The Equine Herpesvirus 1 UL20 Product Interacts with Glycoprotein K and Promotes Egress of Mature Particles

Abstract

The aim of the present study was to identify and functionally characterize the equine herpesvirus 1 (EHV-1) UL20 protein (UL20p). Using a specific antiserum, UL20p was shown to be associated with membranes of infected cells, as well as with envelopes of purified virions. By Western blot analysis, UL20p was detected in two main forms exhibiting M r s of 25,000 and 75,000. Both moieties did not enter the separating gel after heating of protein samples to 99°C. The slower-migrating form of UL20p contains N-linked carbohydrates, and its presence is dependent of that of other viral proteins. Infection of cells that either constitutively express UL20p or a gK-green fluorescent protein (GFP) fusion protein with various EHV-1 deletion mutants revealed a relatively stable hetero-oligomer containing gK and UL20p with an apparent M r of 75,000. As demonstrated by confocal microscopy, UL20p distribution in Rk13 cells changed from a diffuse granular or netlike appearance to a pattern confined to the Golgi network when gK was coexpressed. Analysis of a UL20 deletion mutant of EHV-1 strain RacL11 indicated an involvement of UL20p in cell-to-cell spread, as well as in very late events in virus egress. Based on these and electron microscopic studies we suggest that the EHV-1 UL20 protein might be necessary to avoid fusion of mature virions with membranes of their transport vesicles. Equid herpesvirus 1 (equine herpesvirus 1 [EHV-1]) is one of the most important viral pathogens of horses and classifies as a Varicellovirus within the subfamily Alphaherpesvirinae (21).Alphaherpesviruses express relatively few proteins with the potential to span membranes several times. Accordingly, only four such open reading frames (ORFs) have been identified in the EHV-1 sequence, and they are homologous to the herpes simplex virus type 1 (HSV-1) ORFs UL10, UL20, UL43, and UL53 (31). Of these, the EHV-1 UL10 product glycoprotein M (gM), has been studied most intensively, and it has been shown that gM is involved in virus egress, more precisely in secondary envelopment, and plays a role in cell-to-cell spread (23, 28). The protein encoded by UL53, gK, is less dispensable for virus replication in Rk13 cells than gM. However, like gM, gK influences a late step in virus egress, direct cell-to-cell spread, and the efficiency of virus penetration (20). EHV-1 gK and gM share additional characteristics such as N glycosylation, earlylate expression kinetics, and the fact that they both depend on a viral complex partner for complete processing (20,25). In the present study we sought to characterize the UL20 product to further investigate the specific roles of multiply hydrophobic proteins in EHV-1 replication and to address the question whether the special structural features might be related to common functional properties.In contrast to the EHV-1 UL20 protein, which has not been studied before, the respective homologs of HSV-1 and pseudorabies virus (PRV) are well characterized. The structure of the PRV UL20p is still unknown, since no specific antibodie...

Functional and Physical Interactions of the Herpes Simplex Virus Type 1 UL20 Membrane Protein with Glycoprotein K

“…Importantly, detailed confocal colocalization experiments have shown a strong interdependence of gK and UL20p for intracellular transport, cell surface expression and localization at the TGN, virus-induced cell fusion, and cytoplasmic virion envelopment, suggesting physical interactions between gK and UL20p (11,12,14,18,31). Additional evidence for the formation of heterodimeric or multimeric structures of gK with UL20p was obtained with equine herpesvirus type 1-infected cells (20).…”

Comparative Genomic Sequencing and Pathogenic Properties of Equine Herpesvirus 1 KyA and RacL11

“… b Function is assigned primarily from published data for proteins of herpes simplex virus 1 (HSV-1) ( 23 , 43 , 44 ). The discovery and function of EHV-1 IR2 ( 25 , 26 ), IR3 ( 27 – 29 ), UL1 and UL2 ( 39 ), UL3 ( 45 ), UL11 ( 46 ), UL20 ( 47 ), UL31 ( 48 ), UL34 ( 49 ), US4 (gp2) ( 50 ), and US6 (gI) and US7 (gE) ( 37 ) were reported previously . –, not present .…”

Properties of an equine herpesvirus 1 mutant devoid of the internal inverted repeat sequence of the genomic short region

“…The findings that the vL11ΔIR showed reduced plaque size and delayed growth in RK13 cells clearly suggest that the deletion of sequences including the genes within the IR affects the biological properties of EHV-1 in cell culture. Previous studies showed that the EHV-1 Us4, Us6, Us7, UL6, UL10, and UL20 gene products are involved in cell-to-cell spread of virus and virus egress (Frampton et al, 2002; Guggemoos et al, 2006; Neubauer and Osterrieder, 2004; Osterrieder et al, 1996a; von Einem et al, 2004), but none of these genes is located within sequences deleted here. The smaller plaque size and delayed growth of vL11ΔIR as compared to the parent virus may be explained by the roles of EHV-1 major regulatory genes IE and IR4 that are located within both inverted repeats (Grundy et al, 1989; Holden et al, 1994; Telford et al, 1992).…”