Identification and characterization of the pseudorabies virus UL43 protein

Abstract

Among the least characterized herpesvirus membrane proteins are the homologs of UL43 of herpes simplex virus 1 (HSV-1). To identify and characterize the UL43 protein of pseudorabies virus (PrV), part of the open reading frame was expressed in Escherichia coli and used for immunization of a rabbit. The antiserum recognized in Western blots a 34-kDa protein in lysates of PrV infected cells and purified virions, demonstrating that the UL43 protein is a virion component. In indirect immunofluorescence analysis, the antiserum labeled vesicular structures in PrV infected cells which also contained glycoprotein B. To functionally analyze UL43, a deletion mutant was constructed lacking amino acids 23-332 of the 373aa protein. This mutant was only slightly impaired in replication as assayed by one-step growth kinetics, measurement of plaque sizes, and electron microscopy. Interestingly, the PrV UL43 protein was able to inhibit fusion induced by PrV glycoproteins in a transient expression-fusion assay to a similar extent as gM. Double mutant viruses lacking, in addition to UL43, the multiply membrane spanning glycoproteins K or M did not show a phenotype beyond that observed in the gK and gM single deletion mutants.

Analysis of contributions of herpes simplex virus type 1 UL43 protein to induction of cell-cell fusion

“…However, the PrV UL43 protein was suggested to inhibit membrane fusion in the transient co-expression system involving PrV envelope glycoproteins [10]. These findings seem to contradict results obtained herein with the recombinant viruses lacking UL43 gene.…”

“…The HSV-1 UL43 has been shown to be non-essential for virus growth in cell culture and deletion of UL43 did not impair viral replication in a mouse infection model [9]. It has been reported that Pseudorabies virus (PrV) UL43 protein strongly inhibited membrane fusion induced by the viral fusion machinery in a transient expression-fusion system [10]. Although the transient co-expression assay does not accurately model viral fusion, it is conceivable that UL43 protein may be involved in modulating membrane fusion.…”

A replication defect of pseudorabies virus induced by targeted α-helix distortion in the syntaxin-like bundle of glycoprotein H (V275P) is corrected by an adjacent compensatory mutation (V271A)

“…2b). Although in PrV gL is not required for transport of gH (Klupp et al, 1997), cotransfection of cells with gH and gL expression plasmids enhanced the proportion of the mature, fully glycosylated, approximately 95 kDa form of WT gH (Klupp et al, 1992) compared to cells transfected with the gH plasmid alone (Fig. 2a).…”

Analysis of Synonymous Codon Usage in the newly identified DPV UL43 Gene

“…If it is deleted, plaque size is reduced by approximately 10% and viral titers are decreased by ca. 2-to 5-fold [49]. Furthermore, The DPV UL43 protein has several membrane domains, potential ability to cross a membrane several times suggesting a role in signal transduction or as ion channels, just like VZV gene 15, which corresponds to HSV-1 gene UL43, contains a periodic charge pattern similar to that found in voltage-gated ion channels [50].…”