Fig 1.
Failure of neutralizing antiviral effect of gA3G by SIVcpzPtt Vif.
(A) A phylogenetic tree of the vif gene of great ape lentiviruses. The vif sequences were extracted from Los Alamos HIV sequence database (https://www.hiv.lanl.gov/components/sequence/HIV/search/search.html) and the phylogenetic tree was constructed as described in Materials and Methods. The bootstrap values are indicated as follows: *, >80%; **, >95%. A scale bar indicates 0.1 nucleotide substitutions per site. (B) A scheme of cross-species lentiviral transmission in great apes. (C) Antiviral activity of great ape A3G. HEK293T cells were co-transfected with pNL4-3Δvif (500 ng) and the different amounts of expression plasmids for great ape A3G (0, 50, 100, and 200 ng; the plasmid amount was normalized by empty vector). Cells and supernatants were harvested at two days post-transfection and were used for Western blotting (left) and TZM-bl assay (right). For Western blotting, the band intensity of viral p24 was quantified and the intensity value of the leftmost lane was set to 100%. For TZM-bl assay, the infectivity value without A3G was set to 100%. (D and E) Counteracting ability of great ape lentiviral Vif against great ape A3G. HEK293T cells were co-transfected with pNL4-3Δvif (500 ng) and the expression plasmids for hA3G (D; 10 ng) or gA3G (E; 50 ng) and the Vif of indicated viral strain (500 ng). In C-E, cells and supernatants were harvested at two days post-transfection and were used for Western blotting (left) and TZM-bl assay (right). For Western blotting, the band intensity of viral p24 was quantified and the intensity value of the A3G expressing cells without Vif (second from the left) was set to 100%. For TZM-bl assay, the percentage of the value without A3G are shown. The mean values of three independent experiments ± SEM are shown, and statistically significant differences (P < 0.05) versus "no A3G (C, black asterisks)", "gA3G (C, orange asterisks)", or "no Vif" (D and E, asterisks) are shown. (F) CBF-β-dependent degradation of gA3G by SIVgor Vif. CBFB KD HEK293 cells were co-transfected with the expression plasmids for gA3G (50 ng), SIVgor Vif (500 ng) and CBF-β (400 ng). Cells were harvested at two days post-transfection and were used for Western blotting. For Western blotting, the input of cell lysate was standardized to TUBA, and representative results are shown.
Fig 2.
Gain-of-function experiments of SIVcpzPtt Vif to counteract gA3G.
(A) A scheme of SIVcpzPtt MB897 Vif derivatives used in this study. (B-D) Determination of the amino acid residue of SIV Vif that is responsible to neutralize gA3G. HEK293T cells were co-transfected with pNL4-3Δvif (500 ng) and the expression plasmids for gA3G (10 or 50 ng; the plasmid amount was normalized by empty vector) and the indicated Vif derivatives (500 ng). (E) Infectivity of the infectious viruses of SIVcpzPtt MB897 WT, M16E and E2X (vif deleted) derivatives. These viruses were prepared as described in Materials and Methods and the infectivity was measured by using TZM-bl cells. The mean values of three independent experiments ± SEM are shown. (F-H) (F) Expression of gA3G in SupT11-CCR5 cells. The SupT11-CCR5 cells stably expressing gA3G (SupT11-CCR5-gA3G) was prepared as described in Materials and Methods. For Western blotting, the input of cell lysate was standardized to TUBA, and representative results are shown. (G and H) Multi-round replication assay of SIVcpzPtt. The infectious viruses of SIVcpzPtt MB897 WT, M16E and E2X (vif-deleted) derivatives were inoculated into parental SupT11-CCR5 cells (G) or the SupT11-CCR5-gA3G (H) at MOI 0.1. (Left) Culture supernatant was routinely harvested and the amount of infectious viruses was measured by using TZM-bl cells. (I) Conservation of residue 16 in the Vif proteins of all SIVcpzPtt and SIVgor strains reported. Note that “X” is an undefined amino acid because of nucleotide ambiguity. (J) Importance of M16E substitution in other SIVcpzPtt strains to counteract gA3G. HEK293T cells were co-transfected with pNL4-3Δvif (500 ng) and the expression plasmids for gA3G (50 ng) and the indicated Vif strains and derivatives (500 ng). In B-D and J, cells and supernatants were harvested at two days post-transfection and were used for Western blotting and TZM-bl assay. For Western blotting, the input of cell lysate was standardized to TUBA, and representative results are shown. The band intensity of viral p24 was quantified and the intensity value of the gA3G expressing cells without Vif (second from the left) was set to 100%. For TZM-bl assay, the percentage of the value without gA3G is shown. The mean values of nine independent experiments ± SEM are shown. Statistically significant differences (P < 0.05) versus “CP2139” (B-D), “M16E” (G and H) or “WT” (J) are shown by red asterisks. NS, no statistical significance.
Fig 3.
Importance of acidic residues at position 16 of SIVcpzPtt to counteract gA3G.
(A) Importance of acidic residue at position 16 of SIVcpzPtt to counteract gA3G. HEK293T cells were co-transfected with pNL4-3Δvif (500 ng) and the expression plasmids for gA3G (50 ng) and the indicated Vif strains and derivatives (500 ng). Cells and supernatants were harvested at two days post-transfection and were used for Western blotting (top) and TZM-bl assay (bottom). For Western blotting, the input of cell lysate was standardized to TUBA, and representative results are shown. The band intensity of viral p24 was quantified and the intensity value of the gA3G expressing cells without Vif (second from the left) was set to 100%. For TZM-bl assay, the percentage of the value without gA3G is shown. The mean values of nine independent experiments ± SEM are shown. Statistically significant differences (P < 0.05) versus “CP2139” are shown by red asterisks. (B-D) Structure homology models of Vif. The homology models are constructed based on the crystal structure of HIV-1M Vif (PDB: 4N9F) [29]. (B) Ionizability of each amino acid residue. Basic and acidic residues are indicated in blue and red, respectively. (C and D) Structural comparison. The topological changes of respective residues comparing to SIVcpzPtt MB897 Vif are indicated based on the RMSD values of heavy atom positions. The whole structure model (C) and the cartoon model of the residues between 15–19 (D) are respectively shown.
Fig 4.
Counteraction of antiviral gA3F by SIVgor Vif independently of the F1 and F3 boxes.
(A) Loss of F1 box in SIVgor, HIV-1O and HIV-1P Vif. Logo plots of the Vif residues between 14–17 (corresponding to the F1 box in HIV-1M; left) and the residues between 40–44 (corresponding to the G box in HIV-1M; right) in HIV-1MNOP, SIVcpz and SIVgor are shown. The number in parenthesis (n) indicates the number of viral sequences used in this analysis. (B and C) Counteracting activity against gA3G and gA3F. HEK293T cells were co-transfected with pNL4-3Δvif (500 ng) and the expression plasmids for either gA3G (50 ng, B) or gA3F (200 ng, C) and indicated Vif (500 ng). (D) Scheme of HIV-1M JRCSF Vif derivatives used in this study. (E) Counteracting activity against hA3F. HEK293T cells were co-transfected with pNL4-3Δvif (500 ng) and the expression plasmids for hA3F (50 ng) and indicated HIV-1M JRCSF Vif derivatives (500 ng). (F-H) Counteracting activity of SIVgor and SIVcpz Vif against great ape A3F. HEK293T cells were co-transfected with pNL4-3Δvif (500 ng) and the expression plasmids for either gA3F (F, 200 ng) or hA3F (G, 50 ng) and indicated Vif (500 ng). In B, C, and E-H, cells and supernatants were harvested at two days post-transfection and were used for Western blotting and TZM-bl assay. For Western blotting, the input of cell lysate was standardized to TUBA, and representative results are shown. The band intensity of viral p24 was quantified and the intensity value of the A3 expressing cells without Vif (second from the left) was set to 100%. For TZM-bl assay, the percentage of the value without A3 is shown. The mean values of three independent experiments ± SEM are shown, and statistically significant differences (P < 0.05) versus "no Vif" are shown by asterisks.