Fig 1.
Amino acid changes in Nef that disrupt tetherin antagonism, but not CD4 or MHC class I downregulation.
SIV Nef mutants with individual (A) or combinations of double or triple (B) alanine substitutions were tested for the ability to rescue the release of nef-deleted SIV in the presence of rhesus tetherin. 293T cells were co-transfected with SIVmac239Δnef together with constructs expressing rhesus macaque tetherin and the indicated SIV Nef mutants. Percent maximal virus release was calculated from the accumulation of SIV p27 in the culture supernatant of cells transfected with tetherin relative to cells transfected with empty vector. Controls include virus release in the absence of Nef (Vector), or in the presence of wild-type Nef (WT) or a Nef mutant with a glycine-to-alanine substitution at position 2 (G2A). Differences in virus release were corroborated by western blot analysis. Virion and cell lysate proteins were separated by SDS-PAGE, transferred to PVDF membranes and stained with antibodies to tetherin (BST-2.1), β-actin, the SIV Gag (p55 & p27) and Nef proteins. The SIV Nef mutants were also tested for the ability to downmodulate CD4 (C) and MHC class I (D) molecules. TZM-bl cells and Jurkat cells were transfected with bicistronic constructs that express GFP and either WT Nef (black), Nef G2A (striped) or the indicated Nef mutants (grey). At 48 hours post-transfection, relative levels of CD4 and MHC class I expression were determined by comparing the mean fluorescence intensities of staining on GFP+ cells expressing Nef to GFP+ cells transfected with an empty vector (white). Error bars indicate standard deviation of the mean for at least three independent experiments and significant differences with respect to WT Nef are indicated by asterisks (*p<0.05, ** p<0.01, *** p<0.001 & **** p<0.0001, two-tailed unpaired t-test with Welch’s correction in case of unequal variance).
Fig 2.
Phenotypic characterization an infectious molecular clone of SIV with substitutions in Nef that selectively impair tetherin antagonism.
SIVmac239AAA, SIVmac239 and SIVmac239Δnef were tested for resistance to tetherin (A & B), and for downmodulation of tetherin (C), CD3 (D), CD4 (E) and MHC I (F) from the surface of infected primary CD4+ lymphocytes. (A) 293T cells were co-transfected with full-length clones for SIVmac239, SIVmac239Δnef or SIVmac239AAA and increasing amounts of an expression construct for rhesus tetherin (rBST-2.1). The accumulation of SIV p27 in supernatant was measured by antigen-capture ELISA and percent maximal release was calculated relative to control transfections in the absence of tetherin. (B) Differences in virus release were corroborated by western blot analysis of virions and cell lysates. Proteins were separated by SDS-PAGE, transferred to PVDF membranes and stained with antibodies to tetherin (rBST-2.1), β-actin, and the SIV Gag p27 and p55 proteins. (C-F) Activated CD4+ lymphocytes from six different macaques were infected with SIVmac239AAA, SIVmac239 and SIVmac239Δnef. On day 6 post-infection, the cells were stained for surface expression of tetherin, CD3, CD4 and MHC I, and for intracellular expression of the SIV Gag protein. Relative expression levels of tetherin (C), CD3 (D), CD4 (E) and MHC I (F) were determined by comparing the gMFI of each of these molecules on virus-infected (CD4loGag+) cells to uninfected cells (CD4hiGag-). Error bars indicate standard deviation of the mean and significant differences are indicated by asterisks (**p<0.01 & NS, not significant, two-tailed unpaired t-test with Welch’s correction in case of unequal variance).
Fig 3.
NefAAA substitutions impair SERINC5 antagonism and SIVmac239AAA infectivity.
The infectivity of Nef-trans-complemented SIVmac239Δnef produced in JTAg cells with knock-out mutations in SERINC3 (SERINC3-5+), SERINC5 (SERINC3+5-), both (SERINC3-5-) or neither (SERINC3+5+) (A) and viruses harvested from infected rhesus macaque CD4+ lymphocytes (B) were compared. (A) Nef-trans-complemented virus was collected 48-hours after co-transfection of JTAg cells with SIVmac239Δnef and expression constructs for wild-type Nef (WT), Nef G2A (G2A), NefAAA (AAA) or empty vector (Vector). Nef expression was also verified by western blot analysis of JTAg cell lysates. (B) SIVmac239, SIVmac239Δnef and SIVmac239AAA were collected after six days of replication in activated CD4+ lymphocytes from six different rhesus macaques, virus concentrations were measured by SIV p27 antigen-capture ELISA, and C8166-SEAP cells were infected in triplicate with equivalent doses of each virus (0.5 ng p27 per 1x104 cells). Secreted-alkaline phosphatase (SEAP) activity was measured in the cell culture supernatant 72-hours later. Relative infectivity is shown as a percentage of wild-type Nef or SIVmac239 infection. Error bars indicate standard deviation of the mean for at least four independent experiments (A) or six different animals (B). Significant differences are indicated by asterisks (*** p<0.001 & **** p<0.0001, two-tailed unpaired t-test with Welch’s correction in case of unequal variance).
Fig 4.
SIV loads in plasma and tetherin upregulation on CD4+ lymphocytes in SIVmac239AAA- versus SIVmac239-infected macaques.
(A) Viral loads in plasma are shown during acute (weeks 1–4) and chronic (weeks 5–24) infection following intravenous inoculation of macaques with wild-type SIVmac239 (black) or SIVmac239AAA (red). Viral RNA loads were measured by qRT-PCR using an assay with a threshold of detection of 30 copies/ml. Viral loads were significantly lower in SIVmac239AAA-infected animals during acute infection (p = 0.004, linear mixed-effects model). Longitudinal changes in tetherin upregulation on memory CD95+CCR7-CD4+ T cells (B) and memory CD95+CD4+ T cell counts (C) were monitored by flow cytometry. The mean values for animals infected with SIVmac239 (black) and SIVmac239AAA (red) are indicated by heavier lines.
Fig 5.
Sequence changes in Nef restore resistance to tetherin and SERINC5.
(A) The virus population in plasma of animals infected with SIVmac239AAA was sequenced at the indicated time points post-infection. The predicted amino acid sequences in the flexible loop region of Nef are aligned to the corresponding Nef sequences of SIVmac239AAA and SIVmac239. Positions of identity are indicated by periods and amino acid differences are identified by their single-letter code. (B & C) The Nef variants observed at 22–24 weeks PI in SIVmac239AAA-infected animals were tested for the ability to counteract tetherin (B) and SERINC5 (C). (B) 293T cells were co-transfected with SIVmac239Δnef together with constructs expressing the indicated Nef variants and rhesus macaque tetherin. Percent maximal virus release was calculated from the accumulation of SIV p27 in the culture supernatant of cells transfected with tetherin relative to cells transfected with empty vector. Differences in virus release were corroborated by western blot analysis as described in Fig 1. (C) C8166-SEAP cells were infected with Nef trans-complemented virus produced by co-transfection of parental JTAg cells (SERINC3+5+) with SIVmac239Δnef and the indicated Nef variants. Infectivity was measured as described in Fig 3 and Nef expression was confirmed by western blot analysis. (B & C) Error bars indicate standard deviation of the mean for at least four independent experiments and significant differences relative to NefAAA are indicated by asterisks (*p<0.05, ** p<0.01, *** & p<0.001, two-tailed unpaired t-test with Welch’s correction in case of unequal variance).