For years, long noncoding RNAs (lncRNAs) were considered to be transcriptional noise. When viruses invade host cells, we used to think that they replicate and inhibit host antiviral responses through pathways induced by protein complexes. Nowadays, however, we know that various small RNAs in serum, such as microRNAs and lncRNAs, can be used as markers to determine the presence of viral infection and to obtain reliable laboratory data for differential diagnoses of virus-related diseases.
It was once believed that mRNAs make up the majority of the transcriptome. However, with the development of high-throughput sequencing and ENCODE (1), researchers realized that noncoding RNAs (ncRNAs) are also vital components within cell membranes. NcRNAs originate from transcription from the genome. According to their length, ncRNAs are classified into two subgroups: small ncRNAs and lncRNAs with more than 200 base pairs. LncRNAs are transcribed by RNA polymerase II and are found in nuclear and cytosolic fractions. They take part in chromatin transport, stem cell maintenance, and cell differentiation. According to recent reports, their biological functions are as follows: (I) the upstream promoter region of protein-encoding genes transcription interferes the expression of downstream genes; (II) affecting the expression of downstream genes by inhibiting RNA polymerase II or mediating chromatin remodeling and histone modifications; (III) forming complementary double-stranded chains with transcripts that encode proteins to interfere with mRNA splicing, and then inducing the expression of different splicing variants; (IV) forming complementary double-stranded chains with transcripts that encode proteins to produce endogenous siRNA under the action of the Dicer enzyme; (V) regulating the activity of proteins by combining with specific partners; (VI) forming nucleic acid-protein complexes with proteins as structural components; (VII) binding to specific proteins to alter their cellular location; and (VIII) acting as precursor molecules of small RNAs, such as miRNAs.
Reverse transcription polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH), and microarray are currently employed to detect lncRNAs. Iizuka (2) studied differences in the profiles of lncRNA expression between hepatocellular carcinoma and adjacent healthy tissues. The results showed that lncRNAs with altered expression in HCC included high expression in HCC (HEIH), highly upregulated in liver cancer (HULC), HBx-long interspersed nuclear element 1 (HBx-LINE1), H19, maternally expressed imprinted gene 3, and downregulated expression by HBx (DREH). A higher level of HULC in HBV-related HCC was reported elsewhere and confirmed to be a convenient diagnostic biomarker following amplification (3). In addition, Wang (4) reported that HULC expression levels in hepatocellular carcinoma and hepatocarcinoma cell lines were (7.6±3.1) ×105 and (5.6±3.2) ×106 copies/L, respectively, which were significantly higher than those of other cancer and paracarcinoma tissues (<1.8×103 copies/L). The minimum detection limit of RT-PCR to detect HULC is 1.8×103 copies/L, with a linear range of 1.8×103 to 4.6×109 copies/L [coefficient of variation (CV) <2.0%, batch CV <8.0%]. Recently, Panomics QuantiGene Plex technology developed new assays that enable the simultaneous detection and quantification of lncRNAs of 3–80 nucleotides in length in cultured cells, whole blood samples, and tissue samples without reverse transcription or PCR.
lncRNAs, viruses, and immune responses
Viruses have developed various strategies to fight against the human immune system and to increase the efficiency of their self-replication. Some of these strategies are implemented through lncRNAs.
lncRNA expression of host cells by viral infection
Viral infection induces the abnormal expression of lncRNAs in host cells. The first lncRNA screening in mice and mouse embryonic fibroblasts in response to influenza A virus infection revealed that hundreds of potential lncRNAs were differentially expressed. The study (5) further analyzed the expression levels of lncRNAs in human A549 cells after infection with the A/Victoria/3/75 (H3N2) influenza virus. Total nonribosomal RNA was isolated 6 h postinfection and analyzed by deep sequencing, which revealed that 141 lncRNAs were upregulated. Most of them had not been identified so far. The others have been implicated in a variety of biological processes, including cell differentiation and cancer development.
In a comparison with control cells, X protein (hepatitis B virus x protein, HBx) was found to significantly downregulate the tumor suppressor lncRNA DREH (downregulated expression by HBx) in HBV-infected cultured cells or transgenic mice. DREH was typically low in malignant tissues when compared with its level in patient-matched noncancerous hepatic tissues. The role of HBx in DREH downregulation was further confirmed by transfecting murine hepatic cell lines with plasmids encoding the viral protein (6) and engrafting nude mice with murine cells expressing DREH. DREH inhibition disrupted cell proliferation, apoptosis, and migration. In another study, Braconi (7) applied a microarray and revealed that 174 lncRNAs were highly expressed and 712 lncRNAs were downregulated in HCC cells, of which HULC and HEIH were strongly involved in HBV-induced HCC. HBx binds to the HULC promoter by interacting with the transcription factor CREB (cAMP-response element binding protein) and induces HULC expression, which inhibits the tumor suppressor P18 and promotes cancer cell proliferation (8-10). On the other hand, HEIH recruits PRC2 (polycomb repressive complex 2) through the enhancer of zeste homolog 2 (EZH2). Together, they inhibit EZH2 target gene expression and arrest cells in the G0/G1 phase.
Viral lncRNAs influence host cell function
During infection, viruses maintain themselves in a latent or replicative state by encoding their own lncRNAs. At present, we have some understanding of the specific functions of viral lncRNAs in the process of viral infection (Table 1) (23). For example, β2.7 RNA is a highly conserved lncRNA with 2,700 base pairs transcribed by human cytomegalovirus. It localizes in the cytoplasm of host cells and accounts for more than 20% of the total viral transcripts (11). Along with mitochondrial enzyme complex I, β2.7 RNA stabilizes mitochondrial membrane potential and ensures the ATP production of host cells, in order to avoid stress cascade responses, inhibit host cell apoptosis, and promote viral survival (12).
Viral lncRNAs are epigenetic regulatory factors. For example, during the process of human immunodeficiency virus (HIV) infection, the HIV genome encodes antisense RNA transcripts to suppress the expression of virus-encoding genes (24). Recently, it was found that the 5'-long terminal repeat (LTR) region of the HIV virus encodes an antisense lncRNA named ASPRO5. It recruits DNA methyltransferase 3A (DNMT3a), histone deacetylase HDAC-1 (histone deacetylase 1), and histone methyltransferase EZH2 to the 5'-noncoding region of the virus to form a heterochromatin structure like histone H3 lysine 9 dimethylation (H3K9me2) and trimethyl histone H3K27 (H3K27me3), followed by histone deacetylation and inhibition of virus transcriptional activity. Silencing ASPRO5 activates HIV gene expression (14).
Intensive studies of HCC led to the identification of transcripts in which human and viral sequences are fused (25). This is unsurprising as the HBV genome can integrate into the host genome. Integration preferentially occurs within repetitive sequences, such as LINEs. Results have shown that 23.3% of HCCs express chimeras between the HBx protein and host LINE1 sequences. These chimeric transcripts, named HBx-LINE1, have oncogenic effects as they activate Wnt signaling pathways. Conflicting results have been obtained about whether viruses can benefit from the transcription of chimeric lncRNAs (26).
Immune responses and lncRNAs
In recent years, the roles of lncRNAs in the development of immune cells and the regulation of immune responses have been discussed. The innate immune response is the first line of defense of the body against invading pathogens, mainly implemented by innate immune cells including monocytes/macrophages, dendritic cells (DCs), granulocytes, and natural killer cells. Wang (27) analyzed the lncRNA expression profile of DCs differentiating from monocytes and DCs that had undergone maturation induced by lipopolysaccharide. They found that the expression levels of 76 lncRNAs were significantly changed (>20 times), among which lnc-DC was highly expressed in mature DCs. It facilitates the expression of antigen-presenting molecules and costimulatory molecules on the surface of mature DCs, and stimulates IL-12 secretion.
TMEVPG1 (NeST, Ifng-AS1) was initially identified as an lncRNA transcript in the context of Theiler’s virus infection. Mice lacking TMEVPG1 were unable to control intracranial viral infection. TMEVPG1 is absent from the central nervous system (CNS). However, when Theiler’s virus infected resistant B10.S mice, it began to be expressed in CNS-infiltrating immune cells such as macrophages, microglia, and astrocytes. Similar results for TMEVPG1 were observed in human NK cells and CD4+ and CD8+ T lymphocytes (28). Both Temvpg1 and its human ortholog TMEVPG1 are localized in a cluster of cytokine genes including IL-10 homologs and IFN-γ. Research (29) found that interfering with TMEVPG1 expression significantly inhibited IFN-γ secretion in mouse Th1 cells. The combined action of TMEVPG1 and T-bet upregulated IFN-γ level, while the overexpression of TMEVPG1 alone did not, suggesting that TMEVPG1 regulates IFN-γ gene transcription by interacting with other factors.
Prospects of clinical application
lncRNAs are a new hotspot in clinical research. They regulate the expression of genes through different molecular biological mechanisms, are involved in multiple disease-related signaling pathways, and play an important role in disease prognosis. Disease is the result of multiple molecular dysfunctions in cells and circumstances around them. Protein-coding genes are only one part of the interaction network in the pathophysiological processes of diseases. The identification of more lncRNAs should shed light on pathophysiological mechanisms from an RNA perspective. According to research data, the sensitivity and specificity of lncRNA detection assays are higher than those of assays of protein in body fluid. As new promising biomarkers, lncRNAs thus have broad potential for clinical applications. Although microRNAs are the most dazzling stars at present, lncRNA should become a brighter young member of the revealed complex of molecular networks related to viral infection.
Funding: This work was supported by National Natural Science Youth Foundation of China (81501817 to JX Zhang), and the Nature Science Youth Foundation of Jiangsu Province (BK20151029 to JX Zhang).
Conflicts of Interest: The authors have no conflicts of interest to declare.
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Cite this article as: Yu MY, Zhang JX, Wang F. Long noncoding RNAs and viral infection: promising molecular markers? J Lab Precis Med 2017;2:36.