VP8, the Major Tegument Protein of Bovine Herpesvirus-1, Is Partially Packaged during Early Tegument Formation in a VP22-Dependent Manner

Bovine herpesvirus-1 (BoHV-1) is a major cause of rhinotracheitis and vulvovaginitis in cattle. VP8, the major tegument protein of BoHV-1, is essential for viral replication in the host. VP8 is phosphorylated by the viral kinase US3, mediating its translocation to the cytoplasm. VP8 remains nuclear when not phosphorylated. Interestingly, VP8 has a significant presence in mature BoHV-1YmVP8, in which the VP8 phosphorylation sites are mutated. This suggests that VP8 might be packaged during primary envelopment of BoHV-1. This was investigated by mass spectrometry and Western blotting, which showed VP8, as well as VP22, to be constituents of the primary enveloped virions. VP8 and VP22 were shown to interact via co-immunoprecipitation experiments, in both BoHV-1-infected and VP8-transfected cells. VP8 and VP22 also co-localised with one another and with nuclear lamin-associated protein 2 in BoHV-1-infected cells, suggesting an interaction between VP8 and VP22 in the perinuclear region. In cells infected with VP22-deleted BoHV-1 (BoHV-1ΔUL49), VP8 was absent from the primary enveloped virions, implying that VP22 might be critical for the early packaging of VP8. In conclusion, a novel VP22-dependent mechanism for packaging of VP8 was identified, which may be responsible for a significant amount of VP8 in the viral particle.

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Bovine Herpesvirus 1 UL3.5 Interacts with Bovine Herpesvirus 1 α-Transinducing Factor

Journal of Virology ◽

10.1128/jvi.74.6.2876-2884.2000 ◽

2000 ◽

Vol 74 (6) ◽

pp. 2876-2884 ◽

Cited By ~ 9

Author(s):

Ngan Lam ◽

Geoffrey J. Letchworth

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Bovine Herpesvirus ◽

Mass Spectrometry Analysis ◽

Bovine Herpesvirus 1 ◽

Subcellular Compartment ◽

Transfected Cells ◽

Infected Cells ◽

Herpesvirus 1

ABSTRACT The bovine herpesvirus 1 (BHV-1) UL3.5 gene encodes a 126-amino-acid tegument protein. Homologs of UL3.5 are present in some alphaherpesviruses and have 20 to 30% overall amino acid homology that is concentrated in the N-terminal 50 amino acids. Mutant pseudorabies virus lacking UL3.5 is deficient in viral egress but can be complemented by BHV-1 UL3.5 (W. Fuchs, H. Granzow, and T. C. Mettenleiter, J. Virol. 71:8886–8892, 1997). The function of BHV-1 UL3.5 in BHV-1 replication is not known. To get a better understanding of its function, we sought to identify the proteins that interact with the BHV-1 UL3.5 protein. By using an in vitro pull-down assay and matrix-assisted laser desorption ionization mass spectrometry analysis, we identified BHV-1 α-transinducing factor (αBTIF) as a BHV-1 UL3.5-interacting protein. The interaction was verified by coimmunoprecipitation from virus-infected cells using an antibody to either protein, by indirect immunofluorescence colocalization in both virus-infected and transfected cells, and by the binding of in vitro-translated proteins. In virus-infected cells, UL3.5 and αBTIF colocalized in a Golgi-like subcellular compartment late in infection. In transfected cells, they colocalized in the nucleus. Deletion of 20 amino acids from the N terminus of UL3.5, but not 40 amino acids from the C terminus, abolished the UL3.5-αBTIF interaction both in vitro and in vivo. The interaction between UL3.5 and αBTIF may be important for BHV-1 maturation and regulation of αBTIF transactivation activity.

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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

Journal of Virology ◽

10.1128/jvi.02151-18 ◽

2019 ◽

Vol 93 (6) ◽

Cited By ~ 1

Author(s):

Kuan Zhang ◽

Tara Donovan ◽

Soumya Sucharita ◽

Robert Brownlie ◽

Marlene Snider ◽

...

Keyword(s):

Lipid Metabolism ◽

Cellular Localization ◽

Bovine Herpesvirus ◽

Tegument Protein ◽

Cytoplasmic Localization ◽

Cellular Lipid ◽

Bovine Herpesvirus 1 ◽

Cholesterol Levels ◽

Infected Cells ◽

Herpesvirus 1

ABSTRACTBovine herpesvirus 1 (BoHV-1) infects bovine species, causing respiratory infections, genital disorders and abortions. VP8 is the most abundant tegument protein of BoHV-1 and is critical for virus replication in cattle. In this study, the cellular transport of VP8 in BoHV-1-infected cells and its ability to alter the cellular lipid metabolism were investigated. A viral kinase, US3, was found to be involved in regulating these processes. In the early stages of infection VP8 was localized in the nucleus. Subsequently, presumably after completion of its role in the nucleus, VP8 was translocated to the cytoplasm. When US3 was deleted or the essential US3 phosphorylation site of VP8 was mutated in BoHV-1, the majority of VP8 was localized in the nuclei of infected cells. This suggests that phosphorylation by US3 may be critical for cytoplasmic localization of VP8. Eventually, the cytoplasmic VP8 was accumulated in thecis-Golgi apparatus but not in thetrans-Golgi network, implying that VP8 was not involved in virion transport toward and budding from the cell membrane. VP8 caused lipid droplet (LD) formation in the nuclei of transfected cells and increased cellular cholesterol levels. Lipid droplets were not found in the nuclei of BoHV-1-infected cells when VP8 was cytoplasmic in the presence of US3. However, when US3 was deleted or phosphorylation residues in VP8 were mutated, nuclear VP8 and LDs appeared in BoHV-1-infected cells. The total cholesterol level was increased in BoHV-1-infected cells but not in ΔUL47-BoHV-1-infected cells, further supporting a role for VP8 in altering the cellular lipid metabolism during infection.IMPORTANCENuclear localization signals (NLSs) and nuclear export signals (NESs) are important elements directing VP8 to the desired locations in the BoHV-1-infected cell. In this study, a critical regulator that switches the nuclear and cytoplasmic localization of VP8 in BoHV-1-infected cells was identified. BoHV-1 used viral kinase US3 to regulate the cellular localization of VP8. Early during BoHV-1 infection VP8 was localized in the nucleus, where it performs various functions; once US3 was expressed, phosphorylated VP8 was cytoplasmic and ultimately accumulated in thecis-Golgi apparatus, presumably to be incorporated into virions. The Golgi localization of VP8 was only observed in virus-infected cells and not in US3-cotransfected cells, suggesting that this is mediated by other viral factors. Interestingly, VP8 was shown to cause increased cholesterol levels, which is a novel function for VP8 and a potential strategy to supply lipid for viral replication.

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Glycoprotein M of bovine herpesvirus 1 (BHV-1) is nonessential for replication in cell culture and is involved in inhibition of bovine respiratory syncytial virus F protein induced syncytium formation in recombinant BHV-1 infected cells

Veterinary Microbiology ◽

10.1016/s0378-1135(01)00489-8 ◽

2002 ◽

Vol 86 (1-2) ◽

pp. 37-49 ◽

Cited By ~ 17

Author(s):

Patricia König ◽

Katrin Giesow ◽

Günther M Keil

Keyword(s):

Cell Culture ◽

Respiratory Syncytial Virus ◽

Bovine Herpesvirus ◽

Syncytium Formation ◽

Bovine Respiratory Syncytial Virus ◽

Bovine Herpesvirus 1 ◽

F Protein ◽

Infected Cells ◽

Herpesvirus 1 ◽

Syncytial Virus

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Intercellular trafficking of the major tegument protein VP22 of bovine herpesvirus-1 and its application to improve a DNA vaccine

Archives of Virology ◽

10.1007/s00705-005-0694-7 ◽

2005 ◽

Vol 151 (5) ◽

pp. 985-993 ◽

Cited By ~ 12

Author(s):

C. F. Zheng ◽

R. Brownlie ◽

D. Y. Huang ◽

L. A. Babiuk ◽

S. van Drunen Littel-van den Hurk

Keyword(s):

Dna Vaccine ◽

Bovine Herpesvirus ◽

Tegument Protein ◽

Bovine Herpesvirus 1 ◽

Intercellular Trafficking ◽

Herpesvirus 1 ◽

Protein Vp22

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Tyrosine Phosphorylation of Bovine Herpesvirus 1 Tegument Protein VP22 Correlates with the Incorporation of VP22 into Virions

Journal of Virology ◽

10.1128/jvi.75.19.9010-9017.2001 ◽

2001 ◽

Vol 75 (19) ◽

pp. 9010-9017 ◽

Cited By ~ 13

Author(s):

Xiaodi Ren ◽

Jerome S. Harms ◽

Gary A. Splitter

Keyword(s):

Tyrosine Phosphorylation ◽

Phosphorylation Site ◽

Bovine Herpesvirus ◽

Structural Proteins ◽

Tegument Protein ◽

Bovine Herpesvirus 1 ◽

C Terminus ◽

Herpesvirus 1 ◽

Protein Vp22 ◽

Viral Structural Proteins

ABSTRACT Tyrosine phosphorylation has been shown to play a role in the replication of several herpesviruses. In this report, we demonstrate that bovine herpesvirus 1 infection triggered tyrosine phosphorylation of proteins with molecular masses similar to those of phosphorylated viral structural proteins. One of the tyrosine-phosphorylated viral structural proteins was the tegument protein VP22. A tyrosine 38-to-phenylalanine mutation totally abolished the phosphorylation of VP22 in transfected cells. However, construction of a VP22 tyrosine 38-to-phenylalanine mutant virus demonstrated that VP22 was still phosphorylated but that the phosphorylation site may change to the C terminus rather than be in the N terminus as in wild-type VP22. In addition, the loss of VP22 tyrosine phosphorylation correlated with reduced incorporation of VP22 compared to that of envelope glycoprotein D in the mutant viruses but not with the amount of VP22 produced during virus infection. Our data suggest that tyrosine phosphorylation of VP22 plays a role in virion assembly.

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Major tegument protein VP8 of bovine herpesvirus 1 is phosphorylated by viral US3 and cellular CK2 protein kinases

Journal of General Virology ◽

10.1099/vir.0.013532-0 ◽

2009 ◽

Vol 90 (12) ◽

pp. 2829-2839 ◽

Cited By ~ 18

Author(s):

Shaunivan L. Labiuk ◽

Lorne A. Babiuk ◽

Sylvia van Drunen Littel-van den Hurk

Keyword(s):

Protein Kinase ◽

Protein Kinases ◽

Gene Product ◽

Kinase Inhibitor ◽

Bovine Herpesvirus ◽

Kinase Assay ◽

Bovine Herpesvirus 1 ◽

Infected Cells ◽

Herpesvirus 1

The UL47 gene product, VP8, is one of the major tegument proteins of bovine herpesvirus 1 (BoHV-1) and is subject to phosphorylation. Analysis of protein bands co-immunoprecipitated with VP8 from BoHV-1-infected cells by mass spectroscopy suggested that VP8 interacts with two protein kinases: cellular CK2 and viral US3. CK2 is a highly conserved cellular protein, expressed ubiquitously and known to phosphorylate numerous proteins. The US3 gene product is one of the viral kinases produced by BoHV-1 during infection. Interactions of CK2 and US3 with VP8 were confirmed outside the context of infection when FLAG–VP8 was expressed alone or co-expressed with US3–haemagglutinin tag in Cos-7 cells. Furthermore, VP8 and US3 were found to co-localize in the nucleus during viral infection. To explore the significance of these interactions, an in vitro kinase assay was performed, which demonstrated that VP8 is heavily phosphorylated by CK2. In the presence of the highly specific CK2 kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), phosphorylation of VP8 was significantly reduced. Phosphorylation of VP8 was also inhibited by the presence of kenpaullone, a less specific CK2 inhibitor, but not by protein kinase CK1 or protein kinase C inhibitors. When VP8 and US3 were both included in the kinase assay in the presence of DMAT, phosphorylation of VP8 was again observed. Autophosphorylation of US3 was also detected and was not inhibited by DMAT. Based on these results, it is proposed that VP8 interacts with cellular CK2 and viral US3 in BoHV-1-infected cells, and is in turn subject to kinase activities associated with both of these proteins.

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Qualitative Differences in Capsidless L-Particles Released as a By-Product of Bovine Herpesvirus 1 and Herpes Simplex Virus 1 Infections

Journal of Virology ◽

10.1128/jvi.01259-18 ◽

2018 ◽

Vol 92 (22) ◽

Cited By ~ 6

Author(s):

Tiffany Russell ◽

Ben Bleasdale ◽

Michael Hollinshead ◽

Gillian Elliott

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Cell Biology ◽

Bovine Herpesvirus ◽

Herpes Simplex Virus 1 ◽

Bovine Herpesvirus 1 ◽

Infected Cells ◽

Herpesvirus 1 ◽

Simplex Virus ◽

Hsv 1

ABSTRACTDespite differences in the pathogenesis and host range of alphaherpesviruses, many stages of their morphogenesis are thought to be conserved. Here, an ultrastructural study of bovine herpesvirus 1 (BoHV-1) envelopment revealed profiles similar to those previously found for herpes simplex virus 1 (HSV-1), with BoHV-1 capsids associating with endocytic tubules. Consistent with the similarity of their genomes and envelopment strategies, the proteomic compositions of BoHV-1 and HSV-1 virions were also comparable. However, BoHV-1 morphogenesis exhibited a diversity in envelopment events. First, heterogeneous primary envelopment profiles were readily detectable at the inner nuclear membrane of BoHV-1-infected cells. Second, the BoHV-1 progeny comprised not just full virions but also an abundance of capsidless, noninfectious light particles (L-particles) that were released from the infected cells in numbers similar to those of virions and in the absence of DNA replication. Proteomic analysis of BoHV-1 L-particles and the much less abundant HSV-1 L-particles revealed that they contained the same complement of envelope proteins as virions but showed variations in tegument content. In the case of HSV-1, the UL46 tegument protein was reproducibly found to be >6-fold enriched in HSV-1 L-particles. More strikingly, the tegument proteins UL36, UL37, UL21, and UL16 were depleted in BoHV-1 but not HSV-1 L-particles. We propose that these combined differences reflect the presence of truly segregated “inner” and “outer” teguments in BoHV-1, making it a critical system for studying the structure and process of tegumentation and envelopment.IMPORTANCEThe alphaherpesvirus family includes viruses that infect humans and animals. Hence, not only do they have a significant impact on human health, but they also have a substantial economic impact on the farming industry. While the pathogenic manifestations of the individual viruses differ from host to host, their relative genetic compositions suggest similarity at the molecular level. This study provides a side-by-side comparison of the particle outputs from the major human pathogen HSV-1 and the veterinary pathogen BoHV-1. Ultrastructural and proteomic analyses have revealed that both viruses have broadly similar morphogenesis profiles and infectious virus compositions. However, the demonstration that BoHV-1 has the capacity to generate vast numbers of capsidless enveloped particles that differ from those produced by HSV-1 in composition implies a divergence in the cell biology of these viruses that impacts our general understanding of alphaherpesvirus morphogenesis.

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Nucleocytoplasmic Shuttling of Bovine Herpesvirus 1 UL47 Protein in Infected Cells

Journal of Virology ◽

10.1128/jvi.80.2.1059-1063.2006 ◽

2006 ◽

Vol 80 (2) ◽

pp. 1059-1063 ◽

Cited By ~ 19

Author(s):

Janneke Verhagen ◽

Ian Hutchinson ◽

Gillian Elliott

Keyword(s):

Fluorescent Protein ◽

Bovine Herpesvirus ◽

Nucleocytoplasmic Shuttling ◽

Bovine Herpesvirus 1 ◽

Infected Cells ◽

Herpesvirus 1 ◽

Green Fluorescent ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Previous studies with transfected cells have shown that the herpes simplex virus type 1 (HSV-1) and bovine herpesvirus 1 (BHV-1) UL47 proteins shuttle between the nucleus and the cytoplasm. HSV-1 UL47 has also been shown to bind RNA. Here we examine the BHV-1 UL47 protein in infected cells using a green fluorescent protein-UL47-expressing virus. We show that UL47 is detected in the nucleus early in infection. We use fluorescence loss in photobleaching to show that nuclear UL47 undergoes rapid nucleocytoplasmic shuttling. Furthermore, we demonstrate that actinomycin D inhibits the reaccumulation of UL47 in the nuclei of infected cells. These results suggest that UL47 exhibits behavior similar to that of previously characterized RNA-transporting proteins.

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