Proteomic Characterization of Bovine Herpesvirus 4 Extracellular Virions

Lété, Céline; Palmeira, Léonor; Leroy, Baptiste; Mast, Jan; Machiels, Bénédicte; Wattiez, Ruddy; Vanderplasschen, Alain; Gillet, Laurent

doi:10.1128/jvi.00456-12

Cited by 23 publications

(53 citation statements)

References 59 publications

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“…Sequence analysis using the NetOGlyc 4.0 server (38) revealed that in the BoHV-4 strain, most of the potential virions O-glycans (Ͼ98%) are located on gp180 (ϳ70%) and on glycoprotein B (gB) (ϳ28%). This was confirmed by mass spectrometry analysis (32). Moreover, we showed that O-glycans associated with gp180 account for most of the sensitivity to neutralization by the O-glycanspecific lectin jacalin (26).…”

Section: Resultssupporting

confidence: 61%

“…BoHV-4 virions grown on MDBK cells were purified as described previously (32). Briefly, after removal of the cell debris by low-speed centrifugation (1,000 ϫ g, 10 min), virions present in the infected cell supernatant were harvested by ultracentrifugation (100,000 ϫ g, 2 h) through a 30% (wt/vol) sucrose cushion, then centrifuged through two successive 20 to 50% (wt/vol) potassium tartrate gradients in PBS (100,000 ϫ g, 2 h).…”

Section: Importancementioning

confidence: 99%

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Bovine Herpesvirus 4 Modulates Its β-1,6- N -Acetylglucosaminyltransferase Activity through Alternative Splicing

Lété

Markine-Goriaynoff

Machiels

et al. 2016

J Virol

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Carbohydrates play major roles in host-virus interactions. It is therefore not surprising that, during coevolution with their hosts, viruses have developed sophisticated mechanisms to hijack for their profit different pathways of glycan synthesis. Thus, the Bo17 gene of Bovine herpesvirus 4 (BoHV-4) encodes a homologue of the cellular core 2 protein ␤-1,6-N-acetylglucosaminyltransferase-mucin type (C2GnT-M), which is a key player for the synthesis of complex O-glycans. Surprisingly, we show in this study that, as opposed to what is observed for the cellular enzyme, two different mRNAs are encoded by the Bo17 gene of all available BoHV-4 strains. While the first one corresponds to the entire coding sequence of the Bo17 gene, the second results from the splicing of a 138-bp intron encoding critical residues of the enzyme. Antibodies generated against the Bo17 C terminus showed that the two forms of Bo17 are expressed in BoHV-4 infected cells, but enzymatic assays revealed that the spliced form is not active. In order to reveal the function of these two forms, we then generated recombinant strains expressing only the long or the short form of Bo17. Although we did not highlight replication differences between these strains, glycomic analyses and lectin neutralization assays confirmed that the splicing of the Bo17 gene gives the potential to BoHV-4 to fine-tune the global level of core 2 branching activity in the infected cell. Altogether, these results suggest the existence of new mechanisms to regulate the activity of glycosyltransferases from the Golgi apparatus. IMPORTANCEViruses are masters of adaptation that hijack cellular pathways to allow their growth. Glycans play a central role in many biological processes, and several studies have highlighted mechanisms by which viruses can affect glycosylation. Glycan synthesis is a nontemplate process regulated by the availability of key glycosyltransferases. Interestingly, bovine herpesvirus 4 encodes one such enzyme which is a key enzyme for the synthesis of complex O-glycans. In this study, we show that, in contrast to cellular homologues, this virus has evolved to alternatively express two proteins from this gene. While the first one is enzymatically active, the second results from the alternative splicing of the region encoding the catalytic site of the enzyme. We postulate that this regulatory mechanism could allow the virus to modulate the synthesis of some particular glycans for function at the location and/or the moment of infection.

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Section: Resultssupporting

confidence: 61%

Section: Importancementioning

confidence: 99%

Bovine Herpesvirus 4 Modulates Its β-1,6- N -Acetylglucosaminyltransferase Activity through Alternative Splicing

Lété

Markine-Goriaynoff

Machiels

et al. 2016

J Virol

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“…To address this issue, we treated virions with proteinase K, in the absence of detergent, prior to density centrifugation as described previously [30], [32], [36]. We validated this treatment by western blotting (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To reduce cellular contaminants, the supernatant was harvested after 72 hours post-infection (hpi) before complete cell lysis. Extracellular virions were purified from the cell supernatant as described previously [36]. Briefly, after removal of the cell debris by low-speed centrifugation (1,000 g , 10 min at 4°C), virions present in the infected cell supernatant (∼1–5×10 6 PFU/mL) were harvested by ultracentrifugation (100,000 g , 2 h at 4°C) through a 30% weight / volume (w/v) sucrose cushion.…”

Section: Methodsmentioning

confidence: 99%

Proteomic Characterization of Murid Herpesvirus 4 Extracellular Virions

et al. 2013

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Gammaherpesvirinae, such as the human Epstein-Barr virus (EBV) and the Kaposi’s sarcoma associated herpesvirus (KSHV) are highly prevalent pathogens that have been associated with several neoplastic diseases. As EBV and KSHV are host-range specific and replicate poorly in vitro, animal counterparts such as Murid herpesvirus-4 (MuHV-4) have been widely used as models. In this study, we used MuHV-4 in order to improve the knowledge about proteins that compose gammaherpesviruses virions. To this end, MuHV-4 extracellular virions were isolated and structural proteins were identified using liquid chromatography tandem mass spectrometry-based proteomic approaches. These analyses allowed the identification of 31 structural proteins encoded by the MuHV-4 genome which were classified as capsid (8), envelope (9), tegument (13) and unclassified (1) structural proteins. In addition, we estimated the relative abundance of the identified proteins in MuHV-4 virions by using exponentially modified protein abundance index analyses. In parallel, several host proteins were found in purified MuHV-4 virions including Annexin A2. Although Annexin A2 has previously been detected in different virions from various families, its role in the virion remains controversial. Interestingly, despite its relatively high abundance in virions, Annexin A2 was not essential for the growth of MuHV-4 in vitro. Altogether, these results extend previous work aimed at determining the composition of gammaherpesvirus virions and provide novel insights for understanding MuHV-4 biology.

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“…To reduce contamination by cellular DNA, cell supernatant was harvested at the early stages of viral release, when approximately 10% of cells were exhibiting cytopathic effect. Virions were semipurified as described previously (20). Briefly, after removal of the cell debris by centrifugation (1,000 ϫ g, 10 min, 4°C), viral particles were pelleted by ultracentrifugation through a 30% sucrose cushion (100,000 ϫ g, 2 h, 4°C).…”

Section: Cells and Virusesmentioning

confidence: 99%

The Genome of a Tortoise Herpesvirus (Testudinid Herpesvirus 3) Has a Novel Structure and Contains a Large Region That Is Not Required for Replication In Vitro or Virulence In Vivo

Gandar

Wilkie

Gatherer

et al. 2015

J Virol

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Testudinid herpesvirus 3 (TeHV-3) is the causative agent of a lethal disease affecting several tortoise species. The threat that this virus poses to endangered animals is focusing efforts on characterizing its properties, in order to enable the development of prophylactic methods. We have sequenced the genomes of the two most studied TeHV-3 strains (1976 and 4295). TeHV-3 strain 1976 has a novel genome structure and is most closely related to a turtle herpesvirus, thus supporting its classification into genus Scutavirus, subfamily Alphaherpesvirinae, family Herpesviridae. The sequence of strain 1976 also revealed viral counterparts of cellular interleukin-10 and semaphorin, which have not been described previously in members of subfamily Alphaherpesvirinae. TeHV-3 strain 4295 is a mixture of three forms (m1, m2, and M), in which, in comparison to strain 1976, the genomes exhibit large, partially overlapping deletions of 12.5 to 22.4 kb. Viral subclones representing these forms were isolated by limiting dilution assays, and each replicated in cell culture comparably to strain 1976. With the goal of testing the potential of the three forms as attenuated vaccine candidates, strain 4295 was inoculated intranasally into Hermann's tortoises (Testudo hermanni). All inoculated subjects died, and PCR analyses demonstrated the ability of the m2 and M forms to spread and invade the brain. In contrast, the m1 form was detected in none of the organs tested, suggesting its potential as the basis of an attenuated vaccine candidate. Our findings represent a major step toward characterizing TeHV-3 and developing prophylactic methods against it. IMPORTANCETestudinid herpesvirus 3 (TeHV-3) causes a lethal disease in tortoises, several species of which are endangered. We have characterized the viral genome and used this information to take steps toward developing an attenuated vaccine. We have sequenced the genomes of two strains (1976 and 4295), compared their growth in vitro, and investigated the pathogenesis of strain 4295, which consists of three deletion mutants. The major findings are that (i) TeHV-3 has a novel genome structure, (ii) its closest relative is a turtle herpesvirus, (iii) it contains interleukin-10 and semaphorin genes (the first time these have been reported in an alphaherpesvirus), (iv) a sizeable region of the genome is not required for viral replication in vitro or virulence in vivo, and (v) one of the components of strain 4295, which has a deletion of 22.4 kb, exhibits properties indicating that it may serve as the starting point for an attenuated vaccine. T he orderHerpesvirales contains a large number of enveloped, double-stranded DNA viruses that share structural, genetic, and biological properties and is divided into three families infecting a wide range of hosts (1). One of these families, the Herpesviridae, contains viruses infecting mammals, birds, or reptiles and is subdivided into three subfamilies, the Alpha-, Beta-, and Gammaherpesvirinae (2). Members of these subfamilies are referred to ...

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Proteomic Characterization of Bovine Herpesvirus 4 Extracellular Virions

Cited by 23 publications

References 59 publications

Bovine Herpesvirus 4 Modulates Its β-1,6- N -Acetylglucosaminyltransferase Activity through Alternative Splicing

Bovine Herpesvirus 4 Modulates Its β-1,6- N -Acetylglucosaminyltransferase Activity through Alternative Splicing

Proteomic Characterization of Murid Herpesvirus 4 Extracellular Virions

The Genome of a Tortoise Herpesvirus (Testudinid Herpesvirus 3) Has a Novel Structure and Contains a Large Region That Is Not Required for Replication In Vitro or Virulence In Vivo

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