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.

Fig. 1

Characterization of HHV-6 reactivation. a Trichostatin A (TSA) induced histone acetylation in U2OS cells. TSA (80 ng/ml) was added to U2OS cell culture media for 24 h to induce histone acetylation. Total protein was extracted and used for immunoblotting. Histone H4 pan acetylation (H4ac), Histone 3 K27 acetylation (H3K27ac), Histone 3 K27 methylation (H3K27me) and Histone 3 K4 methylation (H3K4me) were studied using specific antibodies. Actin was used as loading control. U2OS cells without HHV-6 (-HHV-6A) were used as control. b TSA treatment induced expression of RFP in latent HHV-6A carrying U2OS cells. Microscopic evaluation was carried out for RFP expression in U2OS cells carrying latent HHV-6A. Cells were treated with DMSO in parallel as a solvent control. c TSA-induced HHV-6A reactivation was quantified by qRT-PCR analysis of two early viral transcripts (p41 and U86). U2OS cells carrying latent HHV-6A were treated with 80 ng/ml of TSA for three different time intervals. Total RNA was extracted and were used for cDNA synthesis and subsequent RT-PCR. Data represent the mean ± SEM of three independent experiments. dpi, days post infection. d Immunoprecipitation (IP) of HHV-6 IE2 protein was carried out to test immediate early protein synthesis. U2OS cells carrying latent HHV-6A were treated with DMSO or TSA for 2 days. Total cell lysates were extracted and used for immunopreciptation. A smaller ~ 55 kDa fraction of IE2 was detected in IP. e Viral DNA replication was studied by qPCR. U2OS cells carrying latent HHV-6A were treated with 80 ng/ml of TSA or DMSO for two different time intervals. Total genomic DNA was extracted and were used for qPCR analysis. Data represent the mean ± SEM of three independent experiments. dpi, days post infection. f Detection of several different HHV-6A encoded small non-coding RNAs by Northern hybridization. U2OS cells carrying latent HHV-6A were treated with 80 ng/ml of TSA (T) or DMSO (D) for 48 h. 10 μg of total RNA were separated on a denaturing Urea gel for Northern hybridization. Decade marker (DM) was used to verify sizes of identified RNA. Transcription of previously described small non-coding RNAs (labeled as sR) was tested using specific DNA probes. Human U6 RNA was used as loading control. g Detection of newly identified HHV-6A encoded sncRNA-U73 by Northern hybridization as described in figure f. h Sequence details of newly identified sncRNA-U73. Genomic location of the sncRNA-U73 is indicated as mapped to HHV-6A U1102 genome (Genebank X83413.2). All blots or gels (a, d, f and g) derive from the same experiment and they were processed in parallel

Fig. 2

HHV-6 alters host miRNA expression and mitochondrial dynamics. a Transcription dynamics of several human miRNAs were studied by Northern hybridization. U2OS cells having latent HHV-6A were used as described in figure 1f. Decade marker (M) was used to verify sizes of identified RNA. Both matured miRNA and their precursor (pre-miRNA) are indicated. Human U6 RNA was used as loading control. All gels derive from the same experiment and they were processed in parallel. b HHV-6A transactivation induces mitochondrial fragmentation. U2OS cells having soluble GFP (mito GFP) within mitochondria and carrying mCherry encoding latent HHV-6A was reactivated with TSA. DMSO treated cells served as solvent control. 2 days after treatment, cells were fixed and processed for confocal microscopy. The scale bars represent 10 μm. c Mitochondrial size and numbers were quantified using ImageJ. Data represent the mean ± SEM of three independent experiments. SC solvent control, TSA trichostain A, D1 Day1, D2 Day 2

Fig. 3

HHV-6 as a potential cause of DRESS-mediated death. a Brief summary of all the clinical conditions and different clinical analysis carried out in the DRESS patient is presented in the form of a schematic diagram. Blood and tissue biopsies analyzed for HHV-6 at various stages of the treatment are indicated. HHV-6 positive analyses are indicated with red color filled circles. HHV-6 negative analyses are indicated with white color filled circles. b Various types of pre-mortem and post-mortem FFPE tissue biopsies from the DRESS patient were analyzed for sncRNA-U14 by FISH analysis. Blown up images of the positive stained cells are shown within white boxes wherever necessary. Imaging was done on a SP5 confocal microscope. The scale bars represent 200 μm