Associating bovine herpesvirus 1 envelope glycoprotein gD with activated phospho-PLC-γ1(S1248).

Phospholipase C gamma 1 (PLC-γ1) may locate at distinct subcellular locations, such as cytosol, plasma membrane, and nucleus for varied biological functions. Bovine herpesvirus 1 (BoHV-1) productive infection activates PLC-γ1 signaling, as demonstrated by increased protein levels of phosphorylated-PLC-γ1 at Ser1248 [p-PLC-γ1(S1248)], which benefits virus productive infection. Here, for the first time, we reported that Golgi apparatus also contains activated p-PLC-γ1(S1248). And BoHV-1 productive infection at later stages (24 hpi) increased the accumulation of p-PLC-γ1(S1248) in the Golgi apparatus, where p-PLC-γ1(S1248) forms highlighted puncta observed via a confocal microscope. Coimmunoprecipitation studies demonstrated that the Golgi p-PLC-γ1(S1248) is specifically associated with the viral protein gD but not gC. In addition, we found that p-PLC-γ1(S1248) is consistently associated with both the plasma membrane-associated virions and the released virions. When the virus-infected cells were treated with PLC-γ1-specific inhibitor, U73122, for a short duration of 4 hours prior to the endpoint of virus infection, we found that the viral protein gD was trapped in the Golgi apparatus, suggesting that the PLC-γ1 signaling may facilitate trafficking of progeny virions out of this organelle. These findings provide a novel insight into the interplay between PLC-γ1 signaling and BoHV-1 replication. IMPORTANCE Bovine herpesvirus 1 (BoHV-1) productive infection increases protein levels of phosphorylated-phospholipase C gamma 1 at Ser1248 [p-PLC-γ1(S1248)]. However, whether it causes any variations to p-PLC-γ1(S1248) localization is not well understood. Here, for the first time, we found that partial p-PLC-γ1(S1248) is residing in the Golgi apparatus, where the accumulation is enhanced by virus infection. p-PLC-γ1(S1248) is consistently associated with virions, partially via binding to gD, in both the Golgi apparatus and cytoplasm membranes. Surprisingly, it also associates with the released virions. Of note, this is the first evidenced BoHV-1 virion-bound host protein. It seems that p-PLC-γ1(S1248) works as an escort during trafficking of progeny virions out of Golgi apparatus to the plasma membranes as well as releasing outside of the cell membranes. Furthermore, we showed that the activated p-PLC-γ1(S1248) is potentially implicated in the transport of virions out of Golgi apparatus, which may represent a novel mechanism to regulate virus productive infection.

NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures.

Bovine herpesvirus 1 (BoHV-1), an important bovine viral pathogen, causes severe disease in the upper respiratory tract and reproductive system. Tonicity-responsive enhancer-binding protein (TonEBP), also known as nuclear factor of activated T cells 5 (NFAT5), is a pleiotropic stress protein involved in a range of cellular processes. In this study, we showed that the knockdown of NFAT5 by siRNA increased BoHV-1 productive infection and overexpression of NFAT5 via plasmid transfection decreased virus production in bovine kidney (MDBK) cells. Virus productive infection at later stages significantly increased transcription of NFAT5 but not appreciably alter measurable NFAT5 protein levels. Virus infection relocalized NFAT5 protein and decreased the cytosol accumulation. Importantly, we found a subset of NFAT5 resides in mitochondria, and virus infection led to the depletion of mitochondrial NFAT5. In addition to full-length NFAT5, another two isoforms with distinct molecular weights were exclusively detected in the nucleus, where the accumulation was differentially affected following virus infection. In addition, virus infection differentially altered mRNA levels of PGK1, SMIT, and BGT-1, the canonical downstream targets regulated by NFAT5. Taken together, NFAT5 is a potential host factor that restricts BoHV-1 productive infection, and virus infection hijacks NFAT5 signaling transduction by relocalization of NFAT5 molecules in cytoplasm, nucleus, and mitochondria, as well as altered expression of its downstream targets. IMPORTANCE Accumulating studies have revealed that NFAT5 regulates disease development due to infection of numerous viruses, underlying the importance of the host factor in virus pathogenesis. Here, we report that NFAT5 has capacity to restrict BoHV-1 productive infection in vitro. And virus productive infection at later stages may alter NFAT5 signaling pathway as observed by relocalization of NFAT5 protein, reduced accumulation of NFAT5 in cytosol, and differential expression of NFAT5 downstream targets. Importantly, for the first time, we found that a subset of NFAT5 resides in mitochondria, implying that NFAT5 may regulate mitochondrial functions, which will extend our knowledge on NFAT5 biological activities. Moreover, we found two NFAT5 isoforms with distinct molecular weights were exclusively detected in the nucleus, where the accumulation was differentially affected following virus infection, representing a novel regulation mechanism on NFAT5 function in response to BoHV-1infection.

DNA Damage Response Differentially Affects BoHV-1 Gene Transcription in Cell Type-Dependent Manners.

Bovine herpesvirus 1 (BoHV-1), an important pathogen of cattle, is also a promising oncolytic virus. Recent studies have demonstrated that the virus infection induces DNA damage and DNA damage response (DDR), potentially accounting for virus infection-induced cell death and oncolytic effects. However, whether the global DDR network affects BoHV-1 productive infection remains to be elucidated. In this study, we show that global DDR induced by ultraviolet (UV) irradiation prior to BoHV-1 infection differentially affected transcription of immediate early (IE) genes, such as infected cell protein 0 (bICP0) and bICP22, in a cell-type-dependent manner. In addition, UV-induced DDR may affect the stabilization of viral protein levels, such as glycoprotein C (gC) and gD, because the variation in mRNA levels of gC and gD as a consequence of UV treatment were not in line with the variation in individual protein levels. The virus productive infection also affects UV-primed DDR signaling, as demonstrated by the alteration of phosphorylated histone H2AX (γH2AX) protein levels and γH2AX formation following virus infection. Taken together, for the first time, we evidenced the interplay between UV-primed global DDR and BoHV-1 productive infection. UV-primed global DDR differentially modulates the transcription of virus genes and stabilization of virus protein. Vice versa, the virus infection may affect UV-primed DDR signaling.