A Nuclear Localization Signal in Herpesvirus Protein VP1-2 Is Essential for Infection via Capsid Routing to the Nuclear Pore

Abaitua, Fernando; Hollinshead, Mike; Bolstad, M.; Crump, Colin M.; O’Hare, Peter

doi:10.1128/jvi.01209-12

Cited by 73 publications

(81 citation statements)

References 62 publications

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“…Although the targets for the UL48 DUB are not known, several key components of the immune signaling pathways have been identified as cellular targets for other herpesviral DUBs (45): HSV-1 pUL36 inhibits interferon beta production by removing polyubiquitin chains from TRAF3 (46), Epstein-Barr virus (EBV) BPLF1 deubiquitinates TRAF6 to inhibit NF-B signaling (47), and Kaposi's sarcoma-associated herpesvirus (KSHV) ORF64 suppresses RIG-I-mediated interferon signaling by reducing RIG-I ubiquitination (48). Second, a deletion of the DUB domain adjacent to the NLS may affect the pUL48-associated activity in routing the capsid to the nuclear pore, which requires the functional NLS, as shown in a recent study using HSV-1 pUL36 (20). However, it appears that at least the nuclear transport of the newly synthesized pUL48 protein does not require the DUB domain, since the nuclear and cytoplasmic localization patterns of wildtype and ⌬DUB proteins were indistinguishable in our study.…”

Section: Discussionmentioning

confidence: 98%

“…pUL36 is required for capsid transport within the cell by interacting with the microtubule network (15)(16)(17)(18). In HSV-1, the nuclear localization signal (NLS) of pUL36 is required for routing of the capsid to the nuclear pore (19,20) and proteolytic cleavage of pUL36 is necessary for release of HSV-1 DNA into the nucleus (21). Recently, pUL48 was also shown to contain the NLS that is indispensable for viral growth just downstream from the DUB domain (22) and can functionally substitute for the NLS of pUL36 (23).…”

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confidence: 99%

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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: 98%

mentioning

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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“…pUL36 is attached directly to the icosahedral capsid shell of these enveloped viruses (10)(11)(12) and, due to its location between the capsid and envelope, is referred to as a herpesvirus tegument protein. Upon virus entry into a cell, pUL36 becomes exposed to the cytosol (4,13,14) and directs delivery of the capsid and its DNA content to the nucleus (2,(15)(16)(17)(18)(19). Following replication, pUL36 is also essential for viral assembly and egress (20)(21)(22).…”

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confidence: 99%

Dynamic ubiquitination drives herpesvirus neuroinvasion

Huffmaster

Sollars

Richards

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Neuroinvasive herpesviruses display a remarkable propensity to enter the nervous system of healthy individuals in the absence of obvious trauma at the site of inoculation. We document a repurposing of cellular ubiquitin during infection to switch the virus between two invasive states. The states act sequentially to defeat consecutive host barriers of the peripheral nervous system and together promote the potent neuroinvasive phenotype. The first state directs virus access to nerve endings in peripheral tissue, whereas the second delivers virus particles within nerve fibers to the neural ganglia. Mutant viruses locked in either state remain competent to overcome the corresponding barrier but fail to invade the nervous system. The herpesvirus “ubiquitin switch” may explain the unusual ability of these viruses to routinely enter the nervous system and, as a consequence, their prevalence in human and veterinary hosts.

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“…It is a major constituent of the innermost layer of tegument, the complex protein layer between the capsid and the inner surface of the envelope, and connects the capsid to multiple outer tegument proteins (22)(23)(24)(25)(26)(27)(28)(29)(30). During entry, UL36p recruits dynein/dynactin (31)(32)(33)(34) to mediate retrograde traffic of capsids to the microtubule organizing center (35) and nuclear pores (36)(37)(38). UL36p also plays essential but poorly understood roles in assembly and egress: transporting capsids to their site of envelopment (34,39,40), helping drive envelopment (30,39,41), and also supporting subsequent traffic of enveloped particles (42).…”

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confidence: 99%

Herpes Simplex Virus Capsid-Organelle Association in the Absence of the Large Tegument Protein UL36p

Kharkwal

Furgiuele

Smith

et al. 2015

J Virol

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UL36p (VP1/2) is the largest protein encoded by herpes simplex virus 1 (HSV-1) and resides in the innermost layer of the viral tegument, lying between the capsid and the envelope. UL36p performs multiple functions in the HSV life cycle, including an essential role in cytoplasmic envelopment. We earlier described the isolation of a virion-associated cytoplasmic membrane fraction from HSV-infected cells. Biochemical and ultrastructural analyses showed that the organelles in this buoyant fraction contain enveloped infectious HSV particles in their lumens and naked capsids docked to their cytoplasmic surfaces. These organelles can also recruit molecular motors and transport their cargo virions along microtubules in vitro. Here we examine the properties of these HSV-associated organelles in the absence of UL36p. We find that while capsid envelopment is clearly defective, a subpopulation of capsids nevertheless still associate with the cytoplasmic faces of these organelles. The existence of these capsidmembrane structures was confirmed by subcellular fractionation, immunocytochemistry, lipophilic dye fluorescence microscopy, thin-section electron microscopy, and correlative light and electron microscopy. We conclude that capsid-membrane binding can occur in the absence of UL36p and propose that this association may precede the events of UL36p-driven envelopment. IMPORTANCEMembrane association and envelopment of the HSV capsid are essential for the assembly of an infectious virion. Envelopment involves the complex interplay of a large number of viral and cellular proteins; however, the function of most of them is unknown. One example of this is the viral protein UL36p, which is clearly essential for envelopment but plays a poorly understood role. Here we demonstrate that organelles utilized for HSV capsid envelopment still accumulate surface-bound capsids in the absence of UL36p. We propose that UL36p-independent binding of capsids to organelles occurs prior to the function of UL36p in capsid envelopment. Herpesviruses replicate their genomes and assemble DNApackaged capsids in the cell nucleus. It is then generally accepted that capsids bud into the inner nuclear membrane to generate primary enveloped perinuclear virions that subsequently fuse with the outer nuclear membrane, releasing mature nucleocapsids ("naked" capsids) into the cytoplasm (1-3). These naked cytoplasmic capsids subsequently undergo secondary envelopment at a postnuclear organelle to assemble the mature, infectious virion (4-9). Completion of cytoplasmic envelopment requires the coordinated interaction of multiple virally encoded proteins (8,(10)(11)(12)(13) and the participation of components of the cellular endosomal sorting complexes required for transport (ESCRT) machinery (14-19).UL36p (VP1/2) is the largest structural protein encoded by the herpesviridae (20, 21). It is a major constituent of the innermost layer of tegument, the complex protein layer between the capsid and the inner surface of the envelope, and connects the capsid to m...

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A Nuclear Localization Signal in Herpesvirus Protein VP1-2 Is Essential for Infection via Capsid Routing to the Nuclear Pore

Cited by 73 publications

References 62 publications

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

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

Dynamic ubiquitination drives herpesvirus neuroinvasion

Herpes Simplex Virus Capsid-Organelle Association in the Absence of the Large Tegument Protein UL36p

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