The extent of sequence complementarity correlates with the potency of cellular miRNA-mediated restriction of HIV-1

MicroRNAs (miRNAs) are 22-nt non-coding RNAs involved in the regulation of cellular gene expression and potential cellular defense against viral infection. Using in silico analyses, we predicted target sites for 22 human miRNAs in the HIV genome. Transfection experiments using synthetic miRNAs showed that five of these miRNAs capably decreased HIV replication. Using one of these five miRNAs, human miR-326 as an example, we demonstrated that the degree of complementarity between the predicted viral sequence and cellular miR-326 correlates, in a Dicer-dependent manner, with the potency of miRNA-mediated restriction of viral replication. Antagomirs to miR-326 that knocked down this cell endogenous miRNA increased HIV-1 replication in cells, suggesting that miR-326 is physiologically functional in moderating HIV-1 replication in human cells.     

The role of microRNAs in Hepatitis C Virus replication and related liver diseases

Hepatitis C virus (HCV) infection is a worldwide health problem and is one of the main causes of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). However, only limited therapeutic options and no vaccines are currently available against HCV infection. Recent studies of microRNAs (miRNAs), which are able to regulate HCV replication and its related liver diseases by directly interacting with the HCV genome or indirectly controlling virus-associated host pathways, have broadened our understanding of the HCV life cycle. HCV utilizes host cellular miRNAs and modulates expression of miRNAs in infected hepatocytes for its infection and propagation. Moreover, such miRNAs directly or indirectly alter HCV replication efficiency and induce liver diseases including liver fibrosis, cirrhosis, or HCC. Representatively, miR-122 directly modulates the HCV life cycle by increasing HCV translation and genomic RNA stability. Recently, a phase IIa clinical trial with miravirsen, an LNA form of antimiR-122 oligonucleotides, showed significant reduction in serum HCV levels in patients chronically infected with HCV with no detectible evidence of resistance. In addition to miR-122, other miRNAs involved in the regulation of HCV propagation could be targeted in strategies to modulate HCV replication and pathogenesis. In this review, we summarize the features of miRNAs critical for HCV replication and HCV-mediated liver abnormalities and briefly discuss their potential application as therapeutic reagents for the treatment of HCV infection and its related diseases.     

Alterations in microRNA expression profile in HCV-infected hepatoma cells: Involvement of miR-491 in regulation of HCV replication via the PI3 kinase/Akt pathway

The aim of this study was to investigate the role of microRNA (miRNA) on hepatitis C virus (HCV) replication in hepatoma cells. Using miRNA array analysis, miR-192/miR-215, miR-194, miR-320, and miR-491 were identified as miRNAs whose expression levels were altered by HCV infection. Among them, miR-192/miR-215 and miR-491 were capable of enhancing replication of the HCV replicon as well as HCV itself. HCV IRES activity or cell proliferation was not increased by forced expression of miR-192/miR-215 or miR-491. Investigation of signaling pathways revealed that miR-491 specifically suppressed the phosphoinositol-3 (PI3) kinase/Akt pathway. Under inhibition of PI3 kinase by LY294002, the suppressive effect of miR-491 on HCV replication was abolished, indicating that suppression of HCV replication by miR-491 was dependent on the PI3 kinase/Akt pathway. miRNAs altered by HCV infection would then affect HCV replication, which implies a complicated mechanism for regulating HCV replication. HCV-induced miRNA may be involved in changes in cellular properties including hepatocarcinogenesis.     

Expression of microRNA miR-122 facilitates an efficient replication in nonhepatic cells upon infection with hepatitis C virus

Hepatitis C virus (HCV) is one of the most common etiologic agents of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. In addition, HCV infection is often associated with extrahepatic manifestations (EHM), including mixed cryoglobulinemia and non-Hodgkin's lymphoma. However, the mechanisms of cell tropism of HCV and HCV-induced EHM remain elusive, because in vitro propagation of HCV has been limited in the combination of cell culture-adapted HCV (HCVcc) and several hepatic cell lines. Recently, a liver-specific microRNA called miR-122 was shown to facilitate the efficient propagation of HCVcc in several hepatic cell lines. In this study, we evaluated the importance of miR-122 on the replication of HCV in nonhepatic cells. Among the nonhepatic cell lines expressing functional HCV entry receptors, Hec1B cells derived from human uterus exhibited a low level of replication of the HCV genome upon infection with HCVcc. Exogenous expression of miR-122 in several cells facilitates efficient viral replication but not production of infectious particles, probably due to the lack of hepatocytic lipid metabolism. Furthermore, expression of mutant miR-122 carrying a substitution in a seed domain was required for efficient replication of mutant HCVcc carrying complementary substitutions in miR-122-binding sites, suggesting that specific interaction between miR-122 and HCV RNA is essential for the enhancement of viral replication. In conclusion, although miR-122 facilitates efficient viral replication in nonhepatic cells, factors other than miR-122, which are most likely specific to hepatocytes, are required for HCV assembly.     

Competitive virus and host RNAs: the interplay of a hidden virus and host interaction

During virus infection, viral RNAs and mRNAs function as blueprints for viral protein synthesis and possibly as pathogen-associated molecular patterns (PAMPs) in innate immunity. Here, considering recent research progress in microRNAs (miRNAs) and competitive endogenous RNAs (ceRNAs), we speculate that viral RNAs act as sponges and can sequester endogenous miRNAs within infected cells, thus cross-regulating the stability and translational efficiency of host mRNAs with shared miRNA response elements. This cross-talk and these reciprocal interactions between viral RNAs and host mRNAs are termed “competitive viral and host RNAs” (cvhRNAs). We further provide recent experimental evidence for the existence of cvhRNAs networks in hepatitis B virus (HBV), as well as Herpesvirus saimiri (HVS), lytic murine cytomegalovirus (MCMV) and human cytomegalovirus (HCMV) infections. In addition, the cvhRNA hypothesis also predicts possible cross-regulation between host and other viruses, such as hepatitis C virus (HCV), HIV, influenza virus, human papillomaviruses (HPV). Since the interaction between miRNAs and viral RNAs also inevitably leads to repression of viral RNA function, we speculate that virus may evolve either to employ cvhRNA networks or to avoid miRNA targeting for optimal fitness within the host. CvhRNA networks may therefore play a fundamental role in the regulation of viral replication, infection establishment, and viral pathogenesis.     

Functional Analysis of a Crustacean MicroRNA in Host-Virus Interactions

Growing evidence from mammals suggests that host microRNAs (miRNAs) play important roles in the antiviral immune response. However, the roles of invertebrate miRNAs in response to virus infection remain to be investigated. Based on our previous studies, the shrimp miR-7 was found to be upregulated in response to white spot syndrome virus (WSSV) infection. In this study, the results showed that shrimp miR-7 could target the 3′-untranslated region (3′UTR) of the WSSV early gene wsv477, implying that miR-7 was involved in viral DNA replication. In insect High Five cells, the synthesized miR-7 significantly decreased the expression level of the fluorescent construct bearing the 3′UTR of wsv477 compared with the expression of the control constructs. When the activity of transfected miR-7 was blocked by locked-nucleic-acid (LNA)-modified anti-miR-7 oligonucleotide (AMO-miR-7), the repression of luciferase gene expression by miR-7 was relieved. In vivo, when the synthesized miR-7 was injected into shrimp, the numbers of WSSV genome copies/mg gills were 1,000-fold lower than those of WSSV only at 72 and 96 h postinfection. The results indicated that the blocking of endogenous miR-7 by AMO-miR-7 led to about a 10-fold increase of WSSV genome copies/mg gills in WSSV-infected shrimp compared with the control WSSV only. Further, it was revealed that the host Dicer1 was an important component for the biogenesis of miR-7, which had a large effect on virus infection. Therefore, our study revealed a novel regulatory function for an invertebrate miRNA in host-virus interactions by targeting the viral early gene.     

Human cytomegalovirus infection alters the expression of cellular microRNA species that affect its replication

The human genome encodes over 500 microRNAs (miRNAs), small RNAs (19 to 26 nucleotides [nt]) that regulate the expressions of diverse cellular genes. Many cellular processes are altered through a variety of mechanisms by human cytomegalovirus (HCMV) infection. We asked whether HCMV infection leads to changes in the expression of cellular miRNAs and whether HCMV-regulated miRNAs are important for HCMV replication. Levels of most miRNAs did not change markedly during infection, but some were positively or negatively regulated. Patterns of miRNA expression were linked to the time course of infection. Some similarly reregulated miRNAs share identical or similar seed sequences, suggesting coordinated regulation of miRNA species that have shared targets. miRNAs miR-100 and miR-101 were chosen for further analyses based on their reproducible changes in expression after infection and on the basis of having predicted targets in the 3' untranslated regions (3'-UTR) of genes encoding components of the mammalian target of rapamycin (mTOR) pathway, which is important during HCMV infection. Reporter genes that contain the 3'-UTR of mTOR (predicted targets for miR-100 and miR-101) or raptor (a component of the mTOR pathway; predicted site for miR-100) were constructed. Mimics of miR-100 and miR-101 inhibited expression from the mTOR construct, while only miR-100 inhibited the raptor construct. Together, miR-100 and miR-101 reduced mTOR protein levels. While the miR-100 and miR-101 mimics individually modestly inhibited production of infectious progeny, much greater inhibition was achieved with a combination of both (33-fold). Our key finding is that HCMV selectively manipulates the expression of some cellular miRNAs to help its own replication.     

Inhibition of hepatitis C virus using siRNA targeted to the virus and Hsp90

Hepatitis C (HCV) is a viral disease affecting millions of people worldwide, and persistent HCV infection can lead to progressive liver disease with the development of liver cirrhosis and hepatocellular carcinoma. During treatment for hepatitis C, the occurrence of viral resistance is common. To reduce the occurrence of resistance, new viral treatments should target both viral and cellular factors. Many interactions occur between viral and host proteins during the HCV replication cycle and might be used for the development of new therapies against hepatitis C. Heat shock protein 90 (Hsp90) plays a role in the folding of cellular and viral proteins and also interacts with HCV proteins. In the present study, we knocked down the expression of the Hsp90 gene and inhibited viral replication using siRNA molecules. Reducing the expression of Hsp90 successfully decreased HCV replication. All siRNA molecules specific to the viral genome showed the efficient inhibition of viral replication, particularly siRNA targeted to the 5′UTR region. The combination of siRNAs targeting the viral genome and Hsp90 mRNA also successfully reduced HCV replication and reduced the occurrence of viral resistance. Moreover, these results suggest that an approach based on the combination of cellular and viral siRNAs can be used as an effective alternative for hepatitis C viral suppression.     

The Epstein-Barr virus (EBV)-induced tumor suppressor microRNA MiR-34a is growth promoting in EBV-infected B cells

Epstein-Barr virus (EBV) infection of primary human B cells drives their indefinite proliferation into lymphoblastoid cell lines (LCLs). B cell immortalization depends on expression of viral latency genes, as well as the regulation of host genes. Given the important role of microRNAs (miRNAs) in regulating fundamental cellular processes, in this study, we assayed changes in host miRNA expression during primary B cell infection by EBV. We observed and validated dynamic changes in several miRNAs from early proliferation through immortalization; oncogenic miRNAs were induced, and tumor suppressor miRNAs were largely repressed. However, one miRNA described as a p53-targeted tumor suppressor, miR-34a, was strongly induced by EBV infection and expressed in many EBV and Kaposi's sarcoma-associated herpesvirus (KSHV)-infected lymphoma cell lines. EBV latent membrane protein 1 (LMP1) was sufficient to induce miR-34a requiring downstream NF-κB activation but independent of functional p53. Furthermore, overexpression of miR-34a was not toxic in several B lymphoma cell lines, and inhibition of miR-34a impaired the growth of EBV-transformed cells. This study identifies a progrowth role for a tumor-suppressive miRNA in oncogenic-virus-mediated transformation, highlighting the importance of studying miRNA function in different cellular contexts.