to asparagine at position 42 and to either asparagine or glycine at codon 43. The H42/43D mutation in mouse G4 remained intact throughout the infection. (B) HIV vif sequence from the terminal time point of 4 mice infected with JRCSFvifW79S showing that the serine at codon 79 was changed to either tyrosine or phenylalanine. A single GA-to-AA mutation (nucleotide 56) was observed in mouse F3, while a point mutation (changing nucleotide 134 from A to T) was present in a portion of the HIV sequences in mouse F4. *, positions of sequence identity to JRCSF.

FIG 4

Mutations that arose in vivo partially restored Vif function. (A) Proviral DNAs with the designated Vif mutations at codon 79 and an expression vector for either V5-tagged APOBEC3G or APOBEC3F were cotransfected into HEK-293T cells. The cell lysates were resolved by SDS-PAGE and probed with anti-V5 to assess degradation of APOBEC3G or APOBEC3F in the presence of the Vif mutants, revealing that F or Y at amino acid 79 partially restored Vif degradation of APOBEC3F. (B) HEK-293T cells were cotransfected with an expression vector for either V5-tagged APOBEC3G or APOBEC3F and proviral DNA with the designated Vif mutations at codons 42 and 43. Cell lysates were resolved by SDS-PAGE and probed with anti-V5 to assess degradation of APOBEC3G or APOBEC3F in the presence of the Vif mutants, revealing that the N at position 42 with an N or G at amino acid 43 partially restored Vif degradation of APOBEC3G.

FIG 5

Evolution of the APOBEC3 disrupting mutations in the vif gene in vivo. (A) Sequences of codons 41 to 44 of vif amplified from the plasma RNA in two viremic mice infected with JRCSFvifH42/43D. In mouse G3, Vif was subjected to positive selection to replace the aspartic acid residues at positions 42 and 43. In mouse G4, no mutation of the vif gene at amino acid 42 or 43 was observed. G, black; A, green; T, red; C, blue. (B) Sequence of codons 78 to 80 of the vif gene amplified from plasma RNA in 4 mice infected with JRCSFvifW79S revealing replacement of the W79S mutation from the initial infecting virus. In all 4 mice, the serine residue at amino acid 79 was converted to either phenylalanine or tyrosine. wpi, weeks postinfection.

FIG 6

Robust replication of JRCSF in a mouse cohort heterozygous for A3H haplotype V. Individual plots of the wild-type JRCSF viral load versus time are presented. The seven mice are from Fig. 2 (WT), where JRCSF replication is presented in aggregate. WT1 has A3H haplotypes I and V, but viral replication is not diminished. WT2 to WT7 are derived from four separate cohorts.

FIG 7

JRCSF, JRCSFvifW79S, and JRCSFvifH42/43D viral load time courses following a low-dose inoculation. Nine individual viral load time courses are presented following a 10-fold-reduced inoculation of JRCSF (9,000 TCIU) compared to Fig. 2. Two mice were inoculated with JRCSF (WT8 and WT9), three were inoculated with JRCSFvifW79S (F5, F6, and F7), and four mice were inoculated with JRCSFvifH42/43D (G5, G6, G7, and G8). WT8 and WT9 are homozygous A3H inactive (cohorts 3 and 6, respectively), F5 and F6 are homozygous A3H inactive (both cohort 3), F7 is heterozygous A3H active (cohort 7), G5 and G8 are homozygous A3H inactive (cohorts 6 and 3, respectively), and G6 and G7 are heterozygous A3H active (both cohort 7) (Table 3).

FIG 8

HIV DNA amplified from an extinguished infection is hypermutated. (A) Highlighter sequence analysis of the reverse transcriptase region of HIV pol. HIV DNA amplified from the lymph nodes of mouse G2 was G-to-A hypermutated (green lines). HIV-1_JRCSF nucleotide numbers are indicated at the bottom. (B) Alignment of wild-type JRCSF and hypermutated JRCSF reverse transcriptase sequence from panel A identifying G-to-A mutations at both GG and GA sites. GG-to-AG mutations are indicated in blue, GA-to-AA mutations are shown in red, and G-to-A mutations present at GT sites are shown in orange.

FIG 9

Mutations in HIV RNase H are present only in virus from mouse G4. HIV-1 RNase H sequence amplified from the plasma of viremic humanized mice infected with either JRCSFvifH42/43D or JRCSFvifW79S showed that only virus from mouse G4 had mutations in RNase H. One G-to-A mutation was present in RNase H from mouse G4 (highlighted in orange) that resulted in a valine-to-isoleucine amino acid substitution.