Discordant varicella-zoster virus glycoprotein C expression and localization between cultured cells and human skin vesicles.

Because of its very low titer, varicella-zoster virus (VZV) infectivity is usually transferred by passage of trypsin dispersed infected cells. Previously, we observed that gC biosynthesis was markedly delayed in monolayers inoculated with cell free virus. In this report, we investigated the kinetics of gC expression in more detail and included studies of monolayers inoculated with trypsin dispersed infected cells, the more traditional method of VZV infection. Extensive imaging analyses disclosed that gC was detectable in some inoculum cells, but little gC biosynthesis occurred during the first 48 hpi in the newly infected underlying monolayer. In contrast, during the first 24-48 hpi, expression of VZV gE and gB was easily detectable. Using real-time RT-PCR, we found a delay in accumulation of VZV gC transcripts that paralleled the delay in expression of VZV gC protein. Treatment with hexamethylene bisacetamide (HMBA) increased expression of both gC protein and gC mRNA. HMBA treatment also increased virus titer by 4-fold, but paradoxically reduced plaque size in the titration assay. Finally, we examined skin vesicles from cases of chickenpox and zoster in humans and observed abundant amounts of gC expression. In short, this report documents an unexpected delay in both gC mRNA and protein production under all conditions of VZV infection of cultured cells.

Comparative analyses of the 9 glycoprotein genes found in wild-type and vaccine strains of varicella-zoster virus.

The complete DNA sequences of wild-type and vaccine strains of varicella-zoster virus have been published and listed in GenBank. In this comparative genomic analysis, the sequences of the 9 glycoprotein open reading frames (ORFs) were compared. They included gE (ORF68), gI (ORF 67), gC (ORF14), gH (ORF37), gL (ORF60), gB (ORF31), gK (ORF5), gM (ORF50), and gN (ORF8 or ORF9A). After realignment on the basis of newer data, the corrected gB sequence was lengthened to include 931 residues. The data showed that there were glycoprotein polymorphisms that differentiated North American/European strains from Japanese strains-for example, an additional ATG codon in the gL of all Oka strains. Also, there were a small number of coding single-nucleotide polymorphisms present only in glycoproteins of vaccine strains. Because these changes were highly conserved, the structure of the glycoprotein was unlikely to be altered.

Delayed biosynthesis of varicella-zoster virus glycoprotein C: upregulation by hexamethylene bisacetamide and retinoic acid treatment of infected cells.

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.

Complete DNA sequence analyses of the first two varicella-zoster virus glycoprotein E (D150N) mutant viruses found in North America: evolution of genotypes with an accelerated cell spread phenotype.

Varicella-zoster virus (VZV) is considered to be one of the most genetically stable of all the herpesviruses. Yet two VZV strains with a D150N missense mutation within the gE glycoprotein were isolated in North America in 1998 and 2002. The mutant strains have an accelerated cell spread phenotype, which distinguishes them from all wild-type and laboratory viruses. Since the VZV genome contains 70 additional open reading frames (ORFs), the possibility existed that the phenotypic change was actually due to an as-yet-undiscovered mutation or deletion elsewhere in the genome. To exclude this hypothesis, the entire genomes of the two mutant viruses were sequenced and found to contain 124,883 (VZV-MSP) and 125,459 (VZV-BC) nucleotides. Coding single-nucleotide polymorphisms (SNPs) were identified in 14 ORFs. One missense mutation was discovered in gH, but none was found in gB, gI, gL, or gK. There were no coding SNPs in the major regulatory protein ORF 62. One polymorphism was discovered which could never have been anticipated based on current knowledge of herpesvirus genomics, namely, the origins of replication differed from those in the prototype strain but not in a manner expected to affect cell spread. When the two complete mutant VZV sequences were surveyed in their entirety, the most reasonable conclusion was that the increased cell spread phenotype was dependent substantially or solely on the single D150N polymorphism in glycoprotein gE. The genomic results also expanded the evolutionary database by identifying which VZV ORFs were more likely to mutate over time.