HHV-6 encoded small non-coding RNAs define an intermediate and early stage in viral reactivation

Abstract

Human herpesvirus 6A and 6B frequently acquires latency. HHV-6 activation has been associated with various human diseases. Germ line inheritance of chromosomally integrated HHV-6 makes viral DNA-based analysis difficult for determination of early stages of viral activation. We characterized early stages of HHV-6 activation using high throughput transcriptomics studies and applied the results to understand virus activation under clinical conditions. Using a latent HHV-6A cell culture model in U2OS cells, we identified an early stage of viral reactivation, which we define as transactivation that is marked by transcription of several viral small non-coding RNAs (sncRNAs) in the absence of detectable increase in viral replication and proteome. Using deep sequencing approaches, we detected previously known as well as a new viral sncRNAs that characterized viral transactivation and differentiated it from latency. Here we show changes in human transcriptome upon viral transactivation that reflect multiple alterations in mitochondria-associated pathways, which was supported by observation of increased mitochondrial fragmentation in virus reactivated cells. Furthermore, we present here a unique clinical case of DIHS/DRESS associated death where HHV-6 sncRNA-U14 was abundantly detected throughout the body of the patient in the presence of low viral DNA. In this study, we have identified a unique and early stage of viral activation that is characterized by abundant transcription of viral sncRNAs, which can serve as an ideal biomarker under clinical conditions.

Human Herpesvirus-6 Reactivation, Mitochondrial Fragmentation, and the Coordination of Antiviral and Metabolic Phenotypes in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

“…U2-OS cells carrying stable GFP expression within mitochondria were developed as mentioned before (16). U2-OS cells carrying latent HHV-6A is previously described (15).…”

“…However, the exact antecedent to mitochondrial modulation in ME/CFS is largely unknown. We have recently shown that HHV-6A reactivation induces mitochondrial fragmentation (15). In this study, we investigated potential infectious causes and molecular mechanism(s) behind mitochondrial dysfunction, likely resulting in the development and/or progression of ME/CFS, using HHV-6A reactivation as a model.…”

Guadecitabine Plus Ipilimumab in Unresectable Melanoma: The NIBIT-M4 Clinical Trial

“…Library concentrations were quantified with the Qubit Fluorometric Quantitation system (Life Technologies), and the size distribution was assessed using the Experion Automated Electrophoresis System (Bio-Rad). For sequencing, samples were diluted and pooled into NGS libraries in equimolar amounts, as previously described (29).…”

“…Expression profiling libraries were sequenced on Illumina HiSeq 3000/4000 instruments in 75-base-pair-paired-end mode, and base calls provided by the Illumina Real-Time Analysis software were subsequently converted into BAM format (Illumina2bam) before demultiplexing (BamIndexDecoder) into individual, sample-specific BAM files via Illumina2bam tools (1.17.3 https://github.com/wtsi-npg/illumina2bam), as previously described (29).…”

Comparative Analysis of Roseoloviruses in Humans, Pigs, Mice, and Other Species

“…Although previous studies have observed HHV-6A/B reactivation inducing diseases in patients [67][68][69][70], reactivation of the virus genome in vitro is challenging. In most cell lines, induction of reactivation using various reagents only results in the expression of some viral genes without the production of viral particles [55,71,72]. Analysis of the chromatin profile of the integrated HHV-6A genome revealed that the viral DNA was transcriptionally silent, highly condensed, and associated with repressive histone modifications (H3K9me3 and H3K27me3) [73], suggesting that epigenetic factors are involved in the establishment and maintenance of latency.…”