Fig 1.
Identification of IFNα-stimulated genes (ISGs) in macaque lymphocytes.
Dot plot of log2 of average expression (x axis) per log2 of fold change (y axis) for all genes identified upon IFNα treatment (1000 U/ml for 24 hours) of Ptm lymphocytes. 198 genes that were differentially expressed (|logFC| ≥ 0.585 & FDR 5%) are highlighted: the 147 genes that were significantly upregulated are shown with red dots and the 51 genes that were significantly downregulated are shown with green dots. The pie chart shows the fraction of genes that contain transmembrane helices based on translation of open reading frames of the upregulated genes. The fold change of the indicated Ptm IFITM3-like and IFITM1 that were selected for subsequent analysis are shown.
Fig 2.
Characterization of macaque IFITMs.
(A) A comparison of the chromosomal context of IFITM and IFITM-like genes in macaques and human. Top panel: Comparison of the human IFITM locus on chromosome 11 (abbreviated “ch11”) of the human reference genome and the corresponding loci in the indicated macaque genomes (chromosome 14 “ch14” for rhesus and crab-eating macaques, and Mnem_1.0 Scaffold195 for pig-tailed macaque). Exons are indicated as filled rectangles and introns as joined angled lines. Gene names are indicated in italics. Vertical alignment of conserved flanking genes NLRP6, PGGHG, B4GALNT4, and PKP3 across species is indicative of synteny. Humans encode IFITM2 (blue), a duplication of IFITM3, which is not present in the macaque genomes. The parental IFITM3 (orange, LOC105494127 in pig-tailed macaque) is present in human and macaques. Macaques genomes contain an additional copy of IFITM3 (indicated IFITM3A, LOC105494124, green) and an IFITM3 pseudogene (gray). Bottom panel: Two additional IFITM3-like genes identified in macaques in different chromosomal contexts. One IFITM3-like gene (indicated IFITM3L, LOC105473984, brown) is located in the intron of CPFAS on chromosome 16 (ch16). The other gene (indicated IFITM3L, LOC105466036, pink) is located in the intron of MCU on chromosome 9 (ch9). The corresponding loci in the human genome are indicated below and lack IFITM3-like genes. (B) A maximum likelihood tree of IFITM nucleotide sequences shows groupings of IFITM1, IFITM3 pseudogenes, and IFITM3/2. Tree is rooted on the IFITM1 group. Text colors correspond to rectangles depicting the same sequences in panel A. Nodes with bootstrap support greater than 90 are labeled. (C) Western blot analysis of Ptm lymphocytes treated with increasing concentrations of IFNα for 24 hours. The concentration of IFNα is indicated above each lane. Immunoblotting performed using anti-IFITM3/3A and anti-GAPDH antibodies.
Fig 3.
Incorporation of macaque IFITM3 in SHIV virions.
(A) Western blot analysis of virions harvested at nine days post-infection from Ptm lymphocytes infected with indicated SHIV. Color-coding indicates whether the SHIV is adapted (orange) or unadapted (blue). Virions produced in the absence or presence of IFNα (1000 U/ml) is indicated above each lane. The values below the bottom panel indicate the amount of virions (ng of SIV p27) loaded into each lane. (B) Western blot analysis of virions purified using sucrose density gradient. The numbers above each lane indicate the fraction collected from the top of the gradient. (C) Western blot analysis of Ptm lymphocytes infected with indicated SHIV and harvested at nine days post-infection. Immunoblotting performed using anti-IFITM3/3A, anti-SIV p27, and anti-GAPDH antibodies.
Fig 4.
Differences in incorporation of macaque IFITM3 in SHIV virions.
(A) Western blot analysis of virions harvested at nine days post-infection from Ptm lymphocytes infected with indicated SHIV in the presence of IFNα (1000 U/ml). Color-coding indicates whether the SHIVs are adapted (orange) or unadapted (blue). Virions equivalent to 5 ng of SIV p27 was loaded into each lane. IFNα sensitivity as measured by IFNα IC50 assay of each SHIV variant was previously determined [25] and is indicated at the top. (B) Western blot analysis of adapted SHIV virions. The values below the bottom panel indicate the amount of virions (ng of SIV p27) loaded into each lane. Immunoblotting performed using anti-IFITM3/3A and anti-SIV p27 antibodies.
Table 1.
CRISPR guide RNA used in this study.
Fig 5.
Knockout of macaque IFITMs affects IFNα sensitivity of unadapted SHIV.
(A) Western blot analysis of Ptm IFITM-knockout cell pools using anti-IFITM3/3A (left panel) and anti-IFITM1 (right panel) antibodies. CRISPR/Cas9 ribonucleoproteins (crRNPs) that targeted IFITM1 and IFITM3 (indicated “M1+M3”), IFITM3A, IFITM3, or a non-targeting control (indicated “NTC”) is indicated at the top. IFNα-treatment (1000 U/ml) of cells is indicated above each lane. The numbers below the bottom panel indicate the IFITM3/3A:GAPDH (left panel) or IFITM1:GAPDH (right panel) signal relative to IFNα-treated, NTC. (B–C) Effect of IFNα-treatment on replication of SHIV-Q23AE (B) and SHIV-AD8-EO (C) in Ptm IFITM-knockout cell pools over a 9-day time course. The identity of each SHIV is indicated above the chart. Color-coding indicates whether the SHIVs are adapted (orange) or unadapted (blue). The key at the right of the graph indicates the color corresponding to each knockout cell line. Replication in the presence of IFNα (1000 U/ml) is indicated in the dashed lines, whereas control replication in the absence of IFNα is indicated in the solid lines. Data points represent the average of four independent experiments, and error bars represent SD. (D–E) Area under the curve (AUC) ratio for (D) SHIV-Q23AE and (E) SHIV-AD8-EO determined from the replication curves shown in (B) and (C), respectively. Data represent the average of four independent experiments, and error bars represent SEM. AUC values were compared using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test. ** p = 0.0054, * p = 0.0156.
Fig 6.
Knockout of macaque IFITMs increases replication of unadapted SHIV.
(A) Western blot analysis of Ptm IFITM-knockout cell pools using anti-IFITM3/3A antibody. CRISPR/Cas9 ribonucleoproteins (crRNPs) that targeted IFITM1 and IFITM3 (indicated “M1+M3”), IFITM3A, IFITM3, or a non-targeting control (indicated “NTC”) is indicated at the top. The numbers below the bottom panel indicate the IFITM3/3A:GAPDH signal relative to NTC. (B–C) Replication kinetics of (B) SHIV-Q23AE and (C) SHIV-AD8-EO in Ptm IFITM-knockout cell pools over a 9-day time course. The identity of each SHIV is indicated above the chart. Color-coding indicates whether the SHIVs are adapted (orange) or unadapted (blue). The key at the right of the graph indicates the color corresponding to each knockout cell line. Data points represent the average of four independent experiments, and error bars represent SD. (D–E) Area under the curve (AUC) for (D) SHIV-Q23AE and (E) SHIV-AD8-EO determined from the replication curves shown in (B) and (C), respectively. Data represent the average of four independent experiments, and error bars represent SEM. AUC values were compared using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test. *** p = 0.002, * p = 0.011.