Phosphorylation of Bovine Herpesvirus 1 VP8 Plays a Role in Viral DNA Encapsidation and Is Essential for Its Cytoplasmic Localization and Optimal Virion Incorporation

Abstract

VP8 is a major tegument protein of bovine herpesvirus 1 (BoHV-1) and is essential for viral replication in cattle. The protein undergoes phosphorylation after transcription through cellular casein kinase 2 (CK2) and a viral kinase, US3. In this study, a virus containing a mutated VP8 protein that is not phosphorylated by CK2 and US3 (BoHV-1-YmVP8) was constructed by homologous recombination in mammalian cells. When BoHV-1-YmVP8-infected cells were observed by transmission electron microscopy, blocking phosphorylation of VP8 was found to impair viral DNA encapsidation, resulting in release of incomplete viral particles to the extracellular environment. Consequently, less infectious virus was produced by the mutant virus than by wild-type (WT) virus. A comparison of mutant and WT VP8 by confocal microscopy revealed that mutant VP8 is nuclear throughout infection while WT VP8 is nuclear early during infection and is associated with the Golgi apparatus at later stages. This, together with the observation that mutant VP8 is present in virions, albeit in smaller amounts, suggests that the incorporation of VP8 may occur at two stages. The first takes place without the need for phosphorylation and before or during nuclear egress of capsids, whereas the second occurs in the Golgi apparatus and requires phosphorylation of VP8. The results indicate that phosphorylated VP8 plays a role in viral DNA encapsidation and in the secondary virion incorporation of VP8. To perform these functions, the cellular localization of VP8 is adjusted based on the phosphorylation status. IMPORTANCEIn this study, phosphorylation of VP8 was shown to have a function in BoHV-1 replication. A virus containing a mutated VP8 protein that is not phosphorylated by CK2 and US3 (BoHV-1-YmVP8) produced smaller numbers of infectious virions than wild-type (WT) virus. The maturation and egress of WT and mutant BoHV-1 were studied, showing a process similar to that reported for other alphaherpesviruses. Interestingly, lack of phosphorylation of VP8 by CK2 and US3 resulted in reduced incorporation of viral DNA into capsids during mutant BoHV-1 infection, as well as lower numbers of extracellular virions. Furthermore, mutant VP8 remained nuclear throughout infection, in contrast to WT VP8, which is nuclear at early stages and Golgi apparatus associated late during infection. This correlates with smaller amounts of mutant VP8 in virions and suggests for the first time that VP8 may be assembled into the virions at two stages, with the latter dependent on phosphorylation. Bovine herpesvirus 1 (BoHV-1), a member of the Alphaherpesviridae, causes infectious bovine rhinotracheitis (IBR) in cattle, as well as conjunctivitis, vulvovaginitis, and balanoposthitis. The herpesvirus particle is composed of a capsid containing the double-stranded DNA genome, which is surrounded by a tegument layer and an envelope containing viral glycoproteins (1). During the herpesvirus life cycle, genome synthesis, capsid assembly, DNA incorporation (2), and primary tegumentation...

US3 Kinase-Mediated Phosphorylation of Tegument Protein VP8 Plays a Critical Role in the Cellular Localization of VP8 and Its Effect on the Lipid Metabolism of Bovine Herpesvirus 1-Infected Cells

“…As a result, it is less likely to be involved in viral particle transport through the TGN. We have previously shown that inhibiting VP8 from entering the cytoplasm significantly reduced the amount of virion-incorporated VP8 (12). This is consistent with VP8 being localized in the cis-Golgi, which is an important site for protein incorporation into virions.…”

“…This function seems to be exclusive to US3. It does not appear to need an intermediate kinase to phosphorylate VP8, because US3 and CK2 are the only kinases that phosphorylate VP8 in BoHV-1infected cells (12) and CK2 does not affect the nuclear localization of VP8.…”

“…15A), indicating that VP8 expression causes accumulation of cholesterol in cells. To confirm that VP8 increases translocate into the nucleus early during infection to promote early events of virus replication, for instance, viral DNA encapsidation (12), and to redistribute ND10 (8). Subsequently, VP8 was translocated to the cytoplasm and accumulated in the Golgi apparatus.…”

“…Overall, our data show that VP8 translocates from the nucleus to the cytoplasm when BoHV-1 infection progresses to the late stages and that US3-mediated phosphorylation is required for VP8 to exit the nucleus and/or remain in the cytoplasm. During early stages of infection, the majority of VP8 is present in the cell nucleus to promote nuclear events for viral replication, such as redistributing promyelocytic leukemia (PML) protein (8) and promoting viral DNA encapsidation (12). Subsequently, the NLSs appear to be impaired or the NESs enabled by US3-mediated phosphorylation, resulting in VP8 accumulation in the cytoplasm.…”

“…The nuclear localization of VP8 is mediated by arginine-rich nuclear localization signal 1 (NLS1; P 11 RPRR 15 ) and NLS2 (R 48 PRVRRPRP 54 ) (10,11). Subsequently, VP8 is exported into the cytoplasm and accumulates in the Golgi apparatus at later stages of infection (12). At least two nuclear export signals (NESs) have been described for VP8.…”