Confocal microscopic analysis was performed as described64

Confocal microscopic analysis was performed as described64. Plasmid co-transfection, RT-qPCR and immunoblotting Plasmid co-transfection was performed using PEImax (molecular weight 40,000) (Polysciences, Inc.). suppresses ORF61 gene expression in co-transfected cells, Cucurbitacin IIb predominates in latently VZV-infected human TG. The discovery of VLT links VZV with the other better characterized human and animal neurotropic alphaherpesviruses and provides insights into VZV latency. Introduction During primary contamination, neurotropic alphaherpesviruses (HVs) gain access to neurons in sensory, cranial and autonomic ganglia to establish a lifelong latent contamination from which they can reactivate to cause debilitating disease1. For the best-studied HVs, including herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), pseudorabies computer virus and bovine herpesvirus 1 (BHV-1), viral latency has been shown to be accompanied by the expression of a Cucurbitacin IIb single or restricted set of latency-associated transcripts (LATs)2C6. These transcripts map antisense to the gene encoding the conserved multifunctional HSV infected cell protein 0 (ICP0), to which varicella-zoster computer virus (VZV) open reading frame 61 (ORF61) is usually homologous, which besides inhibiting intrinsic cellular antiviral responses is the major transcriptional transactivator of lytic viral genes required for reactivation of latent HSV2C10. While the function of the LATs remains uncertain (examined in ref. 11), there is mounting evidence from work on HSV-1, HSV-2 and BHV-1 that LATs induce generalized transcriptional and/or translational repression12, and several studies have shown that LAT-encoded miRNAs (e.g., miR-H2, in HSV-1) or proteins (e.g., BHV-1 latency-related protein) target viral mRNAs including ICP013C15. The exception has been VZV, a human-restricted HV and causative agent of varicella and herpes zoster, for which no latency transcript mapping antisense to its ICP0 homologue ORF61 has been explained1,16C18. While the absence of a canonical latency transcript in VZV may represent a fundamental difference in the development and biology of this virus, it is notable that a putative LAT, antisense to ORF61, has also been explained for simian varicella computer virus (SVV), the varicellovirus most closely related to VZV. However, neither the transcript nor its function in SVV contamination have been analyzed in detail7,8. Like other herpesviruses, lytic VZV contamination is characterized by full viral gene expression occurring with temporally linked immediate-early (IE), early (E) and late (L) kinetics to generate infectious computer virus progeny19,20. By contrast, VZV gene expression during latency remains poorly defined16,18,21C23. This is largely due to the lack of appropriate animal24 and, until recently, in vitro25,26 models, which accurately mimic VZV pathogenesis24C26. VZV latency has been extensively analyzed in cadaveric human trigeminal ganglia (TG), a prominent anatomic site of both HSV-1 and VZV latency, yielding conflicting results as to which VZV transcripts and proteins are expressed27. Whereas viral protein detection by immunohistochemistry (IHC) can largely be attributed to non-specific binding of anti-VZV antibody preparations23,28, the time interval between death and TG specimen processing (post-mortem interval, PMI) determines the number and quantity of VZV transcripts detected16,18,22. Using PCR primers targeting all canonical VZV genes, only the lytic gene ORF63 is usually occasionally detected in TGs with PMI? ?9?h18, whereas multiple viral transcripts of different kinetic classes are detected in human TGs with PMI? ?9?h16,18,22 and also frequently in animal models29,30. Here we describe the identification of a VZV latency-associated transcript (VLT), consistently detected in VZV and HSV-1 co-infected human TG that lies antisense to ORF61. Although multiple alternatively spliced transcripts are present during productive contamination, the unique VLT isoform that supresses ORF61 gene transcription in co-transfected cells predominates during latency. The discovery of VLT unifies the VZV latent viral transcription programme with that of other better-characterized human and animal neurotropic HVs while removing longstanding barriers to understanding VZV latency. Results Exclusive Cucurbitacin IIb detection of HSV-1 LAT and miRNAs in human TG We first validated our experimental approach by sequencing enriched and unenriched viral transcripts from lytically VZV- or HSV-1-infected human retinal pigmented epithelial (ARPE-19) cells to demonstrate detection of all currently annotated VZV Rabbit Polyclonal to ASC and HSV-1 genes at high specificity and sensitivity (Figs.?1 and?2 and Supplementary Fig.?1). RNA-Seq.