Halofuginone suppresses HSV-2 through direct viral inhibition and host ProRS inhibition

Herpes simplex virus type 2 (HSV-2) is the principal cause of genital herpes, establishes lifelong latency with recurrent mucosal lesions, and facilitates acquisition of human immunodeficiency virus type 1 (HIV-1). Because current therapy still relies largely on nucleoside analogues and resistant isolates continue to emerge, alternative antiviral strategies are needed. In this study, halofuginone (HF), a halogenated analogue of the febrifugine scaffold, inhibited both wild-type and acyclovir-resistant HSV-2 in cultured cells at nanomolar concentrations. In a murine genital challenge model, topical administration before viral exposure reduced viral burden and alleviated local inflammatory responses. Pre-exposure experiments further showed that HF decreased the infectivity of extracellular HSV-2 particles and was associated with virion structural damage, whereas docking analysis suggested a possible interaction with glycoprotein D (gD). In parallel, partial reversal by L-proline supported a host component linked to inhibition of the prolyl-tRNA synthetase (ProRS) domain of glutamyl-prolyl-tRNA synthetase (EPRS). HF also suppressed HSV-2-induced activation of phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and nuclear factor kappa B (NF-κB). Beyond HSV-2, HF remained active against herpes simplex virus type 1 (HSV-1) and C-C chemokine receptor type 5 (CCR5)-tropic HIV-1 and retained antiviral activity in an HSV-2/HIV-1 co-infection model. Overall, the data support a dual antiviral effect of HF, involving extracellular impairment of viral particles together with intracellular host-directed restriction, and warrant further evaluation of HF as a topical prevention candidate against HSV-2.