Long noncoding RNAs and viral infection: promising molecular markers?

Introduction

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.

Meet lncRNAs

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).

Chimeric lncRNAs

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.

Acknowledgments

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).

Footnote

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/jlpm.2017.05.13). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Can R, Yun H. Function of long noncoding RNA in energy metabolism and related diseases. Chinese Journal of Pathophysiology 2015;31:956-60.
2. Iizuka N, Oka M, Yamada-Okabe H, et al. Comparison of gene expression profiles between hepatitis B virus-and hepatitis C virus-infected hepatocellular carcinoma by oligo nucleotide microarray data on the basis of a supervised learning method. Cancer Res 2002;62:3939-44. [PubMed]
3. Moyo B, Nicholson SA, Arbuthnot PB. The role of long non-coding RNAs in hepatitis B virus-related hepatocellular carcinoma. Virus Res 2016;212:103-13. [Crossref] [PubMed]
4. Wang X, Guo B, Liang Q, et al. Establishment of the method of real-time fluorescence quantitative PCR detection of HULC lncRNA and its primary application. Laser Journal 2013;34:94-6.
5. Landeras-Bueno S, Ortin J. Regulation of influenza virus infection by long non-coding RNAs. Virus Res 2016;212:78-84. [Crossref] [PubMed]
6. Huang JF, Guo YJ, Zhao CX, et al. Hepatitis B virus X protein (HBx)-related long noncoding RNA (lncRNA)down-regulated expression by HBx (Dreh) inhibits hepatocellular carcinomametastasis by targeting the intermediate filament protein vimentin. Hepatology 2013;57:1882-92. [Crossref] [PubMed]
7. Braconi C, Kogure T, Valeri N, et al. microRNA-29 can regulate expression of the long non-coding RNA gene MEG3 in hepatocellular cancer. Oncogene 2011;30:4750-6. [Crossref] [PubMed]
8. Du Y, Kong G, You X, et al. Elevation of highly up-regulated in liver cancer (HULC) by hepatitis B virus X protein promotes hepatoma cell proliferation via down-regulating p18. J Biol Chem 2012;287:26302-11. [Crossref] [PubMed]
9. Wang J, Liu X, Wu H, et al. CREB up-regulates long non-coding RNA, HULC expression through interaction with microRNA-372 in liver cancer. Nucleic Acids Res 2010;38:5366-83. [Crossref] [PubMed]
10. Maguire HF, Hoeffler JP, Siddiqui A. HBV X protein alters the DNA binding specificity of CREB and ATE-2 by protein-protein interactions. Science 1991;252:842-4. [Crossref] [PubMed]
11. Greenaway PJ, Wilkinson GW. Nucleotide sequence of the most abundantly transcribed early gene of human cytomegalovirus strain AD169. Virus Res 1987;7:17-31. [Crossref] [PubMed]
12. Reeves MB, Davies AA, Mcsharry BP, et al. Complex I binding by a virally encoded RNA regulates mitochondria-induced cell death. Science 2007;316:1345-8. [Crossref] [PubMed]
13. McSharry BP, Tomasec P, Neale ML, et al. The most abundantly transcribed human cytomegalovirus gene (β2.7) is non-essential for growth in vitro. J Gen Virol 2003;84:2511-6. [Crossref] [PubMed]
14. Saayman S, Ackley A, Turner AM, et al. An HIV-encoded antisense long noncoding RNA epigenetically regulates viral transcription. Mol Ther 2014;22:1164-75. [Crossref]
15. Lau CC, Sun Tingting, Ching AK, et al. Viral-human chimeric transcript predisposes risk to liver cancer development and progression. Cancer Cell 2014;25:335-49. [Crossref] [PubMed]
16. Iwakiri D, Takada K. Role of EBERs in the pathogenesis of EBV infection. Adv Cancer Res 2010;107:119-36. [Crossref] [PubMed]
17. Lee SI, Murthy S, Trimble JJ, et al. Four novel U RNAs are encoded by a herpesvirus. Cell 1988;54:599-607. [Crossref] [PubMed]
18. Buck AH, Perot J, Chisholm MA, et al. Post-transcriptional regulation of miR-27 in murine cytomegalovirus infection. RNA 2010;16:307-15. [Crossref] [PubMed]
19. Mathews MB, Shenk T. Adenovirus virus-associated RNA and translation control. J Virol 1991;65:5657-62. [PubMed]
20. Wahid AM, Coventry VK, Conn GL. The PKR-binding domain of adenovirus V A RNAI exists as a mixture of two functionally non-equivalent structures. Nucleic Acids Res 2009;37:5830-7. [Crossref] [PubMed]
21. Xu N, Segerman B, Zhou X, et al. Adenovirus virus-associated RNAII-derived small RNAs are efficiently incorporated into the RNA-induced silencing complex and associate with polyribosomes. J Virol 2007;81:10540-9. [Crossref] [PubMed]
22. Andersson MG, Haasnoot PJ, Xu N, et al. Suppression of RNA interference by adenovirus virus-associated RNA. J Virol 2005;79:9556-65. [Crossref] [PubMed]
23. Yu J, Wu W, Zhang X, et al. Research progress in long noncoding RNA and its relationship with virus. Journal of Zhejiang Normal University 2015;38:354-60. (Nat Sci).
24. Kobayashi-Ishihara M, Yamagishi M, Hara T, et al. HIV-1-encoded antisense RNA suppresses viral replication for a prolonged period. Retrovirology 2012;9:38. [Crossref] [PubMed]
25. Lau CC, Sun T, Ching AK, et al. Viral-human chimeric transcript predisposes risk to liver cancer development and progression. Cancer Cell 2014;25:335-49. [Crossref] [PubMed]
26. Fortes P, Kevin V. Morris. Long noncoding RNAs in viral infections. Virus Res 2016;212:1-11. [Crossref] [PubMed]
27. Wang P, Cao X. The role of noncoding RNA information regulation of human NK cells, dendritic cells and the underlying machanism. Shanghai: Department of Immunology, Second Military Medical University 2013:1-105.
28. Ding YZ, Zhang ZW, Liu YL, et al. Relationship of long noncoding RNA and viruses. Genomics 2016;107:150-4. [Crossref] [PubMed]
29. Collier SP, Collins PL, Williams CL, et al. Cutting edge: influence of Temvpg1, a long intergenic noncoding RNA, on the expression of Ifng by Th1 cells. J Immunol 2012;189:2084-8. [Crossref] [PubMed]