Phenylalanine residues at the carboxyl terminus of the herpes simplex virus 1 UL20 membrane protein regulate cytoplasmic virion envelopment and infectious virus production.
The herpes simplex virus type 1 (HSV-1) UL20 gene encodes a 222-amino-acid nonglycosylated envelope protein which forms a complex with viral glycoprotein K (gK) that functions in virion envelopment, egress, and virus-induced cell fusion. To investigate the role of the carboxyl terminus of the UL20 protein (UL20p) in cytoplasmic virion envelopment, a cadre of mutant viruses was constructed and characterized. The deletion of six amino acids from the carboxyl terminus of UL20p caused an approximately 1-log reduction in infectious virus production compared to that of the wild-type virus. Surprisingly, a phenylalanine-to-alanine replacement at amino acid position 210 caused a gain-of-function phenotype, increasing infectious virus production up to 1 log more than in the wild-type virus. In contrast, the replacement of two membrane-proximal phenylalanines with alanines caused drastic inhibition of infectious virion production and cytoplasmic virion envelopment. Prediction of the membrane topology of UL20p revealed that these two amino acid changes cause retraction of the carboxyl terminus of UL20p from the intracellular space. Confocal microscopy revealed that none of the engineered UL20 mutations affected intracellular transport of UL20p to trans-Golgi network membranes. In addition, a proximity ligation assay showed that none of the UL20 mutations affected UL20p colocalization and potential interactions with the UL37 protein recently found to interact with the gK/UL20 protein complex. Collectively, these studies show that phenylalanine residues within the carboxyl terminus of UL20p are involved in the regulation of cytoplasmic virion envelopment and infectious virus production.


OBJECTIVE
We have shown previously that the UL20/gK protein complex serves crucial roles in cytoplasmic virion envelopment and that it interacts with the UL37 tegument protein to facilitate cytoplasmic virion envelopment. In this study, we investigated the role of phenylalanine residues within the carboxyl terminus of UL20p, since aromatic and hydrophobic amino acids are known to be involved in protein-protein interactions through stacking of their aromatic structures. Characterization of mutant viruses carrying phenylalanine (Phe)-to-alanine (Ala) mutations revealed that the two membrane-proximal Phe residues were critical for the proper UL20p membrane topology and efficient virion envelopment and infectious virus production. Surprisingly, a Phe-to-Ala change located approximately in the middle of the UL20p carboxyl terminus substantially enhanced cytoplasmic envelopment and overall production of infectious virions. This work revealed that Phe residues within the UL20p carboxyl terminus are involved in the regulation of cytoplasmic virion envelopment and infectious virus production.
Division of Biotechnology and Molecular Medicine and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA.
Anu-Susan
AS
Charles
Division of Biotechnology and Molecular Medicine and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA.
Vladimir N
VN
Chouljenko
Division of Biotechnology and Molecular Medicine and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA.
Nithya
N
Jambunathan
Division of Biotechnology and Molecular Medicine and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA.
Ramesh
R
Subramanian
Division of Biotechnology and Molecular Medicine and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA.
Peter
P
Mottram
Division of Biotechnology and Molecular Medicine and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA vtgusk@lsu.edu.
Konstantin G
KG
Kousoulas
D000818:Animals; D002459:Cell Fusion; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000821:virology*; D006023:Glycoproteins / Q000378:metabolism*; D006561:Herpes Simplex / Q000378:metabolism / Q000821:virology*; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D006801:Humans; D008854:Microscopy, Electron; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism*; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism; D014771:Virion / Q000378:metabolism* / Q000472:pathogenicity; D021601:trans-Golgi Network
D006023:Glycoproteins; C121742:UL20 protein, Herpes simplex virus type 1; C084763:UL37 protein, Human herpesvirus 1; D014764:Viral Proteins; D015678:Viral Structural Proteins; D010649:Phenylalanine