Dataset Information

Pseudorabies virus glycoprotein K requires the UL20 gene product for processing.

ABSTRACT: Glycoprotein K (gK) of pseudorabies virus (PrV) has recently been identified as a virion component which is dispensable for viral entry but required for direct cell-to-cell spread. Electron microscopic data suggested a possible function of gK in virus egress by preventing immediate fusion of released virus particles with the plasma membrane (B. G. Klupp, J. Baumeister, P. Dietz, H. Granzow, and T. C. Mettenleiter, J. Virol. 72:1949-1958, 1998). For more detailed analysis, a PrV mutant with a deletion of the UL53 (gK) open reading frame (ORF) from codons 48 to 275 was constructed, and the protein was analyzed with two monoclonal antibodies directed against PrV gK. The salient findings of this report are as follows. (i) From the PrV UL53 ORF, a functional gK is translated only from the first in-frame methionine. From the second in-frame methionine, a nonfunctional product is expressed which is not incorporated into virions. (ii) When constitutively expressed in a stable cell line without other viral proteins, gK is only incompletely processed. After superinfection with gK-deletion mutants, proper processing is restored and mature gK is incorporated into virions. (iii) The UL20 gene product is specifically required for processing of gK. gK is not correctly processed in a UL20 deletion mutant of PrV, and superinfection of gK-expressing cells with PrV-UL20(-) does not restore processing. However, all other known structural viral glycoproteins appear to be processed normally in PrV-UL20(-)-infected cells. (iv) Coexpression of gK and UL20 restored gK processing at least partially. Thus, our data show that the UL20 gene product is required for proper processing of PrV gK.

SUBMITTER: Dietz P

PROVIDER: S-EPMC110860 | biostudies-literature | 2000 Jun

REPOSITORIES: biostudies-literature

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Pseudorabies virus glycoprotein K requires the UL20 gene product for processing.

Dietz P P Klupp B G BG Fuchs W W Köllner B B Weiland E E Mettenleiter T C TC

Journal of virology 20000601 11

Glycoprotein K (gK) of pseudorabies virus (PrV) has recently been identified as a virion component which is dispensable for viral entry but required for direct cell-to-cell spread. Electron microscopic data suggested a possible function of gK in virus egress by preventing immediate fusion of released virus particles with the plasma membrane (B. G. Klupp, J. Baumeister, P. Dietz, H. Granzow, and T. C. Mettenleiter, J. Virol. 72:1949-1958, 1998). For more detailed analysis, a PrV mutant with a del ...[more]

PMID: 10799582

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Project description:Herpesvirus envelopment is a two-step process which includes acquisition of a primary envelope resulting from budding of intranuclear capsids through the inner nuclear membrane. Fusion with the outer leaflet of the nuclear membrane releases nucleocapsids into the cytoplasm, which then gain their final envelope by budding into trans-Golgi vesicles. It has been shown that the UL34 gene product is required for primary envelopment of the alphaherpesvirus pseudorabies virus (PrV) (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 74:10063-10073, 2000). For secondary envelopment, several virus-encoded PrV proteins are necessary, including glycoproteins E, I, and M (A. R. Brack, J. M. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). We show here that the product of the UL37 gene of PrV, which is a constituent of mature virions, is involved in secondary envelopment. Replication of a UL37 deletion mutant, PrV-DeltaUL37, was impaired in normal cells; this defect could be complemented on cells stably expressing UL37. Ultrastructural analysis demonstrated that intranuclear capsid maturation and budding of capsids into and release from the perinuclear space were unimpaired. However, secondary envelopment was drastically reduced. Instead, apparently DNA-filled capsids accumulated in the cytoplasm in large aggregates similar to those observed in the absence of glycoproteins E/I and M but lacking the surrounding electron-dense tegument material. Although displaying an ordered structure, capsids did not contact each other directly. We postulate that the UL37 protein is necessary for correct addition of other tegument proteins, which are required for secondary envelopment. In the absence of the UL37 protein, capsids interact with each other through unknown components but do not acquire the electron-dense tegument which is normally found around wild-type capsids during and after secondary envelopment. Thus, apposition of the UL37 protein to cytoplasmic capsids may be crucial for the addition of other tegument proteins, which in turn are able to interact with viral glycoproteins to mediate secondary envelopment.

Project description:Conserved across the family Herpesviridae, glycoprotein B (gB) is responsible for driving fusion of the viral envelope with the host cell membrane for entry upon receptor binding and activation by the viral gH/gL complex. Although crystal structures of the gB ectodomains of several herpesviruses have been reported, the membrane fusion mechanism has remained elusive. Here, we report the X-ray structure of the pseudorabies virus (PrV) gB ectodomain, revealing a typical class III postfusion trimer that binds membranes via its fusion loops (FLs) in a cholesterol-dependent manner. Mutagenesis of FL residues allowed us to dissect those interacting with distinct subregions of the lipid bilayer and their roles in membrane interactions. We tested 15 gB variants for the ability to bind to liposomes and further investigated a subset of them in functional assays. We found that PrV gB FL residues Trp187, Tyr192, Phe275, and Tyr276, which were essential for liposome binding and for fusion in cellular and viral contexts, form a continuous hydrophobic patch at the gB trimer surface. Together with results reported for other alphaherpesvirus gBs, our data suggest a model in which Phe275 from the tip of FL2 protrudes deeper into the hydrocarbon core of the lipid bilayer, while the side chains of Trp187, Tyr192, and Tyr276 form a rim that inserts into the more superficial interfacial region of the membrane to catalyze the fusion process. Comparative analysis with gBs from beta- and gamma-herpesviruses suggests that this membrane interaction model is valid for gBs from all herpesviruses.IMPORTANCE Herpesviruses are common human and animal pathogens that infect cells by entering via fusion of viral and cellular membranes. Central to the membrane fusion event is glycoprotein B (gB), which is the most conserved envelope protein across the herpesvirus family. Like other viral fusion proteins, gB anchors itself in the target membrane via two polypeptide segments called fusion loops (FLs). The molecular details of how gB FLs insert into the lipid bilayer have not been described. Here, we provide structural and functional data regarding key FL residues of gB from pseudorabies virus, a porcine herpesvirus of veterinary concern, which allows us to propose, for the first time, a molecular model to understand how the initial interactions by gBs from all herpesviruses with target membranes are established.

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Pseudorabies virus glycoprotein K requires the UL20 gene product for processing.

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Pseudorabies virus glycoprotein K requires the UL20 gene product for processing.

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