Nedd4-mediated increase in HIV-1 Gag and Env proteins and immunity following DNA-vaccination of BALB/c mice.

The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins, are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteins with intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 budding have been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function for Nedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 following SHIVSF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immune modulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4+ and CD8+ T cells following SHIVSF162P3-infection of naïve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4 demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca2+/calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfection of Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gag or Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularly with plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 but not anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFNγ but had a trend of increased Gag-specific IL-6, IL-17A and TNFα that was not seen following Env stimulation. Based on our initial findings, Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants further investigation

Differential Effects on Vaccine-Specific Humoral Immune Responses by Nedd4 Co-Adminstration of BALB/c mice.

<p>Intramuscular immunization of BALB/c mice (n = 5 per group; 5–6 week old females) was performed at weeks 0, 2 and 4 with plasmid DNA expressing human Nedd4 alone (Group 1), HIV-1 gag +env (Group 2) or human Nedd4+ HIV-1 gag+env (Group 3) as described (Materials and Methods). Group 4 mice (n = 3) were left un-immunized and served as naïve controls. Mice were sacrificed at week 6 to evaluate serum antibody responses (A, B). Vaccine-induced anti-p24 (A) and anti-gp120 (B) antibody levels were quantitated and reciprocal mean ELISA titers ± standard errors are graphically depicted. Statistical significance, using the Kruskal-Wallis nonparametric test followed by Dunn’s multiple comparison test, was noted in anti-p24 titers (Gag+Env+Nedd4 vs Gag+Env; P<0.05) but not anti-pg120 titers (Gag+Env vs Gag+Env+Nedd4: p>0.05).</p

Effects of Nedd4 Co-Adminstration on Vaccine-Specific Cellular Immune Responses in BALB/c mice.

<p>Intramuscular immunization of BALB/c mice (n = 5 per group; 5–6 week old females) was performed at weeks 0, 2 and 4 with plasmid DNA expressing human Nedd4 alone (Group 1), HIV-1 gag +env (Group 2) or human Nedd4+ HIV-1 gag+env (Group 3) as described (Materials and Methods). Group 4 mice (n = 3) were left un-immunized and served as naïve controls. Mice were sacrificed at week 6 to evaluate splenocyte T-cell responses. Gag (A) and Env (C) peptide (consensus A, consensus B, consensus C)-specific IFNγ production was quantified by ELISPOT assay and mean spots per million splenocyte values ± standard errors are graphically represented. Gag peptide-specific TNFα (B, left panel), IL-6 (B, middle panel), IL-17A (B, right panel) and Env peptide specific TNFα (D, left panel), IL-6 (D, middle panel), IL-17A (D, right panel) cytokines were quantified by CBA from the supernatants of peptide stimulated splenocytes and are represented graphically as mean pg/ml values ± standard error. Statistical significance was assessed using the Kruskal-Wallis nonparametric test followed by Dunn’s multiple comparison test. No statistical difference in IFNγ, TNFα, IL-6 and IL-17A and values were found (Gag+Env vs Gag+Env+Nedd4: p>0.05).</p

Ectopic expression of Nedd4 augments supernatant HIV-1 p24 levels Independent of HECT Domain Function.

<p>293 cells were transfected with HXB2 and a plasmid encoding either rat Nedd4 (rNedd4) or a rat Nedd4 catalytic domain mutant (rNedd4CSmut) and at 48 hr post-transfection, cell supernatants were collected and p24 assays were performed as described (Material and Methods). Schematics highlighting the Ca²⁺/Calmodulin-regulated phospholipid binding domain (C2), WW protein binding domains (WW1–3) and E3 catalytic domain (HECT) for rNedd4 and rNedd4CSmut are shown (A). Mean p24± standard error values, calculated from data obtained from transfections conducted with rNedd4 (n = 7) and rNedd4CSmut (n = 5), are graphically represented (B). Statistical significance was shown for the increased p24 levels by rNedd4 (rNedd4 vs control at 48 hr: p<0.01; rNedd4CSmut vs control at 48 hr; p<0.01) using a one way ANOVA followed by Tukey’s multiple comparison test. No statistical difference was shown for rNedd4CSmut compared to rNedd4 at 48 hr (p>0.05) using this analysis. Immunoblot analysis, using a rabbit anti-mouse Nedd4 antibody, was performed on detergent soluble cell fractions as described (Materials and Methods). Nedd4 and Nedd4CSmut protein bands are indicated (arrows) (C).</p

Increased Levels of Extracellular and Intracellular HIV-1 Gag and Env Proteins by co-expression of Nedd4.

<p>293 cells were transiently transfected with HXB2 and a plasmid encoding human Nedd4 (hNedd4) as described (Materials and Methods). At 32 hr post-transfection, a radio-immunoprecipitation assay (RIPA) was performed by labeling cells for 12.5 hr with ³⁵S-Met, immunoprecipitating target proteins using anti-HIV⁺ human serum, and resolving bands from cell media (A) and lysates (B) using 10% SDS-PAGE and autoradiography at –70°C. Bands corresponding to HIV-1 gp160, gp120, p55 and p24 proteins are shown.</p

Nedd4 expression stabilizes intracellular HIV-1 protein levels but does not increase LTR promoter activity.

<p>Jurkat T cells were transfected with LTR-luciferase and pRL-TK-renilla reporter constructs in the absence or presence of hNedd4 (A). At 24 hr post-transfection, cells were either left unstimulated or stimulated with PMA, ionomycin (Iono) or with PMA+Iono. Jurkat T cells were transfected with LTR-luciferase and pRL-TK-renilla reporter constructs in the absence or presence of hNedd4. At 24 hr post-transfection, cells were either left unstimulated or stimulated with PMA, ionomycin (Iono) or with PMA+Iono. A dual luciferase assay was performed as per the manufacturer’s protocol (Materials and Methods) and mean ± standard error values for n = 3 experiments are shown graphically (A). No statistical significance, using a one way ANOVA followed by Tukey’s multiple comparison test, was shown for the supernatant LTR activity from cells co-transfected in the absence or presence of hNedd4 (p>0.05). Alternatively, 293 cells were transiently transfected with a GFP-Gag fusion construct in the absence or presence of hNedd4 (B). At 24 hr and 48 hr post-transfection, p24 assays were performed as described (Experimental Procedures). Mean p24± standard error values (n = 2) were calculated, graphically represented and statistical significance was shown for GFP-Gag/hNedd4 versus GFP-Gag (at 24 hr, 48 hr: p<0.05). 293 cells were transiently transfected with a GFP-Gag fusion construct in the absence or presence of hNedd4 were treated with cycloheximide at 24 hr post-transfection. Immunoprecipitations were performed using a rabbit anti-GFP polyclonal antibody followed by immunoblotting using a mouse anti-GFP monoclonal antibody on detergent-soluble cell fractions at 0, 6, 12, and 25 hr post-cycloheximide treatment (C).</p

Increased Levels of Extracellular and Intracellular p24 Gag and gp120 Env Proteins by Nedd4.

<p>293 cells were transiently transfected with a plasmid DNA expressing a clade B Mosaic Gag (A) or HIV-1_BaL gp120 (B) and increasing concentrations of hNedd4 as described (Materials and Methods). At 48 hr post-transfection, cell supernatants or cell lysates were assayed for p24 (A) or gp120 (B) by ELISA. Levels (pg/ml) of p24 (A) and gp120 (B) are represented graphically for supernatants (blue line) or cell lysates (red line).</p

Increased Levels of Extracellular and Intracellular HIV-1 Gag and Env Proteins by co-expression of Nedd4.

<p>293 cells were transiently transfected with HXB2 and a plasmid encoding human Nedd4 (hNedd4) as described (Materials and Methods). At 32 hr post-transfection, a radio-immunoprecipitation assay (RIPA) was performed by labeling cells for 12.5 hr with ³⁵S-Met, immunoprecipitating target proteins using anti-HIV⁺ human serum, and resolving bands from cell media (A) and lysates (B) using 10% SDS-PAGE and autoradiography at –70°C. Bands corresponding to HIV-1 gp160, gp120, p55 and p24 proteins are shown.</p

Increased p24 induced by Nedd4 Mediated by C2 domain.

<p>293 cells were transiently transfected with HXB2 and a plasmid encoding human Nedd4 (hNedd4), human Nedd4 C2 deletion mutant (hNedd4C2mut) or a human Nedd4-2 splice variant that lacks the C2 domain (hNedd4-2) as described (Materials and Methods). Cell supernatants were collected at 48 hr post-transfection and p24 assays were performed. A schematic highlighting the Ca²⁺/Calmodulin-regulated phospholipid binding domain (C2), WW protein binding domains (WW1–4) and E3 catalytic domain (HECT) for hNedd4 is shown (A). Mean p24± standard error values were calculated from data obtained from transfections conducted with hNedd4 (n = 3), hNedd4C2mut (n = 3) and hNedd4-2 (n = 4) and are graphically represented (B). Statistical significance was shown for the increased p24 levels by hNedd4 (hNedd4 vs control at 48 hr: p<0.01) using a one way ANOVA followed by Tukey’s multiple comparison test. Statistical significance was not seen for hNedd4C2mut or hNedd4-2 (hNedd4C2mut or hNedd4-2 vs control at 48 hr: p>0.05). Western blot analyses, using a rabbit anti-mouse Nedd4 antibody, was performed to measure Nedd4, Nedd-2 and Nedd4-C2mut protein levels post-transfection as described (Materials and Methods). Nedd4 (dotted arrow), Nedd-2 (arrow), Nedd4-C2mut (arrow head) and actin protein bands are indicated (C). For siRNA experiments, 293 cells were transiently transfected with HXB2 alone or HXB2 with either Nedd4-targeted siRNA or irrelevant control siRNA (Materials and Methods). Following 24 hrs, supernatant p24 levels were measured and mean ± standard error values are graphically represented (D). Western blot analysis of Nedd4 protein levels in 293 cells transfected with HXB2, HXB2/Nedd4± siRNA is shown (E). Statistical significance, using a one way ANOVA followed by Tukey’s multiple comparison test, was shown for the decreased supernatant p24 levels from cells co-transfected with HXB2/siRNA (p<0.05) but not HXB2/control siRNA (p>0.05) as compared to cells with HXB2 alone.</p