Cell-penetrating porcine single-chain antibodies (transbodies) against nonstructural protein 1ß (NSP1ß) of porcine reproductive and respiratory syndrome virus inhibit virus replication.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS) worldwide, especially in domestic pigs, with an enormous economic impact, estimated at $664 million in losses every year to the pig industry. Current vaccines confer limited protection, and no direct-acting anti-PRRS treatment is available. Non-structural protein (NSP) 1ß, a cysteine-like protease (CLPro) of PRRSV plays an essential role in viral polyprotein processing, subgenomic RNA synthesis, and evasion of host innate immunity. Therefore, agents that interfere with the bioactivity of NSP1ß would be expected to inhibit virus replication. In this study, a porcine single-chain antibody (scFv)-phage display library was constructed and used as a tool for production of NSP1ß-specific porcine scFvs (pscFvs). The pscFvs to NSP1ß were linked to a cell-penetrating peptide to form cell-penetrating pscFvs (transbodies), which could be internalized and inhibit PRRSV replication in infected cells. A computer simulation indicated that the effective pscFvs used several residues in multiple complementarity determining regions (CDRs) to interact with multiple residues in the CLPro and C-terminal motifs, which might explain the mechanism of pscFv-mediated inhibition of virus replication. Although experiments are needed to determine the antiviral mechanism of the transbodies, the current data indicate that transbodies can potentially be applied for treatment and prevention of PRRSV infection.

Reversion of radiosensitivity in azacytidine-treated XRS5 cells does not result in full radioprotection by WR-1065.

A series of cell lines were previously generated from the radiation sensitive Chinese hamster ovary line xrs5 after treatment with azacytidine. Six of these lines have been examined for their resistance to killing by 0 to 20 Gray of 60Co gamma rays and the amount of radioprotection afforded by treatment with the drug 2-[(aminopropyl)amino]ethanethiol (WR-1065). As xrs5 cells have lost the ability to be protected by WR-1065, studies were performed to determine whether reversion to radio-resistance correlated with recovery of aminothiol radioprotection. Treatment of azacytidine-treated, radiation sensitive and resistant cells with four millimolar WR-1065 30 minutes prior to irradiation enhanced survival after exposure to gamma radiation, although the enhancement in survival was less than for wild type Chinese hamster ovary K1 cells. The data suggest that there is not an absolute linkage between recovery of gamma ray radiation resistance and protection by WR-1065 and other factors, such as chromatin organization, must play a role.

Conformational changes in chromatin structure induced by the radioprotective aminothiol, WR 1065.

WR 1065, 2-[(minopropyl) amino] ethanethiol is an effective scavenger of free radicals. When present during irradiation it reduces cellular DNA damage as analysed by alkaline elution from filters. The same technique indicates that without irradiation, WR 1065 has no effect of DNA integrity. Using nucleoid analysis, where DNA damage is detected at the level of replicon clusters, WR 1065 distorts replicon supercoiling without breaking the DNA molecule. This confirmational change in nucleoid structure occurs with no detectable change in nucleoid protein content. It is proposed that perturbation of replicon supercoiling affects the process of normal DNA synthesis and strand break rejoining, allowing a longer time for the accurate repair of DNA damage.