Non-Axial View of the Varicella-Zoster Virus Portal Protein Reveals Conserved Crown, Wing and Clip Architecture

Visalli, Robert J.; Howard, Alexander J.

doi:10.1159/000360225

Cited by 7 publications

(8 citation statements)

References 42 publications

(32 reference statements)

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“…Studies by Newcomb et al showed that pUL6 assembled into ring-like structures in vitro that form a portal of entry for viral DNA into the capsid (15). VZV pORF54 shows 44% amino acid identity with its HSV-1 pUL6 homolog (18,77), and it recently was shown to form portallike structures when expressed in insect cells (18,19). The results reported in this paper suggest that pORF54 performs a functional role similar to that of pUL6 during VZV replication.…”

Section: Discussionmentioning

confidence: 51%

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The Varicella-Zoster Virus Portal Protein Is Essential for Cleavage and Packaging of Viral DNA

Visalli

House

Selariu

et al. 2014

J Virol

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The varicella-zoster virus (VZV) open reading frame 54 (ORF54) gene encodes an 87-kDa monomer that oligomerizes to form the VZV portal protein, pORF54. pORF54 was hypothesized to perform a function similar to that of a previously described herpes simplex virus 1 (HSV-1) homolog, pUL6. pUL6 and the associated viral terminase are required for processing of concatemeric viral DNA and packaging of individual viral genomes into preformed capsids. In this report, we describe two VZV bacterial artificial chromosome (BAC) constructs with ORF54 gene deletions, ⌬54L (full ORF deletion) and ⌬54S (partial internal deletion). The full deletion of ORF54 likely disrupted essential adjacent genes (ORF53 and ORF55) and therefore could not be complemented on an ORF54-expressing cell line (ARPE54). In contrast, ⌬54S was successfully propagated in ARPE54 cells but failed to replicate in parental, noncomplementing ARPE19 cells. Transmission electron microscopy confirmed the presence of only empty VZV capsids in ⌬54S-infected ARPE19 cell nuclei. Similar to the HSV-1 genome, the VZV genome is composed of a unique long region (U L ) and a unique short region (U S ) flanked by inverted repeats. DNA from cells infected with parental VZV (VZV LUC strain) contained the predicted U L and U S termini, whereas cells infected with ⌬54S contained neither. This result demonstrates that ⌬54S is not able to process and package viral DNA, thus making pORF54 an excellent chemotherapeutic target. In addition, the utility of BAC constructs ⌬54L and ⌬54S as tools for the isolation of site-directed ORF54 mutants was demonstrated by recombineering single-nucleotide changes within ORF54 that conferred resistance to VZV-specific portal protein inhibitors. IMPORTANCE Antivirals with novel mechanisms of action would provide additional therapeutic options to treat human herpesvirus infections.Proteins involved in the herpesviral DNA encapsidation process have become promising antiviral targets. Previously, we described a series of N-␣-methylbenzyl-N=-aryl thiourea analogs that target the VZV portal protein (pORF54) and prevent viral replication in vitro. To better understand the mechanism of action of these compounds, it is important to define the structural and functional characteristics of the VZV portal protein. In contrast to HSV, no VZV mutants have been described for any of the seven essential DNA encapsidation genes. The VZV ORF54 deletion mutant described in this study represents the first VZV encapsidation mutant reported to date. We demonstrate that the deletion mutant can serve as a platform for the isolation of portal mutants via recombineering and provide a strategy for more in-depth studies of VZV portal structure and function.H erpesvirus DNA encapsidation is understood only in general terms. The mechanistic events and the function(s) of the proteins involved are still under investigation. During viral replication, progeny DNA genomes are synthesized in the nucleus as long, branched head-to-tail concatemers. The capsid proteins are synt...

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Section: Discussionmentioning

confidence: 51%

“…Previous studies provided evidence that HSV-1 pUL6 forms the portal protein through which DNA can enter the viral procapsid (8,(14)(15)(16)(17). Subsequently, we showed that ORF54 encodes the homologous portal protein for VZV, pORF54 (18,19). Small molecules that target the HSV (20) and VZV (21) portal proteins have been described.…”

mentioning

confidence: 89%

The Varicella-Zoster Virus Portal Protein Is Essential for Cleavage and Packaging of Viral DNA

Visalli

House

Selariu

et al. 2014

J Virol

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“…HSV‐2 pUL6 is encoded by the 2034‐bp UL6 gene. Although pUL6‐2 contains 2 additional aa residues compared with HSV‐1 (Table ), pUL6‐1 and ‐2 share >85% aa sequence identity (Figure ) and therefore are likely to be similar in structure and function . Thiourea compounds showed only modest activity against HSV‐2 strains (Table ) .…”

Section: Herpes Simplex Virus Typementioning

confidence: 99%

“…pORF54 was shown to be the structural and functional homolog of HSV‐1 pUL6. pORF54 expressed in a recombinant baculovirus system formed oligomeric VZV portals containing the typical crown‐wing‐stem‐clip portal architecture observed in phage portals (Figure A). VZV ORF54 mutants created via recombineering were only viable in a complementing pORF54‐expressing cell line .…”

Section: Varicella‐zoster Virusmentioning

confidence: 99%

“…Genes encoding confirmed (HSV‐1, VZV, and HCMV) and putative (HSV‐2, Epstein‐Barr virus [EBV], and HHVs 6, 7, and 8) portal proteins in human HHVs have been identified (Table ). Human herpesvirus portal protein monomers range from Mr 68,000 to 86,800, while the predicted functional dodecamers range from Mr 800,000 to >1,000,000 (Table ) . Despite poorly conserved primary aa sequences, electron microscopy (EM) studies of oligomeric herpesvirus portal proteins show typical architectural features observed in phage portals (Figure ).…”

Section: Introductionmentioning

confidence: 99%

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Human herpesvirus portal proteins: Structure, function, and antiviral prospects

Kornfeind

Visalli

2018

Reviews in Medical Virology

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Herpesviruses (Herpesvirales) and tailed bacteriophages (Caudovirales) package their dsDNA genomes through an evolutionarily conserved mechanism. Much is known about the biochemistry and structural biology of phage portal proteins and the DNA encapsidation (viral genome cleavage and packaging) process. Although not at the same level of detail, studies on HSV-1, CMV, VZV, and HHV-8 have revealed important information on the function and structure of herpesvirus portal proteins. During dsDNA phage and herpesviral genome replication, concatamers of viral dsDNA are cleaved into single length units by a virus-encoded terminase and packaged into preformed procapsids through a channel located at a single capsid vertex (portal). Oligomeric portals are formed by the interaction of identical portal protein monomers. Comparing portal protein primary aa sequences between phage and herpesviruses reveals little to no sequence similarity. In contrast, the secondary and tertiary structures of known portals are remarkable. In all cases, function is highly conserved in that portals are essential for DNA packaging and also play a role in releasing viral genomic DNA during infection. Preclinical studies have described small molecules that target the HSV-1 and VZV portals and prevent viral replication by inhibiting encapsidation. This review summarizes what is known concerning the structure and function of herpesvirus portal proteins primarily based on their conserved bacteriophage counterparts and the potential to develop novel portal-specific DNA encapsidation inhibitors.

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