The virion host shutoff protein (vhs), encoded by the gene UL41, has ribonuclease activity and is the key regulator of the early host shutoff response induced by herpes simplex virus (HSV-1). Despite low amino acid similarity, the vhs protein of the swine herpesvirus, pseudorabies virus (PrV), also exhibits ribonuclease activity. However, the mechanism underlying the action of vhs remains undefined. Here, we report that the RNA degradation profile of PrV vhs is similar, but not identical, to that of HSV-1 vhs. Notably, the presence of a cap structure enhances both the degradation rate and the preferential targeting of the vhs protein towards the 3' end of the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). Furthermore, HSV-1 vhs produces a simple degradation pattern, but PrV vhs gives rise to multiple intermediates. The results of Northern blotting using probes recognizing various regions of the RNA substrate found that PrV vhs also cleaves downstream from the IRES region and this vhs protein overall shows 5' to 3' ribonuclease activity. Moreover, the addition of the translation initiation factors eIF4H and eIF4B significantly increased the RNase activity of recombinant PrV vhs against capped RNA. Nonetheless, these proteins did not fully reconstitute the IRES-directed targeting pattern observed for vhs translated in a rabbit reticular lysate system. The interaction between PrV vhs and eIF4H/eIF4B implies that the translation initiation machinery within the cell is able to stimulate the nuclease activity of PrV vhs. However, this process remains inefficient in terms of the IRES-targeting pattern. This article is protected by copyright. All rights reserved.