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...

The ability of soluble C. albicans 20A (serotype A) mannoprotein (CMP) to serve as a ligand for toll-like receptor 4 (TLR4) and its co-receptors was examined using commercially available and stably-transfected HEK293 cells that express human TLR4, MD2 and CD14, but not MR. These TLR4 reporter cells also express an NF-κB-dependent, secreted embryonic alkaline phosphatase (SEAP) reporter gene. TLR4-reporter cells exhibited a dose-dependent SEAP response to both LPS and CMP, wherein peak activation was achieved after stimulation with 40–50 μg/mL of CMP. Incubation on polymyxin B resin had no effect on CMP’s ligand activity, but neutralized LPS-spiked controls. HEK293 Null cells lacking TLR4 and possessing the same SEAP reporter failed to respond to LPS or CMP, but produced SEAP when activated with TNFα. Reporter cell NF-κB responses were accompanied by transcription of IL-8, TNFα, and COX-2 genes. Celecoxib inhibited LPS-, CMP-, and TNFα-dependent NF-κB responses; whereas, indomethacin had limited effect on LPS and CMP responses. SEAP production in response to C. albicans A9 mnn4Δ mutant CMP, lacking phosphomannosylations on N-linked glycans, was significantly greater (p ≤ 0.005) than SEAP responses to CMP derived from parental A9 (both serotype B). These data confirm that engineered human cells expressing TLR4, MD2 and CD14 can respond to CMP with NF-κB activation and the response can be influenced by variations in CMP-mannosylation. Future characterizations of CMPs from other sources and their application in this model may provide further insight into variations observed with TLR4 dependent innate immune responses targeting different C. albicans strains.

Hemoglobin (Hb) released during red blood cell lysis can initiate TLR4-dependent signaling and trigger NF-κB activation in surrounding cells. Observations of chronic bleeding in various cancers leads us to hypothesize that Hb and Hb degradation products released from lysed RBC near cancer nests might modulate local TLR4-positive cells. We addressed the hypothesis in vitro by measuring Hb- and biliverdin (Bv)-induced NF-κB signaling in an engineered human TLR4 reporter cell model (HEK-BlueTM hTLR4). Therein, TLR4 stimulation was assessed by measuring NF-κB-dependent secreted alkaline phosphatase (SEAP). hTLR4 reporter cells incubated with 8 ηM lipopolysaccharide (LPS) or 20-40 μM fungal mannoprotein (FM) produced significant amounts of SEAP. hTLR4 reporter cells also produced SEAP in response to human, but not porcine or bovine, Hb. HEK-Blue Null2TM reporter cells lacking TLR4 did not respond to LPS, FM, or Hb. Bv was non-stimulatory in reporter cells. When Bv was added to Hb-stimulated reporter cells, SEAP production was reduced by 95%, but when Bv was applied during LPS and FM stimulation, SEAP production was reduced by 33% and 27%, respectively. In conclusion, Hb initiated NF-κB signaling that was dependent upon TLR4 expression and that Bv can act as a TLR4 antagonist. Moreover, this study suggests that hemorrhage and extravascular hemolysis could provide competitive Hb and Bv signaling to nearby cells expressing TLR4, and that this process could modulate NF-κB signaling in TLR4-positive cancer cells and cancer-infiltrating leukocytes.

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 ( During herpesvirus replication, large head-to-tail doublestranded DNA (dsDNA) concatemers accumulate in the infected-cell nucleus and are subsequently packaged into preformed viral capsids. The packaging process is complex and involves a group of proteins that recognize specific viral DNA sequences and simultaneously process and translocate unit-length genomes through the portal vertex of the procapsid (1). The protein complex that docks or interacts with the capsid portal protein consists of the heterotrimeric viral terminase. Terminases of DNA bacteriophages have been well described (2, 3), and those of several members of the Herpesviridae, including herpes simplex virus 1 (HSV-1) (4-14), human cytomegalovirus (HCMV) (15-21), Epstein-Barr virus (EBV) (22), and varicella-zoster virus (VZV) (23)(24)(25), are under investigation.Members of the Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae share a core set of orthologous proteins required for DNA encapsidation. A heterotrimeric viral terminase complex consisting of three of these conserved core proteins was recently isolated from HSV-1-infected cells (4). The HSV-1 terminase consisted of a 1:1:1 equimolar complex of two different terminase protein subunits, pUL15 and pUL28, and a protein encoded by the UL33 gene family. The pUL33 component may act as an essential accessory protein involved in the formation and/or stability of the terminase complex (7). Homologous terminase components have also been identified for HCMV (pUL89, pUL56, and pUL51) (16)(17)(18)(19)(20)(21)(26)(27)(28) and VZV (pORF45/42, pORF30, and