Kmiec D et al. (2018), SIVcol Nef counteracts SERINC5 by promoting its...

XB-ART-55213

PLoS Pathog 2018 Aug 20;148:e1007269. doi: 10.1371/journal.ppat.1007269.

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SIVcol Nef counteracts SERINC5 by promoting its proteasomal degradation but does not efficiently enhance HIV-1 replication in human CD4+ T cells and lymphoid tissue.

Kmiec D , Akbil B , Ananth S , Hotter D , Sparrer KMJ , Stürzel CM , Trautz B , Ayouba A , Peeters M , Yao Z , Stagljar I , Passos V , Zillinger T , Goffinet C , Sauter D , Fackler OT , Kirchhoff F .

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SERINC5 is a host restriction factor that impairs infectivity of HIV-1 and other primate lentiviruses and is counteracted by the viral accessory protein Nef. However, the importance of SERINC5 antagonism for viral replication and cytopathicity remained unclear. Here, we show that the Nef protein of the highly divergent SIVcol lineage infecting mantled guerezas (Colobus guereza) is a potent antagonist of SERINC5, although it lacks the CD4, CD3 and CD28 down-modulation activities exerted by other primate lentiviral Nefs. In addition, SIVcol Nefs decrease CXCR4 cell surface expression, suppress TCR-induced actin remodeling, and counteract Colobus but not human tetherin. Unlike HIV-1 Nef proteins, SIVcol Nef induces efficient proteasomal degradation of SERINC5 and counteracts orthologs from highly divergent vertebrate species, such as Xenopus frogs and zebrafish. A single Y86F mutation disrupts SERINC5 and tetherin antagonism but not CXCR4 down-modulation by SIVcol Nef, while mutation of a C-proximal di-leucine motif has the opposite effect. Unexpectedly, the Y86F change in SIVcol Nef had little if any effect on viral replication and CD4+ T cell depletion in preactivated human CD4+ T cells and in ex vivo infected lymphoid tissue. However, SIVcol Nef increased virion infectivity up to 10-fold and moderately increased viral replication in resting peripheral blood mononuclear cells (PBMCs) that were first infected with HIV-1 and activated three or six days later. In conclusion, SIVcol Nef lacks several activities that are conserved in other primate lentiviruses and utilizes a distinct proteasome-dependent mechanism to counteract SERINC5. Our finding that evolutionarily distinct SIVcol Nefs show potent anti-SERINC5 activity supports a relevant role of SERINC5 antagonism for viral fitness in vivo. Our results further suggest this Nef function is particularly important for virion infectivity under conditions of limited CD4+ T cell activation.

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Species referenced: Xenopus
Genes referenced: cd28 cd3g cd4 cd74 cxcl12 cxcr4 egfr gpi lamp1 lck mhc1a pigy psma5 serinc3 serinc5 shc1

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	Fig 1. Phylogenetic position and functional activity of SIVcol Nef proteins.(A) Phylogenetic relationship of Nef amino acid sequences from different primate lentiviruses. Stars on branches indicate ≥90 percentage estimated posterior probabilities. The length of a branch indicates the phylogenetic distance to its origin. (B) Modulation of various receptors and viral infectivity enhancement by SIVcol Nefs. To measure receptor modulation, human PBMCs, Jurkat (for CXCR4) or THP-1 cells (for Ii) were transduced with HIV-1 NL4-3 IRES-eGFP constructs containing the indicated nef alleles. The mean channel numbers of red fluorescence obtained for cells transfected with the nef-defective HIV-1 construct were divided by the corresponding numbers obtained for cells infected with viral constructs coexpressing Nef and eGFP to calculate n-fold down- or up-modulation, respectively. The lowest panel shows the β-galactosidase activity (RLU/s) obtained after infection of P4-CCR5 cells with virus stocks containing 5 ng p24 antigen produced by transfection of HEK293T cells. Results show mean values (+SEM) derived from six to eight measurements in at least two independent experiments. , p < 0.05; , p < 0.01; **, p < 0.001.
	Fig 2. Y86 is critical for SERINC3/5 antagonism by SIVcol Nef.(A) Alignment of the amino acid sequences of HIV and SIV Nef proteins. Dots indicate gaps. Functional amino acid motifs are highlighted. Y residues in SIVcol Nef that were mutated for functional analyses are indicated in green. (B) TZM-bl (left) or P4-CCR5 (right) indicator cells were infected with HIV-1 NL4-3 constructs containing the indicated HIV and SIV nef genes or a defective nef allele. Infections were performed in triplicate with three different virus stocks. Shown are mean values of the nine measurements +SEM. (C) HEK293T cells were co-transfected with HIV-1 NL4-3 constructs encoding indicated Nef and SERINC5 (left) or SERINC3 (right) or control plasmid. Shown are the mean levels of infectious virus production by the respective IMCs in the presence of transient SERINC5 or SERINC3 expression (+SEM; n = 9) relative to those obtained in cells transfected with the control vector (100%). Results were derived from three experiments, each using triplicate infection of TZM-bl cells to determine infectious virus yield. P-values represent reduction of infectious virus yield by SERINC expression or differences in susceptibility between wt and nef-defective HIV-1 IMCs. , p < 0.05; , p < 0.01; **, p < 0.001. (D) Expression of HIV-1 and SIVcol Nef proteins. Western blot analysis of cell lysates following transfection of HEK293T cells with pCG vectors expressing AU1-tagged versions of the indicated Nef proteins. Actin and eGFP are shown as loading and transfection controls, respectively.
	Fig 3. Effect of SIVcol Nef on antiviral activity of vertebrate SERINC5 proteins.Infectious virus yield from HEK293T cells co-transfected with proviral HIV-1 NL4-3 IRES eGFP constructs encoding the indicated nef alleles and pBJ5 SERINC expression or control plasmid. Shown are the mean levels of infectious virus production by the respective IMCs in the presence of transient SERINC5 expression (+SEM; n = 9) relative to those obtained in cells transfected with the control vector (100%). The results were derived from three independent experiments, each using triplicate infection of TZM-bl cells to determine infectious virus yield.
	Fig 4. Anti-tetherin activity of SIVcol Nef.(A) Alignment of tetherin amino acid sequences from humans (HUM), chimpanzees (CPZ), rhesus macaques (MAC) and colobus monkeys (COL). Amino acid identity is indicated by dots and gaps by dashes. Differences between COL and other tetherins in the TM domain are highlighted by yellow boxes. Some known domains, the serine GPI anchor site (red box), and two potential glycosylation sites (underlined) are indicated. (B, C) Release of p24 capsid antigen (B) and infectious virus yield (C) from HEK293T cells following transfection with a ΔvpuΔnef proviral HIV-1 NL4-3 construct, SIVcol Nef or GFP only (nef*) expression constructs, and varying amounts of plasmids expressing human or Colobus tetherin. Virus release was determined by ELISA of cell-free and cell-associated p24 antigen and infectious virus was determined by infection of TZM-bl indicator cells. Both are shown as percentages of those detected in the absence of tetherin (100%) and all values were derived from triplicate experiments ± SEM.
	Fig 5. Modulation of T cell properties by SIVcol Nef variants.(A) Human PBMCs were transduced with VSV-G pseudotyped HIV-1 NL4-3 constructs coexpressing the indicated nef alleles and eGFP and assayed for surface expression of CXCR4 two days later. The mean APC fluorescence intensity values used to calculate receptor modulation are indicated. (B, C) Quantitative assessment of surface (B) and total (C) cellular levels of CXCR4 in infected PBMCs. Values give means + SEM derived from four donors. (D) Cell surface CXCR4 expression and chemotaxis (E) of Jurkat T (CCR7) cells. CXCR4 modulation was calculated in transfected GFP expressing cells for each condition to their respective untransfected cells (100%). Chemotaxis was determined in a transwell system (5μm pore size) towards SDF-1α (10 ng/mL) for 2 h after starvation in hunger medium (0.5% FCS). The percentage of migrated cells was calculated from % GFP expressing cells after migration relative to % GFP expressing cells given as input per condition. Shown are mean values with standard error mean of three independent experiments each performed in duplicates. (F) Representative widefield images and frequency of Lck retargeting assay in transient Nef expressing Jurkat-TAg T cells and control. Cells were plated onto coverslips, fixed, permeabilized and stained for endogenous Lck. The frequency of cells showing retargeting of Lck to the perinuclear region was determined by counting at least 100 cells per condition. (G) Representative widefield images and quantification of peripheral F-actin ring formation by Jurkat-TAg T cells upon activation on anti-CD3 coated coverslips. The right panel shows quantification of samples shown in the images counting at least 100 cells per condition. () Asterisks indicate GFP-positive cells. Bar diagrams show mean values with standard deviation of three independent experiments. Scale bar = 5 μm. , p < 0.05; , p < 0.01; *, p < 0.001.
	Fig 6. Effect of SIVcol Nef on SERINC5 expression and virion incorporation.(A) The upper panel shows the infectivity of HIV-1 particles produced in HEK293T cells relative to the nef-defective control vector (100%). The lower panel shows Western blot analysis of lysates of the corresponding producer cells and purified virions. (B) Quantification of SERINC5 levels in virions determined by densitometry of Western blot analysis. Depicted are means (+SEM) from at least four independent experiments. (C) Surface and total cellular SERINC5 levels in Jurkat T cells containing an HA-coding sequence in exon 8 of the serinc5 alleles (corresponding to the predicted fourth extracellular loop of SERINC5) introduced by CRISPR/Cas9-assisted gene editing infected with NL4-3 IRES eGFP expressing the indicated Nef proteins. Cells were left untreated or treated with MG132 or ammonium chloride, which inhibit proteasomal and lysosomal degradation, respectively. Protein levels were determined at 2 days post-infection. Shown are means of four experiments (+SEM). , p < 0.05; , p < 0.01; **, p < 0.001.
	Fig 7. SIVcol Nef relocalizes SERINC5 to the proteasome by an Y86 dependent mechanism.(A) Representative Laser scanning confocal microscopy images of JTAg SERINC3/5 knock-out cells transfected with SERINC5–GFP (S5-GFP; green) alone or together with the indicated AU1-tagged Nef proteins (red). Endogenous lysosomes were stained with an anti-LAMP1 antibody (blue). (B) Quantification of internal SERINC-GFP fluorescence versus surface SERINC-GFP fluorescence from (A) displayed as means of n = 5 (+SD). (C) Quantification of the pixel area of LAMP1 staining from (A) in triplicates (+SD). (D) Representative Laser scanning confocal microscopy images of JTAg SERINC3/5 knock-out cells transfected with SERINC5–GFP (green) together with indicated AU1-tagged Nef (red) and endogenous proteasomes (anti-PSMA5, blue). Insets show magnifications of the highlighted areas. Size bar, 2 μm. (E) Calculation of Pearson’s co-localization coefficients using Costes thresholds for PSMA5 (proteasome) and SERINC5-GFP for images in (D). Displayed as means of triplicates (+SD). (F) Representative Laser scanning confocal microscopy images (left) and quantitative analysis (right) of JTAg SERINC3/5 knock-out cells transfected with SERINC5–GFP (green) together with AU1-tagged SIVcol Nef wt (red) and either treated with MG132 (10 μM for 3 h) or mock treated. Nuclei are stained with Hoechst (blue). (G) HEK293T B0166 MaMTH reporter cells were co-transfected with 25 ng Bait (Nef) and 25 ng Prey (SERINC5) DNA. After 24 h, protein expression was induced by adding 0.5 μg/ml tetracycline and 40 h post-transfection, Gaussia luciferase luminescence (left) was measured in three independent experiments, each using triplicates of transfection (+SEM) to determine the level of protein interaction as compared to positive control (transcription factor only, Gal4) and EGFR/SHC1 proteins known to interact with each other. Negative control cells co-transfected with each Nef Bait with Pex7 Prey were used to substract non-specific background interaction signal. Western blot (right) shows the expression of V5-tagged SIVcol Nef Baits (WT or Y86F) and FLAG-tagged SERINC5 Prey (S5) constructs in HEK293T B0166 cells. Mo = mock cells. , p < 0.05; , p < 0.01; **, p < 0.001.
	Fig 8. SERINC5 antagonism does not enhance viral replication in primary human CD4+ T cell cultures.(A) Stimulated primary CD4+ T cells were transduced with equal p24 amounts of VSV-G pseudotyped NL4-3 viruses carrying the indicated HIV/SIV nef gene or mutant thereof. Cells were maintained in culture for up to 10 days post-transduction under static conditions (left panel) or with agitation (right panel) to limit cell-to-cell viral spread. Every 2 days, samples of cell culture medium from triplicate wells were harvested to determine infectious virus yield by TZM-bl reporter cell assay and CD4+ T cell infection rates were assessed by intracellular p24 staining followed by flow cytometric analysis. Data shown represents measurements obtained from 3 donors (mean ±SEM). (B) Correlation between the infection rates of TZM-bl and primary CD4+ T cells infected with HIV-1 IRES-eGFP nef recombinants produced in the presence of transiently expressed SERINC5 in HEK293T cells. (C) The infectious virus and p24 antigen yields at 4, 6 and 8 days post-infection in the standard CD4+ T cell infection experiments shown in panel A were determined by TZM-bl infection and p24 antigen ELISA, respectively. Virion infectivity normalized for p24 content is shown relative to the nef-defective HIV-1 construct (100%). Shown are average values obtained at three time points for the three blood donors (+SEM). p < 0.05; *p < 0.01.
	Fig 9. Effect of SERINC5 antagonism on HIV-1 replication in PBMC cultures.(A) Human PBMC were transduced with equal p24 amounts of VSV-G pseudotyped NL4-3 viruses carrying the indicated HIV/SIV nef gene or mutant thereof. Cells were activated with IL-2 and PHA either 3 days prior to transduction (preactivated) or 3 days and 6 days post infection (dpi). Every 3 days, samples of cell culture medium from triplicate wells were harvested to determine infectious virus yield and RT activity. (B) Relative infectivity of virions produced in PBMCs. Results were calculated by dividing infectious virus yield values obtained from TZM-bl reporter infectivity assay by corresponding RT activity values measured by RT radioactivity-based assay of the cell culture supernatants from day 12. (C) SERINC5 mRNA levels in PBMCs cultured in the presence of 2 μg/ml PHA and/or 10 ng/ml IL-2. Gene expression was measured through qRT-PCR and the values were normalized to internal GAPDH control as well as unstimulated PBMCs (CTRL) of the corresponding donor. Data shown represents measurements obtained from 4 donors (mean ±SEM). , p < 0.01; *, p < 0.001.
	Fig 10. Effect of SIVcol Nef on viral replication in ex vivo HLT and NF-κB or NF-AT activity.(A) Replication kinetics of HIV-1 constructs containing the indicated nef alleles in blocks of human lymphoid tissues ex vivo. Panels A to C show mean values (±SEM) obtained using tissues from seven different donors. (B) Cumulative virus production over 15 days of infection by the indicated HIV-1 constructs (see panel A) relative to the replication of the HIV-1 construct containing the NA7 nef allele (100%). (C) Levels of CD4+ T cell depletion in the tissue blocks (left) and cells that migrated in the gel foams (right) at the end of culture at 15 days post-infection. (D) HEK293T cells were cotransfected with a firefly luciferase reporter construct under the control of three NF-κB binding sites, a Gaussia luciferase construct for normalization, and expression vectors for a constitutively active mutant of IKKβ and the indicated Nef variants. Luciferase activity was determined 40 h post-transfection. The mean value of 9 transfections + SEM is shown. (E) Jurkat cells stably transfected with an NF-AT-dependent luciferase reporter gene were transduced with the indicated HIV-1 Nef-IRES-eGFP variants. The levels of luciferase activity were determined at 16 h post-stimulation. Shown are average values (+SD) derived from triplicate transductions relative to the nef-defective control HIV-1 construct (100%). Similar results were obtained in two independent experiments. , p < 0.05; **, p < 0.001.

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	Fig 1. Phylogenetic position and functional activity of SIVcol Nef proteins.(A) Phylogenetic relationship of Nef amino acid sequences from different primate lentiviruses. Stars on branches indicate ≥90 percentage estimated posterior probabilities. The length of a branch indicates the phylogenetic distance to its origin. (B) Modulation of various receptors and viral infectivity enhancement by SIVcol Nefs. To measure receptor modulation, human PBMCs, Jurkat (for CXCR4) or THP-1 cells (for Ii) were transduced with HIV-1 NL4-3 IRES-eGFP constructs containing the indicated nef alleles. The mean channel numbers of red fluorescence obtained for cells transfected with the nef-defective HIV-1 construct were divided by the corresponding numbers obtained for cells infected with viral constructs coexpressing Nef and eGFP to calculate n-fold down- or up-modulation, respectively. The lowest panel shows the β-galactosidase activity (RLU/s) obtained after infection of P4-CCR5 cells with virus stocks containing 5 ng p24 antigen produced by transfection of HEK293T cells. Results show mean values (+SEM) derived from six to eight measurements in at least two independent experiments. , p < 0.05; , p < 0.01; **, p < 0.001.
	Fig 2. Y86 is critical for SERINC3/5 antagonism by SIVcol Nef.(A) Alignment of the amino acid sequences of HIV and SIV Nef proteins. Dots indicate gaps. Functional amino acid motifs are highlighted. Y residues in SIVcol Nef that were mutated for functional analyses are indicated in green. (B) TZM-bl (left) or P4-CCR5 (right) indicator cells were infected with HIV-1 NL4-3 constructs containing the indicated HIV and SIV nef genes or a defective nef allele. Infections were performed in triplicate with three different virus stocks. Shown are mean values of the nine measurements +SEM. (C) HEK293T cells were co-transfected with HIV-1 NL4-3 constructs encoding indicated Nef and SERINC5 (left) or SERINC3 (right) or control plasmid. Shown are the mean levels of infectious virus production by the respective IMCs in the presence of transient SERINC5 or SERINC3 expression (+SEM; n = 9) relative to those obtained in cells transfected with the control vector (100%). Results were derived from three experiments, each using triplicate infection of TZM-bl cells to determine infectious virus yield. P-values represent reduction of infectious virus yield by SERINC expression or differences in susceptibility between wt and nef-defective HIV-1 IMCs. , p < 0.05; , p < 0.01; **, p < 0.001. (D) Expression of HIV-1 and SIVcol Nef proteins. Western blot analysis of cell lysates following transfection of HEK293T cells with pCG vectors expressing AU1-tagged versions of the indicated Nef proteins. Actin and eGFP are shown as loading and transfection controls, respectively.
	Fig 3. Effect of SIVcol Nef on antiviral activity of vertebrate SERINC5 proteins.Infectious virus yield from HEK293T cells co-transfected with proviral HIV-1 NL4-3 IRES eGFP constructs encoding the indicated nef alleles and pBJ5 SERINC expression or control plasmid. Shown are the mean levels of infectious virus production by the respective IMCs in the presence of transient SERINC5 expression (+SEM; n = 9) relative to those obtained in cells transfected with the control vector (100%). The results were derived from three independent experiments, each using triplicate infection of TZM-bl cells to determine infectious virus yield.
	Fig 4. Anti-tetherin activity of SIVcol Nef.(A) Alignment of tetherin amino acid sequences from humans (HUM), chimpanzees (CPZ), rhesus macaques (MAC) and colobus monkeys (COL). Amino acid identity is indicated by dots and gaps by dashes. Differences between COL and other tetherins in the TM domain are highlighted by yellow boxes. Some known domains, the serine GPI anchor site (red box), and two potential glycosylation sites (underlined) are indicated. (B, C) Release of p24 capsid antigen (B) and infectious virus yield (C) from HEK293T cells following transfection with a ΔvpuΔnef proviral HIV-1 NL4-3 construct, SIVcol Nef or GFP only (nef*) expression constructs, and varying amounts of plasmids expressing human or Colobus tetherin. Virus release was determined by ELISA of cell-free and cell-associated p24 antigen and infectious virus was determined by infection of TZM-bl indicator cells. Both are shown as percentages of those detected in the absence of tetherin (100%) and all values were derived from triplicate experiments ± SEM.
	Fig 5. Modulation of T cell properties by SIVcol Nef variants.(A) Human PBMCs were transduced with VSV-G pseudotyped HIV-1 NL4-3 constructs coexpressing the indicated nef alleles and eGFP and assayed for surface expression of CXCR4 two days later. The mean APC fluorescence intensity values used to calculate receptor modulation are indicated. (B, C) Quantitative assessment of surface (B) and total (C) cellular levels of CXCR4 in infected PBMCs. Values give means + SEM derived from four donors. (D) Cell surface CXCR4 expression and chemotaxis (E) of Jurkat T (CCR7) cells. CXCR4 modulation was calculated in transfected GFP expressing cells for each condition to their respective untransfected cells (100%). Chemotaxis was determined in a transwell system (5μm pore size) towards SDF-1α (10 ng/mL) for 2 h after starvation in hunger medium (0.5% FCS). The percentage of migrated cells was calculated from % GFP expressing cells after migration relative to % GFP expressing cells given as input per condition. Shown are mean values with standard error mean of three independent experiments each performed in duplicates. (F) Representative widefield images and frequency of Lck retargeting assay in transient Nef expressing Jurkat-TAg T cells and control. Cells were plated onto coverslips, fixed, permeabilized and stained for endogenous Lck. The frequency of cells showing retargeting of Lck to the perinuclear region was determined by counting at least 100 cells per condition. (G) Representative widefield images and quantification of peripheral F-actin ring formation by Jurkat-TAg T cells upon activation on anti-CD3 coated coverslips. The right panel shows quantification of samples shown in the images counting at least 100 cells per condition. () Asterisks indicate GFP-positive cells. Bar diagrams show mean values with standard deviation of three independent experiments. Scale bar = 5 μm. , p < 0.05; , p < 0.01; *, p < 0.001.
	Fig 6. Effect of SIVcol Nef on SERINC5 expression and virion incorporation.(A) The upper panel shows the infectivity of HIV-1 particles produced in HEK293T cells relative to the nef-defective control vector (100%). The lower panel shows Western blot analysis of lysates of the corresponding producer cells and purified virions. (B) Quantification of SERINC5 levels in virions determined by densitometry of Western blot analysis. Depicted are means (+SEM) from at least four independent experiments. (C) Surface and total cellular SERINC5 levels in Jurkat T cells containing an HA-coding sequence in exon 8 of the serinc5 alleles (corresponding to the predicted fourth extracellular loop of SERINC5) introduced by CRISPR/Cas9-assisted gene editing infected with NL4-3 IRES eGFP expressing the indicated Nef proteins. Cells were left untreated or treated with MG132 or ammonium chloride, which inhibit proteasomal and lysosomal degradation, respectively. Protein levels were determined at 2 days post-infection. Shown are means of four experiments (+SEM). , p < 0.05; , p < 0.01; **, p < 0.001.
	Fig 7. SIVcol Nef relocalizes SERINC5 to the proteasome by an Y86 dependent mechanism.(A) Representative Laser scanning confocal microscopy images of JTAg SERINC3/5 knock-out cells transfected with SERINC5–GFP (S5-GFP; green) alone or together with the indicated AU1-tagged Nef proteins (red). Endogenous lysosomes were stained with an anti-LAMP1 antibody (blue). (B) Quantification of internal SERINC-GFP fluorescence versus surface SERINC-GFP fluorescence from (A) displayed as means of n = 5 (+SD). (C) Quantification of the pixel area of LAMP1 staining from (A) in triplicates (+SD). (D) Representative Laser scanning confocal microscopy images of JTAg SERINC3/5 knock-out cells transfected with SERINC5–GFP (green) together with indicated AU1-tagged Nef (red) and endogenous proteasomes (anti-PSMA5, blue). Insets show magnifications of the highlighted areas. Size bar, 2 μm. (E) Calculation of Pearson’s co-localization coefficients using Costes thresholds for PSMA5 (proteasome) and SERINC5-GFP for images in (D). Displayed as means of triplicates (+SD). (F) Representative Laser scanning confocal microscopy images (left) and quantitative analysis (right) of JTAg SERINC3/5 knock-out cells transfected with SERINC5–GFP (green) together with AU1-tagged SIVcol Nef wt (red) and either treated with MG132 (10 μM for 3 h) or mock treated. Nuclei are stained with Hoechst (blue). (G) HEK293T B0166 MaMTH reporter cells were co-transfected with 25 ng Bait (Nef) and 25 ng Prey (SERINC5) DNA. After 24 h, protein expression was induced by adding 0.5 μg/ml tetracycline and 40 h post-transfection, Gaussia luciferase luminescence (left) was measured in three independent experiments, each using triplicates of transfection (+SEM) to determine the level of protein interaction as compared to positive control (transcription factor only, Gal4) and EGFR/SHC1 proteins known to interact with each other. Negative control cells co-transfected with each Nef Bait with Pex7 Prey were used to substract non-specific background interaction signal. Western blot (right) shows the expression of V5-tagged SIVcol Nef Baits (WT or Y86F) and FLAG-tagged SERINC5 Prey (S5) constructs in HEK293T B0166 cells. Mo = mock cells. , p < 0.05; , p < 0.01; **, p < 0.001.
	Fig 8. SERINC5 antagonism does not enhance viral replication in primary human CD4+ T cell cultures.(A) Stimulated primary CD4+ T cells were transduced with equal p24 amounts of VSV-G pseudotyped NL4-3 viruses carrying the indicated HIV/SIV nef gene or mutant thereof. Cells were maintained in culture for up to 10 days post-transduction under static conditions (left panel) or with agitation (right panel) to limit cell-to-cell viral spread. Every 2 days, samples of cell culture medium from triplicate wells were harvested to determine infectious virus yield by TZM-bl reporter cell assay and CD4+ T cell infection rates were assessed by intracellular p24 staining followed by flow cytometric analysis. Data shown represents measurements obtained from 3 donors (mean ±SEM). (B) Correlation between the infection rates of TZM-bl and primary CD4+ T cells infected with HIV-1 IRES-eGFP nef recombinants produced in the presence of transiently expressed SERINC5 in HEK293T cells. (C) The infectious virus and p24 antigen yields at 4, 6 and 8 days post-infection in the standard CD4+ T cell infection experiments shown in panel A were determined by TZM-bl infection and p24 antigen ELISA, respectively. Virion infectivity normalized for p24 content is shown relative to the nef-defective HIV-1 construct (100%). Shown are average values obtained at three time points for the three blood donors (+SEM). p < 0.05; *p < 0.01.
	Fig 9. Effect of SERINC5 antagonism on HIV-1 replication in PBMC cultures.(A) Human PBMC were transduced with equal p24 amounts of VSV-G pseudotyped NL4-3 viruses carrying the indicated HIV/SIV nef gene or mutant thereof. Cells were activated with IL-2 and PHA either 3 days prior to transduction (preactivated) or 3 days and 6 days post infection (dpi). Every 3 days, samples of cell culture medium from triplicate wells were harvested to determine infectious virus yield and RT activity. (B) Relative infectivity of virions produced in PBMCs. Results were calculated by dividing infectious virus yield values obtained from TZM-bl reporter infectivity assay by corresponding RT activity values measured by RT radioactivity-based assay of the cell culture supernatants from day 12. (C) SERINC5 mRNA levels in PBMCs cultured in the presence of 2 μg/ml PHA and/or 10 ng/ml IL-2. Gene expression was measured through qRT-PCR and the values were normalized to internal GAPDH control as well as unstimulated PBMCs (CTRL) of the corresponding donor. Data shown represents measurements obtained from 4 donors (mean ±SEM). , p < 0.01; *, p < 0.001.
	Fig 10. Effect of SIVcol Nef on viral replication in ex vivo HLT and NF-κB or NF-AT activity.(A) Replication kinetics of HIV-1 constructs containing the indicated nef alleles in blocks of human lymphoid tissues ex vivo. Panels A to C show mean values (±SEM) obtained using tissues from seven different donors. (B) Cumulative virus production over 15 days of infection by the indicated HIV-1 constructs (see panel A) relative to the replication of the HIV-1 construct containing the NA7 nef allele (100%). (C) Levels of CD4+ T cell depletion in the tissue blocks (left) and cells that migrated in the gel foams (right) at the end of culture at 15 days post-infection. (D) HEK293T cells were cotransfected with a firefly luciferase reporter construct under the control of three NF-κB binding sites, a Gaussia luciferase construct for normalization, and expression vectors for a constitutively active mutant of IKKβ and the indicated Nef variants. Luciferase activity was determined 40 h post-transfection. The mean value of 9 transfections + SEM is shown. (E) Jurkat cells stably transfected with an NF-AT-dependent luciferase reporter gene were transduced with the indicated HIV-1 Nef-IRES-eGFP variants. The levels of luciferase activity were determined at 16 h post-stimulation. Shown are average values (+SD) derived from triplicate transductions relative to the nef-defective control HIV-1 construct (100%). Similar results were obtained in two independent experiments. , p < 0.05; **, p < 0.001.