Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Filters applied. Clear all
. 2018 Sep 5;3:25.
doi: 10.1038/s41525-018-0064-5. eCollection 2018.

HHV-6 Encoded Small Non-Coding RNAs Define an Intermediate and Early Stage in Viral Reactivation

Affiliations
Free PMC article

HHV-6 Encoded Small Non-Coding RNAs Define an Intermediate and Early Stage in Viral Reactivation

Bhupesh K Prusty et al. NPJ Genom Med. .
Free PMC article

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.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
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
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
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

Similar articles

See all similar articles

Cited by 8 articles

See all "Cited by" articles

References

    1. Kaufer BB, Flamand L. Chromosomally integrated HHV-6: impact on virus, cell and organismal biology. Curr. Opin. Virol. 2014;9C:111–118. doi: 10.1016/j.coviro.2014.09.010. - DOI - PubMed
    1. Prusty BK, et al. Possible chromosomal and germline integration of human herpesvirus 7 (HHV-7) J. General. Virol. 2016 - PubMed
    1. Kaufer BB, Jarosinski KW, Osterrieder N. Herpesvirus telomeric repeats facilitate genomic integration into host telomeres and mobilization of viral DNA during reactivation. J. Exp. Med. 2011;208:605–615. doi: 10.1084/jem.20101402. - DOI - PMC - PubMed
    1. Wallaschek N, et al. The Telomeric repeats of human herpesvirus 6A (HHV-6A) are required for efficient virus integration. PLoS Pathog. 2016;12:e1005666. doi: 10.1371/journal.ppat.1005666. - DOI - PMC - PubMed
    1. Rotola A, et al. U94 of human herpesvirus 6 is expressed in latently infected peripheral blood mononuclear cells and blocks viral gene expression in transformed lymphocytes in culture. Proc. Natl. Acad. Sci. USA. 1998;95:13911. doi: 10.1073/pnas.95.23.13911. - DOI - PMC - PubMed

LinkOut - more resources

Feedback