Knockdown of PAK3 and SGK2 by shRNAs.

<p>mRNA levels were determined by quantitative reverse transcription PCR analysis at 72 hours after infection with the respective shRNA vectors; control denotes infection with a vector encoding non-target shRNA. Each bar on the bar graph represents the average ± standard deviation of 4 replicates from one of three independent experiments with similar results. mRNA levels were normalized with GAPDH expression; PAK3 and SGK2 protein expression were determined 72 hours after infection using a Western immunoblot. GAPDH protein acted as a protein loading control for each sample. (A) PAK3 shRNA decreased PAK3 mRNA levels in HeLa cells; (B) PAK3 shRNAs reduced PAK3 mRNA expression in DMS-79 cells; (C) PAK3 shRNAs reduced PAK3 protein expression in DMS-79 cells. DMS-79 cell lysates were analyzed with PAK3 monoclonal antibody N-19; (D) SGK2 shRNAs reduced SGK2 mRNA levels in HeLa cells; (E) SGK2 shRNAs reduced SGK2 mRNA levels in GTL16 cells; (F) SGK2 shRNAs reduced SGK2 protein levels in GTL16 cells. GTL16 cell lysates were analyzed with SGK2 monoclonal antibody 3Q-2.</p

Lethality of PAK3 and SGK2 shRNAs to Human Papillomavirus Positive Cervical Cancer Cells Is Independent of PAK3 and SGK2 Knockdown

<div><p>The p21-activated kinase 3 (PAK3) and the serum and glucocorticoid-induced kinase 2 (SGK2) have been previously proposed as essential kinases for human papillomavirus positive (HPV+) cervical cancer cell survival. This was established using a shRNA knockdown approach. To validate PAK3 and SGK2 as potential targets for HPV+ cervical cancer therapy, the relationship between shRNA-induced phenotypes in HPV+ cervical cancer cells and PAK3 or SGK2 knockdown was carefully examined. We observed that the phenotypes of HPV+ cervical cancer cells induced by various PAK3 and SGK2 shRNAs could not be rescued by complement expression of respective cDNA constructs. A knockdown-deficient PAK3 shRNA with a single mismatch was sufficient to inhibit HeLa cell growth to a similar extent as wild-type PAK3 shRNA. The HPV+ cervical cancer cells were also susceptible to several non-human target shRNAs. The discrepancy between PAK3 and SGK2 shRNA-induced apoptosis and gene expression knockdown, as well as cell death stimulation, suggested that these shRNAs killed HeLa cells through different pathways that may not be target-specific. These data demonstrated that HPV+ cervical cancer cell death was not associated with RNAi-induced PAK3 and SGK2 knockdown but likely through off-target effects.</p></div

Study shRNA lentivirus vectors.

<p>^a NA—not available</p><p>Study shRNA lentivirus vectors.</p

Loss of cell viability induced by PAK3 or SGK2 shRNAs.

<p>(A) HeLa cells were infected with PAK3 shRNA at a 1:10 dilution. Cell apoptosis was determined using a caspase 3/7 glo luciferase assay 72 hours after infection. The bar graph presents fold changes of caspase 3/7 activity (luminescence) induced by various shRNAs compared with a no-shRNA control. Data represent the average ± standard deviation of 4 replicates from one of two separate experiments with similar results; (B) Inhibition of proliferation/viability of HeLa cells by PAK3 shRNAs was assessed using a CellTiter blue assay 5 days after infection. The bar graph presents percent viability (fluorescence) of lentiviral shRNA-infected cells compared with a control lentivirus without shRNA expression. Data represent the mean ± standard deviation of three independent experiments; (C) SGK2 lentiviral shRNAs induced HeLa cell apoptosis. Data represent 4 replicates from one of two separate experiments with similar results; (D) SGK2 shRNAs inhibited proliferation/viability of HeLa cells. Data represent the average ± standard deviation of two independent experiments; (E) SGK2 lentiviral shRNAs induced autophagy of HeLa cells. Cells were infected with SGK2 lentiviral shRNAs at 1:16 and 1:32 dilution, respectively. 1 μM Rapamycin was included as an autophagy control on each plate. Cell plates were fixed 72 hour after infection and immunostained for induction of autophagy with a LC3B primary antibody, followed with an Alexa 488-conjugated secondary antibody. Images were captured using the confocal Opera High Content Imager. Images of SGK2 lentiviral shRNA-infected HeLa cells (~1200 cells/well) were captured and cell numbers counted. The average intensity of LC3B staining per cell was measured and calculated using a modified Capella (Perkin Elmer) algorithm. The bar graph presents the average ± standard deviation of LC3B staining intensity derived from two wells.</p

Rescue of SGK2 shRNA-induced phenotypes.

<p>(A) HeLa cells were transfected with serially diluted SGK2 expressing plasmids harboring silent mutations at the shRNA 2111 annealing site. Six hours after transfection, the cells were infected with SGK2 lentiviral shRNA 2111 (1:15 dilution). SGK2 mRNA expression levels were determined 72 hours after infection. The bar graph presents quantities of SGK2 mRNA normalized with GAPDH mRNA. Data represent the average ± standard deviation of 4 replicates from one of two experiments with similar results; (B) HeLa cell apoptosis induced by SGK2 shRNA 2111 was quantified with a caspase 3/7 glo assay 72 hours after infection. Two variants of SGK2 (alpha and beta) were used for the phenotype rescue analysis. Data represent the average ± standard deviation of two independent experiments; (C) HeLa cell proliferation/ viability inhibition induced by SGK2 shRNA 2111 was determined using the CellTiter Blue assay 5 days after infection. Data represents the average ± standard deviation of two independent experiments.</p

Inhibition of HPV+ cervical cancer cells by lentiviral shRNAs that do not target human genes.

<p>Cell viability was assessed using a CellTiter Blue assay 5 days after infection. Each of the viruses was titrated for infection to determine its potency of inducing HeLa cell proliferation/viability inhibition. Percent cell viability (fluorescence) in the presence of lentiviral shRNAs was calculated by comparing with a control lentivirus without shRNA expression. Data represent the average ± standard deviation of 4 replicates from one of three independent experiments with similar results; (A) Inhibition of HeLa cell proliferation/viability; (B) Inhibition of proliferation/viability of CaSki cells.</p

Mechanistic Studies and Modeling Reveal the Origin of Differential Inhibition of Gag Polymorphic Viruses by HIV-1 Maturation Inhibitors

<div><p>HIV-1 maturation inhibitors (MIs) disrupt the final step in the HIV-1 protease-mediated cleavage of the Gag polyprotein between capsid p24 capsid (CA) and spacer peptide 1 (SP1), leading to the production of infectious virus. BMS-955176 is a second generation MI with improved antiviral activity toward polymorphic Gag variants compared to a first generation MI bevirimat (BVM). The underlying mechanistic reasons for the differences in polymorphic coverage were studied using antiviral assays, an LC/MS assay that quantitatively characterizes CA/SP1 cleavage kinetics of virus like particles (VLPs) and a radiolabel binding assay to determine VLP/MI affinities and dissociation kinetics. Antiviral assay data indicates that BVM does not achieve 100% inhibition of certain polymorphs, even at saturating concentrations. This results in the breakthrough of infectious virus (partial antagonism) regardless of BVM concentration. Reduced maximal percent inhibition (MPI) values for BVM correlated with elevated EC₅₀ values, while rates of HIV-1 protease cleavage at CA/SP1 correlated inversely with the ability of BVM to inhibit HIV-1 Gag polymorphic viruses: genotypes with more rapid CA/SP1 cleavage kinetics were less sensitive to BVM. <i>In vitro</i> inhibition of wild type VLP CA/SP1 cleavage by BVM was not maintained at longer cleavage times. BMS-955176 exhibited greatly improved MPI against polymorphic Gag viruses, binds to Gag polymorphs with higher affinity/longer dissociation half-lives and exhibits greater time-independent inhibition of CA/SP1 cleavage compared to BVM. Virological (MPI) and biochemical (CA/SP1 cleavage rates, MI-specific Gag affinities) data were used to create an integrated semi-quantitative model that quantifies CA/SP1 cleavage rates as a function of both MI and Gag polymorph. The model outputs are in accord with <i>in vitro</i> antiviral observations and correlate with observed <i>in vivo</i> MI efficacies. Overall, these findings may be useful to further understand antiviral profiles and clinical responses of MIs at a basic level, potentially facilitating further improvements to MI potency and coverage.</p></div

Modeling of the rate of CA/SP1 cleavage of HIV-1 WT, V370A, V362I and ΔV370 VLP in the presence of 300 nM MI.

<p>Modeled fractional rate of production of SP1 peptide from Gag VLP cleavage using model 2a at 300 nM MI, as noted in text; no MI: diamonds; BVM: squares; BMS-955176: triangles; y-axis: fraction of CA/SP1 cleavage is a surrogate for production of mature virus, as indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005990#ppat.1005990.g005" target="_blank">Fig 5</a>; A) WT; B) V370A; C)V362I; D) ΔV370</p

Scheme for inhibition of HIV-1 infectivity by MIs.

<p>Models that depict the inhibition of mature virus formation by HIV-1 maturation inhibitors. Immature virus B is in equilibrium for binding to MI to produce MI-bound immature form A. In model 1, HIV protease can only cleave immature virus (form B) in the absence of MIs. The innate cleavage rate constant (<i>k</i>₁) is a function of Gag polymorphs. In model 2, HIV-1 protease may cleave CA/SP1 in either form A (bound) or form B (unbound) to produce mature virus C, thus the former pathway provides an escape mechanism for the formation of mature virus in the presence of MIs with rate constant <i>k</i>₂. This second cleavage rate constant (<i>k</i>₂) is a function of both MIs and Gag polymorph, and is derived from inclusion of MPI values from multiple (model 2a) or single (model 2b) cycle antiviral assays (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005990#ppat.1005990.t002" target="_blank">Table 2</a>).</p

Schematic for Cleavage of CA/SP1.

<p>A) Schematic for the processing HIV Gag at CA/SP1 and SP1/NC sites by HIV-protease. B) Detail of the cleavage region around CA/SP1 showing sites for HIV-1 protease cleavage (H1 and H2) and sites for subsequent cleavage by trypsin (T).</p