Anti-HIV Activity of Snake Venom Phospholipase A2s: Updates for New Enzymes and Different Virus Strains

Abstract

Since the beginning of the HIV epidemic, lasting more than 30 years, the main goal of scientists was to develop effective methods for the prevention and treatment of HIV infection. Modern medicines have reduced the death rate from AIDS by 80%. However, they still have side effects and are very expensive, dictating the need to search for new drugs. Earlier, it was shown that phospholipases A2 (PLA2s) from bee and snake venoms block HIV replication, the effect being independent on catalytic PLA2 activity. However, the antiviral activity of human PLA2s against Lentiviruses depended on catalytic function and was mediated through the destruction of the viral membrane. To clarify the role of phospholipolytic activity in antiviral effects, we analyzed the anti-HIV activity of several snake PLA2s and found that the mechanisms of their antiviral activity were similar to that of mammalian PLA2. Our results indicate that snake PLA2s are capable of inhibiting syncytium formation between chronically HIV-infected cells and healthy CD4-positive cells and block HIV binding to cells. However, only dimeric PLA2s had pronounced virucidal and anti-HIV activity, which depended on their catalytic activity. The ability of snake PLA2s to inactivate the virus may provide an additional barrier to HIV infection. Thus, snake PLA2s might be considered as candidates for lead molecules in anti-HIV drug development.

Keywords: antiviral activity; human immunodeficiency virus; phospholipase A2; pseudovirus; recombinant form; retrovirus; snake venom; syncytium.

Figure 1

The antiviral activity of PLA2s against HIV-1 IIIB. PLA2s at different concentrations were added to MT-4 cells, followed by HIV-1 inoculation at 100 TCID₅₀ (fifty percent tissue culture infective dose). Inhibition of the cytopathic effect (CPE) was determined using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) test 5 days after infection. (A) Activity of five investigated PLA2s against HIV-induced CPE. (B) Antiviral activity of dimeric HDP-2 and its subunits against HIV-1. Results are representative of at least three independent experiments with the mean ± SEM.

Figure 2

Antiviral activity of PLA2s against different strains and molecular clones of HIV. (A–D) PLA2s at different dilutions were added to the MT-4 cells; after which, the cells were infected with the corresponding HIV strain at 100 TCID₅₀. CPE inhibition was determined after 48–72 h of infection using the MTT method. (E,F) The inhibition of the replication of infectious molecular clones using PLA2s was determined on TZM-bl cells after 48 h of infection by the activity of the β-Gal reporter gene. Data are the mean ± SEM (n = 3).

Figure 3

Antiviral activity of various PLA2s against the HIV-1 pseudoviruses. Various concentrations of PLA2s were added to TZM-bl target cells and transduced with pseudotyped HIV-1 virions, representing (A) sub-subtype A6 or (B) the recombinant form of CRF02_AG/A6. Infectivity was assessed with a β-Gal reporter gene activity after 48 h of infection. Data are the mean ± SEM from triplicates.

Figure 4

Effect of PLA2s on syncytium formation. Sup-T1 cells and HIV-1 chronically infected cells were mixed and treated with PLA2s. The levels of cell fusion were estimated after 24 h under a microscope. The inhibition of syncytium formation by PLA2s is shown for (A) CEM cells infected with HIV-1 Bru, (B) H9 cells infected with HIV-1 RF, and (C) H9 cells infected with HIV-1 IIIB. (D) The blockage of BF-PLA2-II enzymatic activity resulted in the decrease of the inhibition of syncytium formation by H9 cells infected with HIV-1 IIIB. The data represent the mean ± SEM from three independent experiments. Student’s t-test: *** p < 0.001, ^ns p > 0.05.

Figure 5

Virucidal activity of HDP-2 and the blocking of HIV-1 adsorption. (A) The HIV-1 stock was treated with different concentrations of HDP-2 and then used to infect MT-4 cells for 5 days. The antiviral effect of HDP-2 was assessed by determining the titer of the virus based on the CPE. Data represent the means ± SEM for three independent experiments. One-way ANOVA with Tukey’s post hoc test: * p ˂ 0.05 and ** p ˂ 0.01. (B) Inhibitory effects of HDP-2 and its subunits on HIV-1 IIIB binding to MT-4 cells. Data are presented as the mean ± SEM (n = 3).

Figure 6

Combinations of HDP-2 with (A) 3TC or (B) TDF inhibit HIV-1-mediated CPE in MT-4 cells. The interaction landscapes of the drug combinations are presented as dose–response matrices. Synergy distribution and 3D synergy landscapes are shown. The most synergistic area is displayed on the matrices.