Identification and Characterization of the UL56 Gene Product of Herpes Simplex Virus Type 2

Abstract

The UL56 gene product of herpes simplex virus (HSV) has been shown to play an important role in viral pathogenicity. However, the properties and functions of the UL56 protein are little understood. We raised rabbit polyclonal antisera specific for the UL56 protein of HSV type 2 (HSV-2) and examined its expression and properties. The gene product was identified as three polypeptides with apparent molecular masses ranging from 32 to 35 kDa in HSV-2-infected cells, and at least one species was phosphorylated. Studies of their origins showed that the UL56 protein of HSV-2 is also translated from the upstream in-frame methionine codon that is not present in the HSV-1 genome. Synthesis was first detected at 6 h postinfection and was not abolished by the viral DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies revealed that the UL56 protein localized to both the Golgi apparatus and cytoplasmic vesicles in HSV-2-infected and single UL56-expressing cells. Deletion mutant analysis showed that the C-terminal hydrophobic region of the protein was required for association with the cytoplasmic membrane and that the N-terminal proline-rich region was important for its translocation to the Golgi apparatus and cytoplasmic vesicles. Moreover, the results of protease digestion assays and sucrose gradient fractionation strongly suggested that UL56 is a tail-anchored type II membrane protein associated with lipid rafts. We thus hypothesized that the UL56 protein, as a tail-anchored type II membrane protein, may be involved in vesicular trafficking in HSV-2-infected cells.

Copatching and Lipid Raft Association of Different Viral Glycoproteins Expressed on the Surfaces of Pseudorabies Virus-Infected Cells

“…The HSV type 1 vhs tegument protein and, to a lesser extent, gH glycoprotein have also been shown to be associated with rafts in infected cells (26). Further, the HSV type 2 UL56 protein, a protein with significant similarities to the PRV Us9 type II-anchored membrane protein (3), has also been shown to be raft associated (24). Recently, it has been shown that the PRV UL11 tegument protein is membrane associated and is a diacylated protein, a hallmark of many raft-associated proteins, and may therefore be raft associated (23).…”

“…Lipid rafts are enriched in glycosyl-phosphatidylinositol-linked proteins, specific transmembrane proteins, signal transduction molecules, and the ganglioside GM1. Recently it has been shown that the HSV gB glycoprotein, as well as two HSV tegument-membrane proteins (vhs and UL56), are lipid raft associated in infected cells or become lipid raft associated during virus entry (2,24,26).…”

Oncolytic virotherapy with an HSV amplicon vector expressing granulocyte–macrophage colony-stimulating factor using the replication-competent HSV type 1 mutant HF10 as a helper virus

“…UL56 associates with the kinesin motor protein KIF1A, and the absence of UL56 causes decreased pathogenicity without affecting viral replication in tissue culture. [44][45][46] Thus, the lack of UL56 may be responsible for the attenuated nature of HF10.…”

Oncolytic herpes simplex virus 1 (HSV-1) vectors: Increasing treatment efficacy and range through strategic virus design

“…A mutation in UL56 is thought to be responsible for attenuation in noncancerous cells (50). However, while experiments using HSV-2 suggest that the gene product of UL56 plays a critical role in vesicular trafficking, its precise function in HSV-1 is unclear (51,52). Ultimately, although several genetic rearrangements have been identified in the strain, and phenotypically it was notable for its tendency toward syncytium formation upon infection, the specific mutations and mechanisms accounting for the efficacy of HF-10 as a viral oncolytic agent remain unclear (37).…”