Antitumor and antibacterial properties of virally encoded cationic sequences

Abstract

Objective: The objective of this study was to test our Viral Quinta Columna Strategy (VQCS), a new biological hypothesis predicting that specific multifunctional virally encoded cationic domains may have the capacity to penetrate human cells and interact with PP2A proteins to deregulate important human intracellular pathways, and may display LL37 cathelicidin-like antagonistic effects against multiple pathogens such as bacteria or viruses. Methods: We comparatively analyzed the host defense properties of adenodiaphorins and of some specific cationic sequences encoded by different viruses using two distinct biological models: U87G, a well-characterized cell tumor model; and a group B Streptococcus agalactiae NEM316 ΔdltA, highly sensitive to LL37 cathelicidin. Results: We found that the adenovirus type 2 E4orf4 is a cell-permeable protein containing a new E4orf4_64-95 protein transduction domain, named large adenodiaphorin or LadD_64-95. Interestingly, the host defense LL37 peptide is the unique cathelicidin in humans. In this context, we also demonstrated that similarly to LL37 LadD_64-95, several virally encoded cationic sequences including the C-terminus HIV-1 89.6 Vpr_77-92, shorter adenodiaphorins AdD_67-84/AdD/_69-84/AdD_69-83, as well as HIV-2 Tat_67-90 and JC polyomavirus small t_115-134, displayed similar toxicity against Gram-positive S. agalactiae NEM316 ΔdltA strain. Finally, LadD_64-95, adenodiaphorin AdD_67-84, AdD_69-84, and LL37 and LL_17-32 cathelicidin peptides also inhibited the survival of human U87G glioblastoma cells. Conclusion: In this study, we demonstrated that specific cationic sequences encoded by four different viruses displayed antibacterial activities against S. agalactiae NEM316 ΔdltA strain. In addition, HIV-1 Vpr_71-92 and adenovirus 2 E4orf4_64-95, two cationic penetrating sequences that bind PP2A, inhibited the survival of U87G glioblastoma cells. These results illustrate the host defense properties of virally encoded sequences and could represent an initial step for future complete validation of the VQCS hypothesis.

Keywords: PP2A; bacteria; cancer; cationic sequences; viruses.

Figure 1

Effects of human adenovirus type 2 E4orf4 peptides on intracellular delivery of streptavidin–peroxidase in human U87G glioblastoma cells and in human DHF cells. Streptavidin–peroxidase coupled with 125 nM of biotinylated peptides was incubated at 37°C for 6 hours (upper panel) or 0–6 hours (middle panel) with U87G, and for 6 hours (lower panel) with DHF cells. Internalized complexes were visualized by a colorimetric test, OPD, as described previously., HRP internalization of E4orf4 peptides is expressed as % of Tat-mediated HRP peptide (incubated for 6 hours) used as positive control. SD is shown for n=3. For negative control (Control), no peptide, no HRP, HRP alone, or cargo-inactive DPT-sh1 peptide (VKKKIKREIKI) was used, giving similar results. 6.88±0.96 ng and 4.29±0.82 ng of HRP, respectively, were internalized by 105 U87G (upper and middle panels) and by 105 DHF (lower panel) cells following 6-hour incubation with 125 ng of biotinylated-Tat peptide complexed with streptavidin–peroxidase. Abbreviations: adD, adenodiaphorin; DHF, dermal human fibroblast; HRP, horseradish peroxidase; LadD, large adenodiaphorin; OPD, O-phenylenediamine dihydrochloride.

Figure 2

LadD inhibits a constitutively active PI3K/Akt survival pathway in U87G cells. Upper panel: cells were treated for 24 hours with LadD and adD peptides (0–150 μM), and cell viability was assessed by the MTT test (n=3). The lower panel shows a Western blot probed with the antibody specifically recognizing phosphorylated Akt-pSer473 (D9E rabbit mAb from cell signaling). The same blot was reprobed with the antibody specifically recognizing total Akt (C67E rabbit mAb from cell signaling) and with the antibody specifically recognizing HP1γ (2MOD-1G6 mouse mAb from Euromedex) that was used as loading control. Cells were untreated (control) or treated for 5 hours at 37°C with 100 μM or 150 μM of LadD, or with 150 μM of AdD peptides. The Western blot was quantified with ImageJ software, and AKT/HP1γ and pAKT/HP1γ normalized ratios, corresponding to the quotient of AKT or pAKT versus HP1γ expression, are illustrated in a histogram shown in the lower panel (n=3). Abbreviations: adD, adenodiaphorin; DHF, dermal human fibroblast; LadD, large adenodiaphorin; mAb, monoclonal antibody.

Figure 3

Effect of cathelicidin LL-37/LL_17–32, adenodiaphorin_{67–84/69–84/69–83}/deletion mutants, HIV-2 Tat_67–90, and JC polyomavirus small t_115–134 sequences, on viability of U87G glioblastoma cells. U87G cells were treated for 24 hours with the LL37 and LL_17–32 cathelicidin peptides (0–50 μM) (upper panel) or with virally encoded LadD deletion mutants (AdD_67–84, AdD_69–84, and AdD_69–83), HIV-2 Tat_67–90, and JC polyomavirus small t_115–134 cationic peptides (0–150 μM) (lower panel). Cell viability was assessed by the MTT test (n=3). Abbreviations: adD, adenodiaphorin; LadD, large adenodiaphorin.