All HHV-6B miRNAs are conserved within HHV-6A at both the structural and sequence levels, although they display minor variations in their sequences (see Fig. In contrast to the case for other betaherpesviruses, HHV-6B miRNAs are expressed from direct repeat regions (DRLand DRR) located at either side of the genome. All miRNAs are conserved in the closely related HHV-6A variant, and one of them is a seed Anlotinib ortholog of the human miRNA miR-582-5p. Similar to alphaherpesvirus Anlotinib miRNAs, they are expressed RAC3 in antisense orientation relative to immediate-early open reading frames (ORFs) and thus have the potential to regulate key viral genes. == INTRODUCTION == Human herpesvirus 6 (HHV-6) is one of eight herpesviruses known to infect humans and was first isolated in 1986 from immunocompromised patients suffering from lymphoproliferative disorders (43). HHV-6 is a ubiquitous human pathogen with seroprevalence rates exceeding 95% in industrialized nations. Together with HHV-7, it belongs to theRoseolovirusgenus within the betaherpesvirus subfamily. It exists as two variants, HHV-6A and HHV-6B, which differ with respect to their biological properties, tropisms, and clinical manifestations (14). In 1988, HHV-6B was identified as the causative agent of the childhood disease exanthem subitum Anlotinib (roseola or 3-day fever) (57). Primary infection typically presents as an acute febrile illness, sometimes followed by a red rash. Additional symptoms may include otitis, meningitis, and seizures (42). While severe complications resulting from primary infection, such as encephalitis and encephalopathy, are rare, reactivation of HHV-6 in transplant patients, particularly those receiving hematopoietic stem cells, can present with life-threatening complications, most notably due to encephalitis (31,58). In addition, HHV-6 may facilitate the progression of other viral infections,trans-activating HHV-7, human cytomegalovirus (HCMV), and HIV-1, which generally results in a poorer prognosis (reviewed in reference14). Finally, HHV-6 has been implicated in a range of neurological diseases, most prominently multiple sclerosis (MS), although a causative role of HHV-6 in MS pathology remains controversial (36). The HHV-6 genome consists of a linear, double-stranded DNA molecule of 160 to 162 kb, comprising a unique region (U) of 145 kb flanked by terminal direct repeats (DRLand DRR) of 8 to 9 kb. HHV-6 has a broad cellular tropism, as its receptor (CD46) is expressed on all nucleated cells. The sites of latency are not precisely defined yet but could consist of CD4+T cells, monocytes, and early bone marrow progenitor cells (14,32,49). HHV-6, like HCMV (although it is less well studied), uses a multitude of approaches to modulate the host-pathogen environment and establish latency. Previous studies have focused on the role of viral proteins that modulate major histocompatibility complex (MHC) class I gene expression and of viral chemokines and receptors that interact with the host (34). Recently, many members of the herpesvirus family have been shown to express their own microRNAs (miRNAs) (11,19,40,41). Since the seminal discovery of miRNAs in Epstein-Barr virus (EBV) (41), more than 235 miRNAs of viral origin have been identified and listed in the miRNA repository miRBase (v.17) (25,26). Conserved throughout metazoan evolution, miRNAs derive from a large primary transcript (pri-miRNA) which is sequentially processed by the nuclear and cytoplasmic RNase III enzymes Drosha and Dicer to generate the mature, 22-nucleotide (nt) miRNA. This miRNA becomes functional only after its incorporation into an Argonaute (Ago)-containing RNA-induced silencing complex (RISC), where it will mediate the regulation of target messenger Anlotinib RNAs via binding to partially complementary sites, usually located Anlotinib in the 3-untranslated regions (3-UTRs) (3). The vast majority of virus-encoded miRNAs derive from herpesviruses, and so far HHV-3, HHV-6, and HHV-7 are the only herpesviruses for which miRNAs have not been characterized. For HHV-3, deep sequencing of latently infected trigeminal ganglia failed to identify any miRNAs (55). To resolve the question of whether HHV-6 encodes miRNAs, we performed small RNA cloning followed by massively parallel sequencing to identify the small RNAs deriving from the virus during lytic infection. We cloned small RNAs both from total RNA isolated from a CD4+T lymphocyte cell line (Sup-T-1) infected with a laboratory strain of HHV-6B and from RNAs coimmunoprecipitated with Ago2. Here we report that HHV-6B encodes at least four.
All HHV-6B miRNAs are conserved within HHV-6A at both the structural and sequence levels, although they display minor variations in their sequences (see Fig