Human Cytomegalovirus UL97 Phosphorylates the Viral Nuclear Egress Complex

Sharma, Madhu; Bender, Brian J.; Kamil, Jeremy P.; Lye, Ming F.; Pesola, Jean M.; Reim, Natalia I.; Hogle, James M.; Coen, Donald M.

doi:10.1128/jvi.02426-14

Cited by 57 publications

(78 citation statements)

References 57 publications

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“…Furthermore, a related yet distinct mechanism by which ORF36 may play a role in nuclear egress is by phosphorylating components of the viral nuclear egress complex (NEC). This has recently been suggested as an additional mechanism by which HCMV UL97 modulates nuclear egress (38,42). The KSHV NEC, comprised of ORF67 and ORF69, has only recently begun to be studied (43,44).…”

Section: Discussionmentioning

confidence: 99%

Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36

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Tepper

Pifer

et al. 2016

J Virol

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Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human malignancies. KSHV ORF36 encodes a serine/threonine viral protein kinase, which is conserved throughout all herpesviruses. Although several studies have identified the viral and cellular substrates of conserved herpesvirus protein kinases (CHPKs), the precise functions of KSHV ORF36 during lytic replication remain elusive. Here, we report that ORF36 interacts with another lytic protein, ORF45, in a manner dependent on ORF36 kinase activity. We mapped the regions of ORF36 and ORF45 involved in the binding. Their association appears to be mediated by electrostatic interactions, since deletion of either the highly basic N terminus of ORF36 or an acidic patch of ORF45 abolished the binding. In addition, the dephosphorylation of ORF45 protein dramatically reduced its association with ORF36. Importantly, ORF45 enhances both the stability and kinase activity of ORF36. Consistent with previous studies of CHPK homologs, we detected ORF36 protein in extracellular virions. To investigate the roles of ORF36 in the context of KSHV lytic replication, we used bacterial artificial chromosome mutagenesis to engineer both ORF36-null and kinase-dead mutants. We found that ORF36-null/mutant virions are moderately defective in viral particle production and are further deficient in primary infection. In summary, our results uncover a functionally important interaction between ORF36 and ORF45 and indicate a significant role of ORF36 in the production of infectious progeny virions. IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus with a significant public health burden. KSHV ORF36 encodes a serine/threonine viral protein kinase, whose functions throughout the viral life cycle have not been elucidated. Here, we report that ORF36 interacts with another KSHV protein, ORF45. We mapped the regions of ORF36 and ORF45 involved in their association and further characterized the consequences of this interaction. We engineered ORF36 mutant viruses in order to investigate the functional roles of ORF36 in the context of KSHV lytic replication, and we confirmed that ORF36 is a component of KSHV virions. Moreover, we found that ORF36 mutants are defective in virion production and primary infection. In summary, we discovered and characterized a functionally important interaction between KSHV ORF36 and ORF45, and our results suggest a significant role of ORF36 in the production of infectious progeny virions, a process critical for KSHV pathogenesis. K aposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus and the causative agent of Kaposi's sarcoma (KS), as well as two lymphoproliferative disorders (1-3). All herpesviruses encode at least one serine/threonine protein kinase that is conserved throughout the three subfamilies (alpha-, beta-, and gammaherpesviruses), collectively referred to as conserved herpesvirus protein kinases (CHPKs) (reviewed in references 4 and 5). Orthologs of CHPKs include UL13 of herpes simp...

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Section: Discussionmentioning

confidence: 99%

Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36

Avey

Tepper

Pifer

et al. 2016

J Virol

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“…Hence, the NEC represents an attractive and promising target for antiviral drug development. It has been shown previously that inhibition of pUL97 activity, in particular pUL97-mediated core NEC phosphorylation, led to a significant reduction of viral titres, underlining the importance of proper NEC formation (Prichard et al, 1999;Wolf et al, 2001;Krosky et al, 2003;Marschall et al, 2005;Sharma et al, 2015).…”

Section: Introductionmentioning

confidence: 96%

Cytomegalovirus pUL50 is the multi-interacting determinant of the core nuclear egress complex (NEC) that recruits cellular accessory NEC components

Sonntag

Hamilton

Hanife

et al. 2016

Journal of General Virology

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Nuclear egress of herpesvirus capsids through the nuclear envelope is mediated by the multimeric nuclear egress complex (NEC). The human cytomegalovirus (HCMV) core NEC is defined by an interaction between the membrane-anchored pUL50 and its nuclear co-factor pUL53, tightly associated through heterodimeric corecruitment to the nuclear envelope. Cellular proteins, such as p32/gC1qR, emerin and protein kinase C (PKC), are recruited by direct interaction with pUL50 for the multimeric extension of the NEC. As a functionally important event, the recruitment of both viral and cellular protein kinases leads to site-specific lamin phosphorylation and nuclear lamina disassembly. In this study, interaction domains within pUL50 for its binding partners were defined by co-immunoprecipitation. The interaction domain for pUL53 is located within the pUL50 N-terminus (residues 10-169), interaction domains for p32/gC1qR (100-358) and PKC (100-280) overlap in the central part of pUL50, and the interaction domain for emerin is located in the C-terminus (265-397). Moreover, expression and formation of core NEC proteins at the nuclear rim were consistently detected in cells permissive for productive HCMV replication, including two trophoblast-cell lines. Importantly, regular nuclear-rim formation of the core NEC was blocked by inhibition of cyclin-dependent kinase (CDK) activity. In relation to the recently published crystal structure of the HCMV core NEC, our findings result in a refined view of NEC assembly. In particular, we suggest that CDKs may play an important regulatory role in NEC formation during HCMV replication.

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“…Thus, it is of no surprise that the NEC of HCMV and other herpesviruses is currently considered a promising target for the development of novel antiviral strategies. Such strategies might aim either at a direct block of protein-protein interactions via in silico-designed small molecule inhibitors or at an interference with regulatory mechanisms that control NEC assembly, such as the inhibition of NEC protein phosphorylation through kinase inhibitors (6,43). We anticipate that the structural information presented here for the pUL50-pUL53 complex will accelerate the validation of the NEC as a unique antiherpesviral target and might promote the development of a novel type of NEC-directed drugs.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of the Human Cytomegalovirus pUL50-pUL53 Core Nuclear Egress Complex Provides Insight into a Unique Assembly Scaffold for Virus-Host Protein Interactions

Walzer

Egerer-Sieber

Sticht

et al. 2015

Journal of Biological Chemistry

139

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Background:The conserved cytomegalovirus proteins pUL50 and pUL53 heterodimerize and form a core nuclear egress complex. Results: The crystal structure of pUL50-pUL53 was solved at 2.44 Å resolution, revealing an N-terminal hooklike extension of pUL53. Conclusion: Data unravel the core NEC architecture, providing a scaffold for viral-cellular NEC protein interactions. Significance: The identified NEC structure will stimulate the development of novel antiviral strategies.

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Human Cytomegalovirus UL97 Phosphorylates the Viral Nuclear Egress Complex

Cited by 57 publications

References 57 publications

Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36

Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36

Cytomegalovirus pUL50 is the multi-interacting determinant of the core nuclear egress complex (NEC) that recruits cellular accessory NEC components

Crystal Structure of the Human Cytomegalovirus pUL50-pUL53 Core Nuclear Egress Complex Provides Insight into a Unique Assembly Scaffold for Virus-Host Protein Interactions

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