Analysis of the Early Steps of Herpes Simplex Virus 1 Capsid Tegumentation

Henaff, Daniel; Rémillard-Labrosse, Gaudeline; Loret, Sandra; Lippé, Roger

doi:10.1128/jvi.03292-12

Cited by 32 publications

(36 citation statements)

References 118 publications

(203 reference statements)

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“…This conclusion is also supported by data from a previous study using VP8-specific immunogold staining that showed accumulation of VP8 protein around the nuclear capsids, as well as along the outer nuclear membrane (9). The addition of certain tegument components of herpesviruses to capsids prior to or during nuclear egress is referred to as primary tegumentation (30). In HHV-1, U L 36, U L 37, ICP0, and ICP4 have been identified as primary tegument proteins that are present on the capsids before they reach the cytoplasm (30).…”

Section: Discussionsupporting

confidence: 76%

“…The addition of certain tegument components of herpesviruses to capsids prior to or during nuclear egress is referred to as primary tegumentation (30). In HHV-1, U L 36, U L 37, ICP0, and ICP4 have been identified as primary tegument proteins that are present on the capsids before they reach the cytoplasm (30). The U L 31 and U L 34 gene products of PrV are incorporated in the primary enveloped capsids and eventually dissociate from the capsids during the later process (31).…”

Section: Discussionmentioning

confidence: 99%

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

Brownlie

Snider

Hurk

2016

J Virol

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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...

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Section: Discussionsupporting

confidence: 76%

Section: Discussionmentioning

confidence: 99%

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

Brownlie

Snider

Hurk

2016

J Virol

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“…Assembly of herpesviruses commences with nuclear procapsids that package viral genomes and mature into icosahedral capsids that diffuse to the inner nuclear membrane for primary budding (9,11,12). The primary envelopes fuse with the outer nuclear membrane and release the capsids into the cytosol, where they associate with inner tegument proteins: e.g., pUL36 and pUL37 of the Alphaherpesviri-nae (10,13,14). Cytosolic capsids are transported along microtubules to the cytoplasmic organelles of secondary envelopment (15,16; K. Döhner, A. Buch, L. Ivanova, A. Binz, A. Pohlmannn, M. Capucci, M. Sandbaumhüter, B. Sodeik, and R. Bauerfeind, submitted for publication).…”

mentioning

confidence: 99%

Conserved Tryptophan Motifs in the Large Tegument Protein pUL36 Are Required for Efficient Secondary Envelopment of Herpes Simplex Virus Capsids

Ivanova

Buch

Döhner

et al. 2016

J Virol

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Herpes simplex virus (HSV) replicates in the skin and mucous membranes, and initiates lytic or latent infections in sensory neurons. Assembly of progeny virions depends on the essential large tegument protein pUL36 of 3,164 amino acid residues that links the capsids to the tegument proteins pUL37 and VP16. Of the 32 tryptophans of HSV-1-pUL36, the tryptophan-acidic motifs 1766 WD 1767 and 1862 WE 1863 are conserved in all HSV-1 and HSV-2 isolates. Here, we characterized the role of these motifs in the HSV life cycle since the rare tryptophans often have unique roles in protein function due to their large hydrophobic surface. The infectivity of the mutants HSV-1(17 ؉ )Lox-pUL36-WD/AA-WE/AA and HSV-1(17 ؉ )Lox-CheVP26-pUL36-WD/AA-WE/AA, in which the capsid has been tagged with the fluorescent protein Cherry, was significantly reduced. Quantitative electron microscopy shows that there were a larger number of cytosolic capsids and fewer enveloped virions compared to their respective parental strains, indicating a severe impairment in secondary capsid envelopment. The capsids of the mutant viruses accumulated in the perinuclear region around the microtubule-organizing center and were not dispersed to the cell periphery but still acquired the inner tegument proteins pUL36 and pUL37. Furthermore, cytoplasmic capsids colocalized with tegument protein VP16 and, to some extent, with tegument protein VP22 but not with the envelope glycoprotein gD. These results indicate that the unique conserved tryptophan-acidic motifs in the central region of pUL36 are required for efficient targeting of progeny capsids to the membranes of secondary capsid envelopment and for efficient virion assembly. IMPORTANCEHerpesvirus infections give rise to severe animal and human diseases, especially in young, immunocompromised, and elderly individuals. The structural hallmark of herpesvirus virions is the tegument, which contains evolutionarily conserved proteins that are essential for several stages of the herpesvirus life cycle. Here we characterized two conserved tryptophan-acidic motifs in the central region of the large tegument protein pUL36 of herpes simplex virus. When we mutated these motifs, secondary envelopment of cytosolic capsids and the production of infectious particles were severely impaired. Our data suggest that pUL36 and its homologs in other herpesviruses, and in particular such tryptophan-acidic motifs, could provide attractive targets for the development of novel drugs to prevent herpesvirus assembly and spread. T he diversity of clinical herpesvirus isolates is shaped to a large extent by the long coevolution between virus and host as well as by homologous recombination among different strains (1-4). The subfamilies of the Herpesviridae are characterized by similar properties: for example, fast replication of members of the Alphaherpesvirinae in keratinocytes, epithelial cells, or fibroblasts and latency in sensory neurons. Infections with the herpes simplex viruses (HSV-1 or HSV-2) or varicella-zoster virus occur...

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“…nucleus by packaging of the viral genomes into capsids but is completed at cytoplasmic membranes with the secondary envelopment of the capsids. Nuclear capsids seem to associate with several partially characterized tegument proteins, 1,2 final tegumentation, however, occurs during secondary envelopment. HSV1 virions released to the extracellular milieu contain more than 25 tegument proteins 1 Christina Funk, Verena Raschbichler and Diana Lieber should be considered joint first authors.…”

Section: Morphogenesis Of Herpesviral Virions Is Initiated In the Hostmentioning

confidence: 99%

Comprehensive analysis of nuclear export of herpes simplex virus type 1 tegument proteins and their Epstein‐Barr virus orthologs

Funk

Raschbichler

Lieber

et al. 2019

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Morphogenesis of herpesviral virions is initiated in the nucleus but completed in the cytoplasm. Mature virions contain more than 25 tegument proteins many of which perform both nuclear and cytoplasmic functions suggesting they shuttle between these compartments. While nuclear import of herpesviral proteins was shown to be crucial for viral propagation, active nuclear export and its functional impact are still poorly understood. To systematically analyze nuclear export of tegument proteins present in virions of Herpes simplex virus type 1 (HSV1) and Epstein‐Barr virus (EBV), the Nuclear EXport Trapped by RAPamycin (NEX‐TRAP) was applied. Nine of the 22 investigated HSV1 tegument proteins including pUL4, pUL7, pUL11, pUL13, pUL21, pUL37d11, pUL47, pUL48 and pUS2 as well as 2 out of 6 EBV orthologs harbor nuclear export activity. A functional leucine‐rich nuclear export sequence (NES) recognized by the export factor CRM1/Xpo1 was identified in six of them. The comparison between experimental and bioinformatic data indicates that experimental validation of predicted NESs is required. Mutational analysis of the pUL48/VP16 NES revealed its importance for herpesviral propagation. Together our data suggest that nuclear export is an important feature of the herpesviral life cycle required to co‐ordinate nuclear and cytoplasmic processes.

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Analysis of the Early Steps of Herpes Simplex Virus 1 Capsid Tegumentation

Cited by 32 publications

References 118 publications

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

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

Conserved Tryptophan Motifs in the Large Tegument Protein pUL36 Are Required for Efficient Secondary Envelopment of Herpes Simplex Virus Capsids

Comprehensive analysis of nuclear export of herpes simplex virus type 1 tegument proteins and their Epstein‐Barr virus orthologs

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