Targeted Genome Sequencing Reveals Varicella-Zoster Virus Open Reading Frame 12 Deletion.

The neurotropic herpesvirus varicella-zoster virus (VZV) establishes a lifelong latent infection in humans following primary infection. The low abundance of VZV nucleic acids in human neurons has hindered an understanding of the mechanisms that regulate viral gene transcription during latency. To overcome this critical barrier, we optimized a targeted capture protocol to enrich VZV DNA and cDNA prior to whole-genome/transcriptome sequence analysis. Since the VZV genome is remarkably stable, it was surprising to detect that VZV32, a VZV laboratory strain with no discernible growth defect in tissue culture, contained a 2,158-bp deletion in open reading frame (ORF) 12. Consequently, ORF 12 and 13 protein expression was abolished and Akt phosphorylation was inhibited. The discovery of the ORF 12 deletion, revealed through targeted genome sequencing analysis, points to the need to authenticate the VZV genome when the virus is propagated in tissue culture.IMPORTANCE Viruses isolated from clinical samples often undergo genetic modifications when cultured in the laboratory. Historically, VZV is among the most genetically stable herpesviruses, a notion supported by more than 60 complete genome sequences from multiple isolates and following multiple in vitro passages. However, application of enrichment protocols to targeted genome sequencing revealed the unexpected deletion of a significant portion of VZV ORF 12 following propagation in cultured human fibroblast cells. While the enrichment protocol did not introduce bias in either the virus genome or transcriptome, the findings indicate the need for authentication of VZV by sequencing when the virus is propagated in tissue culture.

Cytokines Produced in Response to Varicella-Zoster Virus Infection of ARPE-19 Cells Stimulate Lymphocyte Chemotaxis.

Posterior uveitis is an ocular complication that can occur with reactivation of varicella-zoster virus (VZV). It may lead to loss of vision due to retinal detachment and chronic inflammation, which often causes more severe disease than the virus infection itself. To increase our understanding of the immune response, we infected the retinal pigment epithelial (RPE) cell line, ARPE-19, with cell-associated VZV and compared its response to that of the MeWo cell line using multiplex assays. We observed (1) a difference in the magnitude and kinetics of cytokine responses between the 2 cell types and (2) differential migration of CD4+ and CD8+ T cells towards these cytokines. Thus, our data provide information about the cytokine and lymphocytic responses to VZV infection of RPE cells, thereby providing a useful platform for future studies to address mechanisms underlying the immunopathology of VZV-associated posterior uveitis.

Ordered multisite phosphorylation of lethal giant larvae by atypical protein kinase C.

In Par complex-mediated cell polarity, phosphorylation by atypical protein kinase C (aPKC) is coupled to substrate cortical displacement. Polarized substrates often contain multiple phosphorylation sites, but the role of multisite phosphorylation in Par-mediated polarity remains unclear. Here, we have dissected the role of the three aPKC phosphorylation sites within the tumor suppressor Lethal giant larvae. Using a cultured Drosophila S2 cell cortical displacement assay, we observed that phosphorylation at any one site causes only partial displacement. Complete displacement requires that all three sites be modified. We undertook a kinetic analysis to determine if aPKC phosphorylates each site equivalently. As the sites are closely spaced, we observed not only differences in the rate of phosphorylation but also interaction between the sites. A complete description of the rates reveals a preferential order of phosphorylation. Our results provide new insights into how multiple phosphorylations and phosphorylation rates could regulate localization behaviors of fate determinants at the cortex.

Partitioning-defective protein 6 (Par-6) activates atypical protein kinase C (aPKC) by pseudosubstrate displacement.

Atypical protein kinase C (aPKC) controls cell polarity by modulating substrate cortical localization. Aberrant aPKC activity disrupts polarity, yet the mechanisms that control aPKC remain poorly understood. We used a reconstituted system with purified components and a cultured cell cortical displacement assay to investigate aPKC regulation. We find that aPKC is autoinhibited by two domains within its NH(2)-terminal regulatory half, a pseudosubstrate motif that occupies the kinase active site, and a C1 domain that assists in this process. The Par complex member Par-6, previously thought to inhibit aPKC, is a potent activator of aPKC in our assays. Par-6 and aPKC interact via PB1 domain heterodimerization, and this interaction activates aPKC by displacing the pseudosubstrate, although full activity requires the Par-6 CRIB-PDZ domains. We propose that, along with its previously described roles in controlling aPKC localization, Par-6 allosterically activates aPKC to allow for high spatial and temporal control of substrate phosphorylation and polarization.

Varicella-zoster virus inhibits autophagosome-lysosome fusion and the degradation stage of mTOR-mediated autophagic flux.

Macroautophagy (herein referred to as autophagy) is a lysosomal degradation mechanism that is important for maintaining homeostasis and for coping with cellular stress such as nutrient deprivation. Previously, varicella-zoster virus (VZV) was reported to modulate the autophagy pathway in the host. However, how VZV affects the autophagy pathway is still unclear. In this study, we examined how wild-type rOka and attenuated vOka strains of cell-associated VZV affect autophagy in MRC-5 fibroblasts by using ratiometric flow cytometry and immunoblotting methods. While VZV does not prevent autophagosome formation, we demonstrate that, particularly when autophagy is upregulated, VZV inhibits late-stage autophagic flux, likely at the point where autophagosomes and lysosomes fuse or where vesicle contents are degraded. Importantly, inhibition of autophagy yields higher VZV titers. These results substantially contribute to the current view of the interaction between VZV and autophagy, and to a better understanding of VZV pathogenesis.