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Varicella zoster virus ORF25 gene product: an essential hub protein linking encapsidation proteins and the nuclear egress complex.

ABSTRACT: Varicella zoster virus (VZV) ORF25 is a 156 amino acid protein belonging to the approximately 40 core proteins that are conserved throughout the Herpesviridae. By analogy to its functional orthologue UL33 in Herpes simplex virus 1 (HSV-1), ORF25 is thought to be a component of the terminase complex. To investigate how cleavage and encapsidation of viral DNA links to the nuclear egress of mature capsids in VZV, we tested 10 VZV proteins that are predicted to be involved in either of the two processes for protein interactions against each other using three independent protein-protein interaction (PPI) detection systems: the yeast-two-hybrid (Y2H) system, a luminescence based MBP pull-down interaction screening assay (LuMPIS), and a bioluminescence resonance energy transfer (BRET) assay. A set of 20 interactions was consistently detected by at least 2 methods and resulted in a dense interaction network between proteins associated in encapsidation and nuclear egress. The results indicate that the terminase complex in VZV consists of ORF25, ORF30, and ORF45/42 and support a model in which both processes are closely linked to each other. Consistent with its role as a central hub for protein interactions, ORF25 is shown to be essential for VZV replication.

SUBMITTER: Vizoso Pinto MG

PROVIDER: S-EPMC3230707 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Varicella zoster virus ORF25 gene product: an essential hub protein linking encapsidation proteins and the nuclear egress complex.

Vizoso Pinto Maria G MG Pothineni Venkata R VR Haase Rudolf R Woidy Mathias M Lotz-Havla Amelie S AS Gersting Søren W SW Muntau Ania C AC Haas Jürgen J Sommer Marvin M Arvin Ann M AM Baiker Armin A

Journal of proteome research 20111026 12

Varicella zoster virus (VZV) ORF25 is a 156 amino acid protein belonging to the approximately 40 core proteins that are conserved throughout the Herpesviridae. By analogy to its functional orthologue UL33 in Herpes simplex virus 1 (HSV-1), ORF25 is thought to be a component of the terminase complex. To investigate how cleavage and encapsidation of viral DNA links to the nuclear egress of mature capsids in VZV, we tested 10 VZV proteins that are predicted to be involved in either of the two proce ...[more]

PMID: 21988664

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Project description:The herpesviral terminase complex is part of the intricate machinery that delivers a single viral genome into empty preformed capsids (encapsidation). The varicella-zoster virus (VZV) terminase components (pORF25, pORF30, and pORF45/42) have not been studied as extensively as those of herpes simplex virus 1 and human cytomegalovirus (HCMV). In this study, VZV bacterial artificial chromosomes (BACs) were generated with small (?30S), medium (?30M), and large (?30L) ORF30 internal deletions. In addition, we isolated recombinant viruses with specific alanine substitutions in the putative zinc finger motif (30-ZF3A) or in a conserved region (region IX) with predicted structural similarity to the human topoisomerase I core subdomains I and II (30-IXAla, 30-620A, and 30-622A). Recombinant viruses replicated in an ORF30-complementing cell line (ARPE30) but failed to replicate in noncomplementing ARPE19 and MeWo cells. Transmission electron microscopy of 30-IXAla-, 30-620A-, and 30-622A-infected ARPE19 cells revealed only empty VZV capsids. Southern analysis showed that cells infected with parental VZV (VZVLUC) or a repaired virus (30R) contained DNA termini, whereas cells infected with ?30L, 30-IXAla, 30-620A, or 30-622A contained little or no processed viral DNA. These results demonstrated that pORF30, specifically amino acids 619 to 624 (region IX), was required for DNA encapsidation. A luciferase-based assay was employed to assess potential intermolecular complementation between the zinc finger domain and conserved region IX. Complementation between 30-ZF3A and 30-IXAla provided evidence that distinct pORF30 domains can function independently. The results suggest that pORF30 may exist as a multimer or participate in higher-order assemblies during viral DNA encapsidation.Antivirals with novel mechanisms of action are sought as additional therapeutic options to treat human herpesvirus infections. Proteins involved in the viral DNA encapsidation process have become promising antiviral targets. For example, letermovir is a small-molecule drug targeting HCMV terminase that is currently in phase III clinical trials. It is important to define the structural and functional characteristics of proteins that make up viral terminase complexes to identify or design additional terminase-specific compounds. The VZV ORF30 mutants described in this study represent the first VZV terminase mutants reported to date. Targeted mutations confirmed the importance of a conserved zinc finger domain found in all herpesvirus ORF30 terminase homologs but also identified a novel, highly conserved region (region IX) essential for terminase function. Homology modeling suggested that the structure of region IX is present in all human herpesviruses and thus represents a potential structurally conserved antiviral target.

Dataset Information

Varicella zoster virus ORF25 gene product: an essential hub protein linking encapsidation proteins and the nuclear egress complex.

Publications

Varicella zoster virus ORF25 gene product: an essential hub protein linking encapsidation proteins and the nuclear egress complex.

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