Abstract
Purpose : To investigate whether UL43 protein, which is highly conserved in alpha- and gamma herpes viruses, and a non-glycosylated transmembrane protein, is involved in virus entry and virus-induced cell fusion. Methods : Mutagenesis was accomplished by a markerless two-step Red recombination mutagenesis system implemented on the Herpes simplex virus 1 (HSV-1) bacterial artificial chromosome (BAC). Growth properties of HSV-1 UL43 mutants were analyzed using plaque morphology and one-step growth kinetics. SDS-PAGE and Western blot was employed to assay the synthesis of the viral glycoproteins. Virus-penetration was assayed to determine if UL43 protein is required for efficient virus entry. Results : Lack of UL43 expression resulted in significantly reduced plaque sizes of syncytial mutant viruses and inhibited cell fusion induced by gBΔ28 or gKsyn20 (p < 0.05). Deletion of UL43 did not affect overall expression levels of viral glycoproteins gB, gC, gD, and gH on HSV-1(F) BAC infected cell surfaces. Moreover, mutant viruses lacking UL43 gene exhibited slower kinetics of entry into Vero cells than the parental HSV-1(F) BAC. Conclusion : Thus, these results suggest an important role for UL43 protein in mediating virus-induced membrane fusion and efficient entry of virion into target cells. Keywords : Herpes simplex virus type 1, UL43 protein, Membrane fusion, Mutant viruses, Virion, Mutagenesis
Highlights
Herpes simplex virus type 1 (HSV-1) is a common human pathogen that causes most forms of non-genital herpes simplex infection
To assess the effect of UL43 on virus-induced cell fusion, we constructed a set of recombinant viruses containing either the gBΔ28 or gKsyn20 mutation in the presence or absence of mutations lacking the UL43 gene by using the HSV-1 F genome cloned as a bacterial artificial chromosome (BAC)
GB- or gKmediated syncytia formation and virus spread were restored to nearly HSV-1 (F) BAC levels (Figure 2B)
Summary
Herpes simplex virus type 1 (HSV-1) is a common human pathogen that causes most forms of non-genital herpes simplex infection. Most mutations that cause extensive virus-induced cell fusion (syncytial or syn mutations) have been mapped to the four HSV genes, UL20 [4], UL24 [5], UL27 encoding glycoprotein B (gB) [6], and UL53 coding for glycoprotein K (gK) [7]. 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]. The transient co-expression assay does not accurately model viral fusion, it is conceivable that UL43 protein may be involved in modulating membrane fusion
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