Evolution of HIV-1 isolates that use a novel Vif-independent mechanism to resist restriction by human APOBEC3G

Abstract

The human APOBEC3G protein restricts the replication of Vif-deficient HIV-1 by deaminating nascent viral cDNA cytosines to uracils, leading to viral genomic strand G-to-A hypermutations. However, the HIV-1 Vif protein triggers APOBEC3G degradation, which helps to explain why this innate defense does not protect patients. The APOBEC3G-Vif interaction is a promising therapeutic target, but the benefit of the enabling of HIV-1 restriction in patients is unlikely to be known until Vif antagonists are developed. As a necessary prelude to such studies, cell-based HIV-1 evolution experiments were done to find out whether APOBEC3G can provide a long-term block to Vif-deficient virus replication and, if so, whether HIV-1 variants that resist restriction would emerge. APOBEC3G-expressing T cells were infected with Vif-deficient HIV-1. Virus infectivity was suppressed in 45/48 cultures for more than five weeks, but replication was eventually detected in three cultures. Virus-growth characteristics and sequencing demonstrated that these isolates were still Vif-deficient and that in fact, these viruses had acquired a promoter mutation and a Vpr null mutation. Resistance occurred by a novel tolerance mechanism in which the resistant viruses packaged less APOBEC3G and accumulated fewer hypermutations. These data support the development of antiretrovirals that antagonize Vif and thereby enable endogenous APOBEC3G to suppress HIV-1 replication.

Figure 1. Selecting Vif-Deficient HIV-1 Isolates that Resist APOBEC3G

(A) Short-term spreading infection data for Vif-deficient (circles) or Vif-proficient (inverted triangles) viruses. Throughout the manuscript, a unique symbol represents each virus, open symbols and dashed lines represent virus growth on vector control cells (e.g., V2) and filled symbols and solid lines represent virus growth on APOBEC3-expressing cells (e.g., G1, G2). (B) An immunoblot showing APOBEC3G levels in CEM, vector control CEM-SS clones (V1 and V2), CEM-SS (--) and three independently derived APOBE3G-expressing CEM-SS clones (G1, G2 and G3). The same membrane was stripped and probed for Tubulin (TUB). Complementary immunoblot data comparing model cell lines to primary cells are shown in Figure S1. (C) Highlights of the long-term spreading infection experiments for Vif-deficient viruses on vector control or APOBEC3G-expressing CEM-SS cell lines. 45/48 cultures showed no virus replication on APOBEC3G-expressing cells (flat lines not graphed). The 3 APOBEC3G-resistant isolates that eventually emerged were A3G-R1 (squares), A3G-R2 (triangles) and A3G-R3 (diamonds). (D) Spreading infections of A3G-R1, -R2 or -R3 on CEM-SS clones expressing APOBEC3G (G1, G2) or a vector control (V2). Parental viruses were also analyzed in parallel and these data resembled those in Figure 1A (not shown). See the APOBEC3G panels in Figure S2 for complementary data and Table S1 additional information.

Figure 2. APOBEC3G-Resistant Isolates Are Still Susceptible to APOBEC3F

(A & B) Replication kinetics of the parental viruses and A3G-R1, -R2 and -R3 on CEM cells or APOBEC3F-expressing CEM-SS cells (F1 or F2). These experiments were done in parallel, so the vector control data (V2) shown in the CEM panels are also applicable to the APOBEC3F panels. The A3G-R2 and -R3 data on APOBEC3F lines are presented in one panel to make space for Figure 2C. A3G-R1, -R2 and -R3 also spread with rapid kinetics on APOBEC3G-expressing cells (G2; not shown). The higher overall titers of the APOBEC3G resistant viruses warranted expanded Y-axes. Symbol and line designations are identical those in Figure 1. See Figures S1 & S2 for complementary data. (C) An immunoblot of APOBEC3F levels in two APOBEC3F-expressing CEM-SS clones (F1 and F2), parental CEM-SS cells (--) and two non-permissive cell lines (H9 and CEM). The same membrane was stripped and probed for Tubulin (TUB).

Figure 3. HIV-1 Mutations that Confer Vif-independent Resistance to APOBEC3G

(A) A schematic of HIV-1_IIIB indicating the coding regions and relevant restriction sites (GenBank Accession EU541617). Asterisks denote the tandem stop mutations in vif. (B) Summaries of the types of base substitutions found in A3G-R1, -R2 and -R3 proviral DNA. Each horizontal line is a composite of 4 independent proviral DNA sequences from large or small PCR amplicons (solid and dashed lines, respectively). G-to-A substitutions are depicted by vertical bars and other substitutions by closed circles. Vertical arrows highlight the common A-to-T/C and vpr-inactivating mutations. See Figure S4 for an overview of 800 kb of sequencing data. (C) Replication kinetics of molecular clone-derived viruses with the indicated genotypes. Symbol and line designations are identical those in Figure 1, except the double mutants share the same symbols as the APOBEC3G-resistant triple mutant (diamonds for A3G-R3M). See Figure S3 for supporting data, including experiments with A3G-R1M, A3G-R2M and their double mutant derivatives.

Figure 4. A200T-or-C Contributes to APOBEC3G Resistance by Enhancing HIV-1 Transcription, Increasing Virus Titers and Diminishing APOBEC3G Encapsidation

(A) Immunoblots showing the level of virus particle-associated APOBEC3G-E259Q or p24^Gag from a spreading infection experiment with the indicated viruses. Relative to the Vif⁽⁻⁾ virus with an APOBEC3G/p24^Gag ratio normalized to 1 (n=3), the Vif⁽⁻⁾/Vpr⁽⁻⁾, A200T-orC/Vif⁽⁻⁾ and A200T-orC/Vif⁽⁻⁾/Vpr⁽⁻⁾ viruses had mean ratios of 1.1 ± 0.36 (n=9), 0.25 ± 0.10 (n=9) and 0.32 ± 0.17 (n=8), respectively. See Figure S5 for additional quantification and Figure S6 for complementary single-cycle data. (B) Relative titers of the indicated viruses produced after a single-round of replication on CEM-SS cells expressing APOBEC3G-E259Q. Each histogram bar shows the mean and s.d. of 3 independently derived supernatants (some error bars were too small to graph). The titer for the Vif-proficient virus was normalized to 1 to facilitate comparisons. (C) Expression level of the indicated LTR-GFP reporter constructs in HEK-293 cells. Each histogram bar shows the mean and s.d. of 3 independent replicas. The GFP expression level of the A200 HIV-1_IIIB construct was normalized to 1 to facilitate comparisons. The inset depicts the LTR-GFP expression construct, with position of nucleotide 200 marked by an asterisk.