Singapore grouper iridovirus (SGIV) encoded SGIV-miR-13 attenuates viral infection via modulating major capsid protein expression
Introduction
microRNAs (miRNAs) are ∼22-nucleotide (nt) noncoding RNA molecules that regulate gene expression at either the post-transcriptional or translational level (Bartel, 2004). Since firstly identified in Caenorhabditis elegans in last 1990s, miRNAs are now well known to be encoded by all metazoan eukaryotes and plant species, and modulate a range of fundamental biological processes such as embryonic development, cell differentiation, apoptosis, and oncogenesis (Bushati and Cohen, 2007, Ghosh et al., 2009). Given several unique features miRNAs possess, such as minimal genomic space, non-immunogenicity and the capacity to regulate specific but also multiple targets, it is no surprise that viruses also encode miRNAs as ideal mediators to facilitate viral replication (Skalsky and Cullen, 2010).
Recent studies have provided plenty of evidence that the biogenesis of viral miRNA is solely dependent on cellular miRNA biogenesis mechanism, which also initiates from the transcription of primary miRNAs in the nucleus, followed by sequential processing under two endogenous RNase IIIs Drosha and Dicer (Boss and Renne, 2010). Therefore, viral miRNAs are to date investigated and experimentally confirmed to be encoded by DNA viruses of which the genome replication occurs in the nucleus, but not RNA viruses and cytoplasmic DNA viruses like poxviruses (Skalsky and Cullen, 2010). Till now, more than 200 viral miRNAs have been identified, and most of them are herpesvirus origin, including Marek's disease virus (Burnside et al., 2006, Yao et al., 2007), herpes simplex virus HSV-1 and HSV-2 (Tang et al., 2009), human and murine cytomegalovirus (hCMV and mCMV) (Dolken et al., 2007, Grey et al., 2005), Kaposi's sarcoma-associated herpesvirus (KSHV) (Samols et al., 2005) and Epstein-Barr virus (EBV) (Pfeffer et al., 2004). Apart from herpesviruses, some other dsDNA viruses, such as polyomavirus, adenovirus, ascovirus and baculovirus, are also able to encode miRNAs, suggesting that the encoding of miRNAs during replication is a general phenomenon in DNA virus families. (Grundhoff and Sullivan, 2011).
Compared to the well studied miRNAs in metazoan eukaryotes and plant species, information about the exact functions of most viral miRNAs is still imperfect. Generally, viral miRNAs are thought to favor the virus propagation and pathogenesis by evading immune response or promoting cell survival to support persistent infection (Ghosh et al., 2009, Gottwein and Cullen, 2008). To accomplish this, viral miRNAs may target cellular or viral transcripts with full-length or seed sequence match through Watson-crick base-pairing mechanisms, resulting in gene repression at post-transcriptional level. For example, the major histocompatibility complex class I-related chain B (MICB) was selectively targeted by hCMV miR-UL112-1, and its repression benefits the persistent viral infection via preventing infected cells from killing by natural killer (NK) cells (Stern-Ginossar et al., 2007). Two subsequent studies lent support to this finding that both KSHV miR-K7 and EBV miR-BART2 also suppressed MICB expression by targeting its 3′UTR but at different sites (Nachmani et al., 2009). Besides, the role of viral miRNAs in regulating viral functional genes has also begun to be appreciated. To date, most of the well-defined viral targets recognized by viral miRNAs are predominantly involved in persistent/latent viral infection. One of the best examples is the discovery that EBV miR-BART2 conducted negative regulation of a lytic gene–DNA polymerase BALF5 and hence led to enhanced latent infection (Barth et al., 2008). Other instances arised from the studies on HSV-1 and HSV-2, which utilized their miRNAs such as miR-H2, miR-H3 and miR-H4 to downregulate the expression of two immediate early genes, ICP0 and ICP34.5 in a siRNA-like manner to establish the latent viral infection state (Tang et al., 2008, Tang et al., 2009, Umbach et al., 2008). These findings suggest that viral miRNAs play indispensible roles during viral infection. An in-depth and systematic study of the interaction of viral miRNAs with cellular or viral targets will help us better understand the molecular mechanism of viral pathogenesis.
Singapore grouper iridovirus (SGIV) was firstly isolated from diseased brown-spotted grouper, Epinephelus tauvina, and subsequently characterized as a novel member of the family Iridoviridae, genus Ranavirus (Qin et al., 2003, Qin et al., 2001). In recent years, SGIV has gained increasing attentions because of the high mortality and huge economic losses it posed to aquaculture in China and Southeast Asia (Song et al., 2004). In the past decade, the SGIV genome has been sequenced (Song et al., 2004), the whole genome transcriptional profiles of SGIV in virus-infected grouper cells and tissues, as well as the viral-infection induced cellular transcriptome alteration have been broadly studied (Teng et al., 2008). However, a comprehensive understanding of SGIV still remains largely unknown due to the limited information about defined roles of SGIV-encoded genes and molecular mechanisms of viral pathogenesis (Yan et al., 2013).
Recently, by employing Illumina/Solexa deep-sequencing, we identified 16 viral miRNAs in SGIV infected grouper cells. These viral miRNAs are dispersed throughout the SGIV genome, and some showed marked sequence and length heterogeneity at their 3′ and/or 5′ end that could modulate their functions (Yan et al., 2011). Moreover, we found that 11 of these miRNAs possess potential biological activities, and one of our recent studies provides experimental evidence that SGIV encoded miR-homoHSV, which shares 57% sequence identity with HSV2-miR-H4-5p, was able to attenuate virus-cell death through targeting the pro-apoptotic viral gene SGIV-LITAF (Yan et al., 2011, Guo et al., 2013).
Among the SGIV miRNAs with potential biological activities, SGIV-miR-13, which maps antisense to the transcripts of SGIV major capsid protein gene (SGIV-MCP), also raised our great interest. In the present study, we found that SGIV-miR-13 could directly target SGIV-MCP. Acting like siRNAs, SGIV-miR-13 promotes the cleavage/degradation of SGIV-MCP transcripts and thus attenuates SGIV propagation in the early stage after viral infection. Our data suggest that iridovirus encoded miRNAs could function in a negative regulatory mechanism to fine-tune the process of viral assembly during the course of infection.
Section snippets
Cells and virus
Two fish cell lines, Grouper spleen cells (GS) and fathead minnow cells (FHM) were cultured as previously described (Cui et al., 2011, Gravell and Malsberg, 1965). SGIV (strain A3/12/98) was originally isolated from diseased brown-spotted grouper, E. tauvina, and the propagation of SGIV was performed as reported previously (Qin et al., 2003).
Plasmids, oligoribonucleotides and transfection
Plasmids used in this study were constructed and stored in our laboratory as reported previously (Yan et al., 2011, Ou-Yang et al., 2012). Briefly, to
SGIV-miR-13 was implicated in SGIV infection
To ascertain whether SGIV-miR-13 possesses biological functions in SGIV infection, we employed vector-based overexpression of SGIV-miR-13 (pLL-miR-13) in FHM cells followed by SGIV infection for 28 h. Comparing with control group, pLL-miR-13 transfected cells exhibited weak cytopathic effect (CPE) phenotypically, with obvious reduction in the amount of detached cells and viral plaques (Fig. 1A). Furthermore, we also examine the transcriptional kinetics of SGIV genes, including immediate early
Discussion
In the past decade, miRNAs have attracted tremendous attentions due to their regulatory roles in almost all biological processes in eukaryotes (Bartel, 2004). Of note, since the first discovery of viral origin miRNA in EBV, mounting evidence illustrates divergent viral families possess the ability to encode miRNAs which share similar biogenesis with cellular origin miRNAs (Cullen, 2006, Umbach and Cullen, 2009). However, compared to the well studied miRNAs of cellular origin, the information
Acknowledgments
We particularly thank Dr. Xiujie Wang (The Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for her help in the bioinformatic analysis of putative targets of SGIV miRNAs. This work was supported by the grants from the National Natural Science Foundation of China (41206137, 31330082), National Basic Research Program of China (973) (2012CB114402), and the Knowledge Innovation Program of Chinese Academy of Sciences (SQ201116).
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