Delayed Biosynthesis of Varicella-Zoster Virus Glycoprotein C: Upregulation by Hexamethylene Bisacetamide and Retinoic Acid Treatment of Infected Cells

Abstract

In the course of examining the trafficking pathways of varicella-zoster virus (VZV) glycoproteins gE, gI, gH, and gB, we discovered that all four are synthesized within 4 to 6 h postinfection (hpi) in cultured cells. Thereafter, they travel via the trans-Golgi network to the outer cell membrane. When we carried out a similar analysis on VZV gC, we observed little gC biosynthesis in the first 72 hpi. Further examination disclosed that gC was present in the inocula of infected cells, but no new gC biosynthesis occurred during the first 24 to 48 h thereafter, during which time new synthesis of gE, gH, and major capsid protein was easily detectable. Similarly, delayed gC biosynthesis was confirmed with three different VZV strains and two different cell lines. Bioinformatics analyses disclosed the presence of PBX/HOX consensus binding domains in the promoter/ enhancer regions of the genes for VZV gC and ORF4 protein (whose orthologs transactivate gC in other herpesviruses). Bioinformatics analysis also identified two HOXA9 activation regions on ORF4 protein. Treatment of infected cultures with chemicals known to induce the production of PBX/HOX transcription proteins, namely, hexamethylene bisacetamide (HMBA) and retinoic acid, led to more rapid gC biosynthesis. Immunoblotting demonstrated a fivefold increase in the HOXA9 protein after HMBA treatment. In summary, these results documented that gC was not produced during early VZV replication cycles, presumably related to a deficiency in the PBX/HOX transcription factors. Furthermore, these results explain the apparent spontaneous loss of VZV gC in some passaged viruses, as well as other anomalous gC results.Varicella-zoster virus (VZV) is a very cell-associated virus in cell culture. In addition, the infectivity titers are extremely low, usually less than 1,000,000 U per 25-cm 2 monolayer. Further, the virions produced in cell culture have an aberrant appearance. Explanations for these observations are a subject of continuing research. To this end, we have been investigating the biosynthesis and maturation of several VZV structural glycoproteins found in the envelope of the virion, especially the predominant gE/gI complex. These two glycoproteins are synthesized in the Golgi and then traffic through the trans-Golgi network en route to the outer plasma membrane within the expected 12-h replication cycle of an alphaherpesvirus. After endocytosis via their tyrosine-and dileucine-based motifs, they travel back to the trans-Golgi network (12). At this location, the glycoproteins appear to be incorporated into the virion assembly vacuoles. Two other major VZV glycoproteins, gH and gB, have similar endocytosis motifs and similar trafficking profiles.During the glycoprotein trafficking studies, VZV gC was used as a control because there are no obvious trafficking motifs in its short cytoplasmic tail (20). There have been clues that gC is important for virion production. The severe combined immunodeficient (SCID) mouse has become an important animal model for the investigation...

Aberrant Virion Assembly and Limited Glycoprotein C Production in Varicella-Zoster Virus-Infected Neurons

“…The low abundance of VZV gC protein in infected neurons was paralleled by low levels of true late VZV gC transcripts compared to transcription of the VZV IE62 gene and the early-late VZV gE gene in infected neurons, suggesting that low VZV gC abundance in infected neurons is due to a block in virus transcription rather than translation. Reduced transcription of VZV gC compared to that of VZV IE62 and VZV gE in neurons and fibroblasts is consistent with previous observations of reduced VZV gC transcription in human melanoma cells (8,19). While VZV gC production is significantly reduced in both virus-infected neurons and nonneuronal cells in tissue culture, VZV gC is nevertheless abundant in vesicles (20).…”

“…Cells were blocked with 5% nonfat dry milk in PBS, incubated with primary antibodies (mouse anti-VZV gC or anti-VZV gE) for 2 h at room temperature, washed three times with PBS, and further incubated with Alexa Fluor GAM 488-conjugated anti-mouse antibody for 1 h. Hoechst H33342 was used to stain cell DNA. Images of the labeled infected cells were collected on a laser-scanning confocal microscope as described previously (8). A three-dimensional (3D) image of a single infected cell was generated from a z-stack using Imaris software as described previously (9).…”

Role of the IE62 Consensus Binding Site in Transactivation by the Varicella-Zoster Virus IE62 Protein

“…Even late in infection, few cells express gC, and this expression occurs at various levels. The VZV ORF14 promoter, which drives the expression of VZV glycoprotein C, is responsive to IE62-mediated activation and has previously been reported to contain an atypical TATA box near the transcriptional start site and sites for inducible cellular Hox family factors far upstream (47). Our computer analysis identified an IE62 consensus site 76 bp upstream from the putative TATA box within the gC promoter (Fig.…”

“…The IE62 protein is the major viral transactivator encoded by VZV (18,39,47,54). Despite the importance of IE62 in VZV replication and the intense and extensive study of which it has been the subject, the fundamental question of the function of IE62 DNA binding and role played by the IE62 consensus binding site (5Ј-ATCGT-3Ј) has remained largely unexplored.…”

The Attenuated Genotype of Varicella-Zoster Virus Includes an ORF0 Transitional Stop Codon Mutation

“…When we compared all our data about ORF0 with three previously examined VZV proteins-IE62, gE, and gC-the ORF0 results most closely resembled those of VZV gC (31,32). In the VZV system, the gC product is expressed late in the infectious cycle in cultured cells, at least 24 h after abundant IE62 and gE expression.…”