Dynamic ubiquitination drives herpesvirus neuroinvasion

Huffmaster, Nicholas J; Sollars, Patricia J.; Richards, Alexsia; Pickard, Gary E.; Smith, Greg

doi:10.1073/pnas.1512559112

Cited by 47 publications

(46 citation statements)

References 45 publications

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“…Recently, Huffmaster et al reported that pUL36, a homolog of pUL48, of PRV contains ubiquitin addition sites and that both the DUB activity and modification by ubiquitin at a specific lysine reside play a role in neuroinvasion (50). They demonstrated that the DUB activity was dispensable for neurotropism but essential for neuroinvasion and that ubiquitin addition at a lysine residue (K442) was required for retrograde axonal transport.…”

Section: Discussionmentioning

confidence: 99%

Involvement of the N-Terminal Deubiquitinating Protease Domain of Human Cytomegalovirus UL48 Tegument Protein in Autoubiquitination, Virion Stability, and Virus Entry

Kim

Kwon

et al. 2016

J Virol

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Human cytomegalovirus (HCMV) protein pUL48 is closely associated with the capsid and has a deubiquitinating protease (DUB) activity in its N-terminal region. Although this DUB activity moderately increases virus replication in cultured fibroblast cells, the requirements of the N-terminal region of pUL48 in the viral replication cycle are not fully understood. In this study, we characterized the recombinant viruses encoding UL48(⌬DUB/NLS), which lacks the DUB domain and the adjacent nuclear localization signal (NLS), UL48(⌬DUB), which lacks only the DUB, and UL48(⌬360 -1200), which lacks the internal region (amino acids 360 to 1200) downstream of the DUB/NLS. While ⌬DUB/NLS and ⌬360 -1200 mutant viruses did not grow in fibroblasts, the ⌬DUB virus replicated to titers 100-fold lower than those for wild-type virus and showed substantially reduced viral gene expression at low multiplicities of infection. The DUB domain contained ubiquitination sites, and DUB activity reduced its own proteasomal degradation in trans. Deletion of the DUB domain did not affect the nuclear and cytoplasmic localization of pUL48, whereas the internal region (360 -1200) was necessary for cytoplasmic distribution. In coimmunoprecipitation assays, pUL48 interacted with three tegument proteins (pUL47, pUL45, and pUL88) and two capsid proteins (pUL77 and pUL85) but the DUB domain contributed to only pUL85 binding. Furthermore, we found that the ⌬DUB virus showed reduced virion stability and less efficiently delivered its genome into the cell than the wild-type virus. Collectively, our results demonstrate that the N-terminal DUB domain of pUL48 contributes to efficient viral growth by regulating its own stability and promoting virion stabilization and virus entry. IMPORTANCEHCMV pUL48 and its herpesvirus homologs play key roles in virus entry, regulation of immune signaling pathways, and virion assembly. The N terminus of pUL48 contains the DUB domain, which is well conserved among all herpesviruses. Although studies using the active-site mutant viruses revealed that the DUB activity promotes viral growth, the exact role of this region in the viral life cycle is not fully understood. In this study, using the mutant virus lacking the entire DUB domain, we demonstrate that the DUB domain of pUL48 contributes to viral growth by regulating its own stability via autodeubiquitination and promoting virion stability and virus entry. This report is the first to demonstrate the characteristics of the mutant virus with the entire DUB domain deleted, which, along with information on the functions of this region, is useful in dissecting the functions associated with pUL48. H uman cytomegalovirus (HCMV) belongs to the Betaherpesvirus subfamily. HCMV infection is usually asymptomatic and causes latent or persistent infections in healthy people. However, congenital infection and reactivation from latent infection in immunocompromised individuals can cause severe disease (1). The HCMV virion is composed of an icosahedral capsid containing a 235-kb linea...

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Section: Discussionmentioning

confidence: 99%

Involvement of the N-Terminal Deubiquitinating Protease Domain of Human Cytomegalovirus UL48 Tegument Protein in Autoubiquitination, Virion Stability, and Virus Entry

Kim

Kwon

et al. 2016

J Virol

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“…This region has been suggested to be of functional importance since deletion of a larger region of PRV-pUL36 of residues 1294 to 2025 including the homologous residues cannot complement virus replication (90). The HSV-1 lysine at position 1799 located between our tryptophan-acidic motifs is ubiquitinated and targeted by the autocatalytic USP activity of pUL36; however it is not conserved among the alphaherpesviruses (91). The novel HSV-1 mutants were amplified to the same titers as their parental strains in the complementing Vero-HS30 cells.…”

Section: Figmentioning

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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“…Although the DUBs of various herpesviruses collectively have a diversity of cellular substrates (Whitehurst et al 2009, 2012; Gastaldello et al 2010, 2013; Inn et al 2011; Saito et al 2013; Wang et al 2013; van Gent et al 2014; Kim et al 2016), the alphaherpesvirus neuroinvasive property proved to function by an autocatalytic mechanism that switches the virus between two invasive states (Bolstad et al 2011; Huffmaster et al 2015). The first invasive state promotes transmission of infection from epithelial tissues to peripheral nerves, and the second state subsequently promotes sustained retrograde transport in axons.…”

Section: 6 Deliverymentioning

confidence: 99%

“…VP1/2 lacking ubiquitin modification at a lysine near the N-terminus of the protein (Fig. 8.4) moves bidirectionally on microtubules, and the addition of ubiquitin switches transport to unidirectional transport toward the minus-ends of microtubules (Huffmaster et al 2015). The modified state is critical upon entering axon terminals, where sustained long-distance transport is required to deliver capsids to neural soma in peripheral ganglia.…”

Section: 6 Deliverymentioning

confidence: 99%