A Human Herpesvirus 6A-Encoded MicroRNA: Role in Viral Lytic Replication

Abstract

Human herpesvirus 6A (HHV-6A), a member of the betaherpesvirus family, is associated with several human diseases. Like all herpesviruses, HHV-6A establishes a lifelong, latent infection in its host. Reactivation of HHV-6A is frequent within the immunosuppressed and immunocompromised populations and results in lytic viral replication within multiple organs, often leading to severe disease. MicroRNAs (miRNAs) are key regulators of multiple cellular processes that regulate the translation of specific transcripts. miRNAs carried by herpesviruses play important roles in modulating the host cell, thereby facilitating a suitable environment for productive viral infection and/or latency. Currently, there are approximately 150 known human herpesvirusencoded miRNAs, although an miRNA(s) encoded by HHV-6A has yet to be reported. We hypothesized that HHV-6A, like other members of the human herpesvirus family, encodes miRNAs, which function to promote viral infection. We utilized deep sequencing of small RNA species isolated from cells harboring HHV-6A to identify five novel small noncoding RNA species that originate from the viral genome, one of which has the characteristics of a viral miRNA. These RNAs are expressed during productive infection by either bacterial artificial chromosome (BAC)-derived virus in Jjhan cells or wild-type HHV-6A strain U1102 virus in HSB2 cells and are associated with the RNA induced silencing complex (RISC) machinery. Growth analyses of mutant viruses that lack each individual miRNA revealed that a viral miRNA candidate (miR-U86) targets the HHV-6A IE gene U86, thereby regulating lytic replication. The identification and biological characterization of this HHV-6A-specific miRNA is the first step to defining how the virus regulates its life cycle. IMPORTANCEA majority of the human population is infected with human herpesvirus 6A (HHV-6A), a betaherpesvirus family member. Infections usually occur in young children, and upon resolution, the virus remains in a latent state within the host. Importantly, during times of weakened immune responses, the virus can reactivate and is correlated with significant disease states. Viruses encode many different types of factors that both undermine the host antiviral response and regulate viral replication, including small RNA species called microRNAs (miRNAs). Here we report that HHV-6A encodes at least one miRNA, which we named miR-U86. We have characterized the requirement of this viral miRNA and its impact on the viral life cycle and found that it functions to regulate a viral protein important for efficient viral replication. Our data suggest that viral miRNAs are important for HHV-6A and that they may serve as an important therapeutic target to inhibit the virus. Human herpesvirus 6 (HHV-6), a ubiquitous pathogen in the general population, is a member of the betaherpesvirus family. HHV-6 consists of two variants: HHV-6A and HHV-6B. Even though they are nearly 90% homologous at the genomic level and they have common cell tropism (e.g., T-lymphocytes)...

Hepatitis B Virus-Encoded MicroRNA Controls Viral Replication

“…For instance, HCMV-encoded miR-UL112-1 regulates a variety of genes by binding to the 3= untranslated region (UTR) of viral transcripts and inhibiting translation (9). Human herpesvirus 6A (HHV-6A)-encoded miR-U86 targets the HHV-6A U86 immediate-early (IE) gene, thereby regulating lytic replication (35). These findings suggested that a virus-encoded miRNA modulates its own replication process and infection status.…”

“…Although previous computational analysis predicted an HBV miRNA (34), we did not get the predicted miRNA sequence. Generally, the tools for identifying novel miRNAs were Northern blotting and stem-loop RT-qPCR (7,(35)(36)(37)(38) and bioinformatics analysis for predicating the secondary structure of candidate miR precursors. Bioinformatics analysis predicted the precursor structure and found that one hairpin structure contained HBV sncRNA-3 and -5 at two stems, which were named HBV-miR-3-3p and HBV-miR-3, and they are conserved in various HBV strains.…”

The putative U94 integrase is dispensable for human herpesvirus 6 (HHV-6) chromosomal integration

“…Cell-free virus stocks were generated by concentrating the supernatant of highly infected JJHan cells and titrated by analysing the genome copies in newly infected cells by qPCR. Multi-step growth kinetics (Oyaizu et al, 2012;Nukui et al, 2015) revealed that the DU94 mutant had a significant growth defect compared to the wt and revertant virus (Fig. 1c), suggesting that the impaired HHV-6A replication is either due to the abrogation of U94 expression or effects on neighbouring genes such as U95 caused by deletion of the U94 sequences.…”

“…1b) as described previously (Kaufer et al, 2011). The recombinant viruses were subsequently reconstituted by nucleofection of JJHan cells with the HHV-6A BAC DNA as described previously (Nukui et al, 2015). Cell-free virus stocks were generated by concentrating the supernatant of highly infected JJHan cells and titrated by analysing the genome copies in newly infected cells by qPCR.…”

Small RNAs growing tall: miRNAs as drug targets in herpesvirus infections

“…Although viral miRNAs evolved independently in the three herpesvirus subfamilies, repression of viral immediate early (IE) genes is a common theme [17,36,37,[48][49][50]. Repression is best exemplified by herpes simplex virus 1 and 2 (HSV-1 and HSV-2), where viral miRNAs derived from the latency-associated-transcript (LAT) are expressed antisense to key IE genes, thereby allowing for strong suppression of ICP0, ICP4 and ICP34.5 [17,51].…”