Expression, purification and virucidal activity of two recombinant isoforms of phospholipase A2 from Crotalus durissus terrificus venom

Abstract

The global emergence and re-emergence of arthropod-borne viruses (arboviruses) over the past four decades have become a public health crisis of international concern, especially in tropical and subtropical countries. A limited number of vaccines against arboviruses are available for use in humans; therefore, there is an urgent need to develop antiviral compounds. Snake venoms are rich sources of bioactive compounds with potential for antiviral prospection. The major component of Crotalus durissus terrificus venom is a heterodimeric complex called crotoxin, which is constituted by an inactive peptide (crotapotin) and a phospholipase A 2 (PLA 2-CB). We showed previously the antiviral effect of PLA 2-CB against dengue virus, yellow fever virus and other enveloped viruses. The aims of this study were to express two PLA 2-CB isoforms in a prokaryotic system and to evaluate their virucidal effects. The sequences encoding the PLA 2-CB isoforms were optimized and cloned into a plasmid vector (pG21a) for recombinant protein expression. The recombinant proteins were expressed in the E. coli BL21(DE3) strain as insoluble inclusion bodies; therefore, the purification was performed under denaturing conditions, using urea for protein solubilization. The solubilized proteins were applied to a nickel affinity chromatography matrix for binding. The immobilized recombinant proteins were subjected to an innovative protein refolding step, which consisted of the application of a decreasing linear gradient of urea and dithiothreitol (DTT) concentrations in combination with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) as a protein stabilizer. The refolded recombinant proteins showed phospholipase activity and virucidal effects against chikungunya virus, dengue virus, yellow fever virus and Zika virus.

Organometallic Complex Strongly Impairs Chikungunya Virus Entry to the Host Cells

“…The virucidal effect was the strongest activity observed for RcP which might suggest that an anti-CHIKV mechanism of action for this complex might be related to a direct action on the viral particle envelope. Previous studies have shown that the virucidal effect of several compounds is closely related to an action on the virus envelope (Tang et al, 1990;Schuhmacher et al, 2003;Carravilla et al, 2017;Kong et al, 2019;Russo et al, 2019). This suggests the existence of interactions between CHIKV envelope glycoproteins and RcP.…”

What do secreted phospholipases A2 have to offer in combat against different viruses up to SARS-CoV-2?

“…Crotoxin and its CB subunit had antiviral activity not only against Flaviviridae viruses (such as dengue, yellow fever and Zika) but also against Peribunyaviridae and Togaviridae viruses whose envelopes are different, originating from Golgi and plasma membranes, respectively. For example, two recombinant enzymatically active CB variants, CB1 and CB2, showed antiviral effects, not only against dengue, yellow fever and Zika viruses, but also against chikungunya virus ( Togaviridae ) [ 99 ]. In a separate study [ 100 ], crotoxin and its CB subunit were effective against in vitro infection with hepatitis C virus ( Flaviviridae ).…”

Antiviral Strategies against Arthritogenic Alphaviruses

“…Several natural products have been reported to exert virucidal activity against arthritogenic alphaviruses [ 44 , 45 , 46 ]. Such compounds directly interact with the viral particle, leading to virus inactivation.…”

“…Such compounds directly interact with the viral particle, leading to virus inactivation. The phospholipases A2 (PLA2s) from snake ( Crotalus durissus terrificus ) venom have been reported to have a virucidal effect against several enveloped viruses, including CHIKV [ 44 ]. Another example is ginkgolic acid, isolated from the Ginkgo biloba plant, which has been recently shown to inhibit the in vitro replication of CHIKV and MAYV through a direct virucidal effect [ 45 ].…”