The UL21 Tegument Protein of Herpes Simplex Virus 1 Is Differentially Required for the Syncytial Phenotype

Abstract

The initial goal of this study was to reexamine the requirement of UL21 for herpes simplex virus 1 (HSV-1) replication. Previous studies suggested that UL21 is dispensable for replication in cell cultures, but a recent report on HSV-2 challenges those findings. As was done for the HSV-2 study, a UL21-null virus was made and propagated on complementing cells to discourage selection of compensating mutations. This HSV-1 mutant was able to replicate in noncomplementing cells, even at a low multiplicity of infection (MOI), though a reduction in titer was observed. Also, increased proportions of empty capsids were observed in the cytoplasm, suggesting a role for UL21 in preventing their exit from the nucleus. Surprisingly, passage of the null mutant resulted in rapid outgrowth of syncytial (Syn) variants. This was unexpected because UL21 has been shown to be required for the Syn phenotype. However, earlier experiments made use of only the A855V syncytial mutant of glycoprotein B (gB), and the Syn phenotype can also be produced by substitutions in glycoprotein K (gK), UL20, and UL24. Sequencing of the syncytial variants revealed mutations in the gK locus, but UL21 was shown to be dispensable for UL20 and UL24 To test whether UL21 is needed only for the A855V mutant, additional gB derivatives were examined in the context of the null virus, and all produced lytic rather than syncytial sites of infection. Thus, UL21 is required only for the gB phenotype. This is the first example of a differential requirement for a viral protein across the four loci. UL21 is conserved among alphaherpesviruses, but its role is poorly understood. This study shows that HSV-1 can replicate without UL21, although the virus titers are greatly reduced. The null virus had greater proportions of empty (DNA-less) capsids in the cytoplasm of infected cells, suggesting that UL21 may play a role in retaining them in the nucleus. This is consistent with reports showing UL21 to be capsid associated and localized to the nuclei of infected cells. UL21 also appears to be needed for viral membrane activities. It was found to be required for virus-mediated cell fusion, but only for mutants that harbor syncytial mutations in gB (not variants of gK, UL20, or UL24). The machinery needed for syncytial formation is similar to that needed for direct spread of the virus through cell junctions, and these studies show that UL21 is required for cell-to-cell spread even in the absence of syncytial mutations.

Proteomics Analysis Identifies IRSp53 and Fascin as Critical for PRV Egress and Direct Cell–Cell Transmission

“…The cell–cell spreading assay was performed as reported for HSV‐1 . Briefly, BHK21 cells and IRSp53 KO cells were cultured on 12‐well glass coverslips coated with poly‐lysine, infected with PRV325 at ≈30 PFU per well for 1.5 h. After rinsing with DMEM, the cells were incubated in low FBS DMEM (2% FBS) containing 5 mg mL –1 swine IgG (Solarbio, China, SP037).…”

“…The cell-cell spreading assay was performed as reported for HSV-1. [28,29] Briefly, BHK21 cells and IRSp53 KO cells were cultured on 12-well glass coverslips coated with poly-lysine, infected with PRV325 at ≈30 PFU per well for 1.5 h. After rinsing with DMEM, the cells were incubated in low FBS DMEM (2% FBS) containing 5 mg mL -1 swine IgG (Solarbio, China, SP037). At 40 hpi, immunochemistry analysis was performed following incubation with anti-GFP (Abcam, ab290, 1:1000) and Alexa Fluor 488 secondary antibody (ABclonal, 1:200) for visualization.…”

Duck enteritis virus UL21 is a late gene encoding a protein that interacts with pUL16

“…The total RNA was isolated from the DEV-infected DEFs at different time points (3,6,13,17,24,36,48 and 60 hpi), and then, reverse transcription was performed; an uninfected control was included. The primers were designed with Oligo 7 (Additional file 1: Table S1).…”

“…In addition, the HSV-1, DEV, bovine herpesvirus 1 (BHV-1), gazelle herpesvirus 2 (GHV-2), GHV-3, PRV, equine herpesvirus 4 (EHV-4) and varicella-zoster virus (VZV) pUL21 proteins exhibit high similarity in the region comprising amino acids 73-92 [12]. The UL21 gene has been considered both a late (L) gene and an early (E)/L gene because it possesses the features of both, and its functions are related to virus particle replications, virulence, transmission and immunization [13][14][15][16]. Moreover, pUL21 contains numerous sites for modifications, such as Nglycosylation and phosphorylation [17], suggesting that the protein undergoes posttranslational modification.…”

Duck enteritis virus UL21 is a late gene and encodes a protein that interacts with pUL16

“…In addition, the HSV-1, DEV, bovine herpesvirus 1 (BHV-1), gazelle herpesvirus 2 (GHV-2), GHV-3, PRV, equine herpesvirus 4 (EHV-4) and varicella-zoster virus (VZV) pUL21 proteins exhibit high similarity in the region comprising amino acids 73-92 (13). The UL21 gene has been considered both a late (L) gene and an early (E)/L gene because it possesses features of both, the functions of which are related to virus particles replications, virulence, transmission and immunization (14)(15)(16)(17). Moreover, pUL21 contains numerous sites for modifications such as N-glycosylation and phosphorylation (18), suggesting that the protein undergoes posttranslational modification.…”