Bovine herpesvirus 1 tegument protein UL21 plays critical roles in viral secondary envelopment and cell-to-cell spreading

Abstract

Bovine herpesvirus 1 (BoHV-1) UL21 is a tegument protein thought to be indispensable for efficient viral growth but its precise function in BoHV-1 is currently unknown. To determine the function of UL21 in BoHV-1 replication, we constructed a mutant virus bearing a UL21 deletion (vBoHV-1-∆UL21) and its revertant virus, vBoHV-1-∆UL21R, in which the UL21 gene was restored using a bacterial artificial chromosome system. The replication of vBoHV-1-∆UL21 was 1,000-fold lower and its plaque size was 85% smaller than those of the wild-type virus (BoHV-1). An ultrastructural analysis showed that deletion of UL21 led to an un-enveloped capsid accumulation in the cytoplasm, whereas nucleocapsid egress was not impaired, suggesting that UL21 is critical for secondary envelopment in BoHV-1. Co-immunoprecipitation assays revealed that HA-tagged UL21 pulled down UL16, suggesting that these two proteins form a complex, and this was further confirmed by a co-immunofluorescence assay. Taken together, these data provide evidence that UL21 plays critical roles in BoHV-1 secondary envelopment, and UL16 is likely to be involved in these activities.

Ivermectin Inhibits Bovine Herpesvirus 1 DNA Polymerase Nuclear Import and Interferes With Viral Replication

“…MDBK cells were grown and maintained in complete Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 IU/mLpenicillin and 100 µg/mLstreptomycin at 37 • C in 5% CO 2 . The IBRV HB06 strain was propagated and maintained as described previously [28].…”

Disruption of Herpes Simplex Virus Type 2 pUL21 Phosphorylation Impairs Secondary Envelopment of Cytoplasmic Nucleocapsids

“…Additionally, unlike what was observed in pUL21 deletion strains (6), the phosphorylation profiles of pUL31 in lysates from cells infected with pUL21 phosphorylation-deficient viruses were indistinguishable from WT infected cells (Figure 3D) suggesting that pUL21-mediated regulation of nuclear egress complex components is unperturbed by pUL21 phosphorylation. The importance of pUL21 in secondary envelopment has been demonstrated for bovine herpesvirus (4), however, the importance of HSV pUL21 in secondary envelopment was speculative prior to this study. Our ultrastructure analysis of cells infected with multiple HSV strains lacking pUL21 now confirms the importance of HSV pUL21 in secondary envelopment as well as a requirement for phosphorylated pUL21 in the case of HSV-2.…”

“…The ratio of non-enveloped to enveloped cytoplasmic nucleocapsids in WT HSV-2 strain 186 infected cells was approximately 1 and this value increased 7 to 26-fold in viruses deficient in pUL21 phosphorylation supporting the notion that secondary envelopment was defective in the absence of pUL21 phosphorylation. To contextualize this result, we consulted the literature on the role of pUL21 in secondary envelopment and found that this has only been demonstrated experimentally for bovine herpesvirus 1 (4). It has been speculated that HSV pUL21 plays a role in secondary envelopment (32, 33), however this has not been quantified.…”

“…With so many important roles played by pUL21 during viral infection, it is not surprising that viruses deficient in pUL21 display a small plaque phenotype and reduced viral replication in comparison to wild type (WT) virus (1,4,8,(12)(13)(14)(15). The defects in cell-cell spread of virus infection and replication of pUL21 deficient viruses are more pronounced in herpes simplex virus type 2 (HSV-2) than they are in herpes simplex virus type 1 (HSV-1) even though the pUL21s of these species are about 84% identical (13).…”

“…The capsid-associated tegument component pUL21 is a multifunctional protein unique to the alphaherpesvirus subfamily of the Herpesviridae . Known functions of pUL21 include facilitating transport of capsids along microtubules (1–3), promoting secondary envelopment of capsids (4), regulating nuclear egress of capsids (5, 6), directing protein phosphatase 1 alpha (PP1α) to specific substrates for dephosphorylation (7, 8), triggering autophagic degradation of cyclic GMP-AMP synthase (cGAS) (9), enabling retention of viral genomes within capsids (10) and, most recently, preventing capsids from docking to nuclear pores after nuclear egress (11). With so many important roles played by pUL21 during viral infection, it is not surprising that viruses deficient in pUL21 display a small plaque phenotype and reduced viral replication in comparison to wild type (WT) virus (1, 4, 8, 12–15).…”