Excessive scar formation caused by myofibroblast aggregations is of great clinical importance during skin wound healing. Studies have shown that mesenchymal stem cells (MSCs) can promote skin regeneration, but whether MSCs contribute to scar formation remains undefined. We found that umbilical cord-derived MSCs (uMSCs) reduced scar formation and myofibroblast accumulation in a skin-defect mouse model. We found that these functions were mainly dependent on uMSC-derived exosomes (uMSC-Exos) and especially exosomal microRNAs. Through high-throughput RNA sequencing and functional analysis, we demonstrated that a group of uMSC-Exos enriched in specific microRNAs (miR-21, -23a, -125b, and -145) played key roles in suppressing myofibroblast formation by inhibiting the transforming growth factor-b2/SMAD2 pathway. Finally, using the strategy we established to block miRNAs inside the exosomes, we showed that these specific exosomal miRNAs were essential for the myofibroblast-suppressing and anti-scarring functions of uMSCs both in vitro and in vivo. Our study revealed a novel role of exosomal miRNAs in uMSC-mediated therapy, suggesting that the clinical application of uMSC-derived exosomes might represent a strategy to prevent scar formation during wound healing. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:1425-1439 SIGNIFICANCEExosomes have been identified as a new type of major paracrine factor released by umbilical cordderived mesenchymal stem cells (uMSCs). They have been reported to be an important mediator of cell-to-cell communication. However, it is still unclear precisely which molecule or group of molecules carried within MSC-derived exosomes can mediate myofibroblast functions, especially in the process of wound repair. The present study explored the functional roles of uMSC-exosomal microRNAs in the process of myofibroblast formation, which can cause excessive scarring. This is an unreported function of uMSC exosomes. Also, for the first time, the uMSC-exosomal microRNAs were examined by high-throughput sequencing, with a group of specific microRNAs (miR-21, miR-23a, miR-125b, and miR-145) found to play key roles in suppressing myofibroblast formation by inhibiting excess a-smooth muscle actin and collagen deposition associated with activity of the transforming growth factor-b/SMAD2 signaling pathway.

Hepatitis C virus (HCV) is a significant global public health problem, causing more than 350,000 deaths every year. Although the development of direct-acting antivirals has improved the sustained virological response rate in HCV patients, novel anti-HCV agents with higher efficacy as well as better tolerance and cheaper production costs are still urgently needed. Cell-based therapy, especially its unique and strong paracrine ability to transfer information to other cells via extracellular vesicles such as exosomes, has become one of the most popular therapeutic methods in recent years. In our study, exosomes secreted from umbilical mesenchymal stem cells (uMSCs), which are widely used in regenerative medicine, inhibited HCV infection in vitro, especially viral replication, with low cell toxicity. Our analysis revealed that microRNAs (miRNAs) from uMSC-derived exosomes (uMSC-Exo) had their unique expression profiles, and these functional miRNAs, mainly represented by let-7f, miR-145, miR-199a, and miR-221 released from uMSC-Exo, largely contributed to the suppression of HCV RNA replication. These four miRNAs possessed binding sites in HCV RNA as demonstrated by the target prediction algorithm. In addition, uMSC-Exo therapy showed synergistic effect when combined with U.S. Food and Drug Administration-approved interferon-a or telaprevir, enhancing their anti-HCV ability and thus improving the clinical significance of these regenerative substances for future application as optimal adjuvants of anti-HCV therapy. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:1190-1203 SIGNIFICANCEThis work reported, for the first time, the identification of stem cell-derived exosomes of antiviral activity. Umbilical mesenchymal stem cell-secreted exosomes inhibited hepatitis C virus infection through transporting a mixture of microRNAs complementing the viral genomes to the host cells. This finding provides insights and prospects for physiologically secreted substances for antiviral therapy.

bJapanese encephalitis virus (JEV) is a mosquito-borne flavivirus and one of the most common agents of viral encephalitis. The infectious entry process of JEV into host cells remains largely unknown. Here, we present a systemic study concerning the cellular entry mechanism of JEV to B104 rat neuroblastoma cells. It was observed that JEV internalization was inhibited by chloroquine and ammonium chloride, both of which can elevate the pH of acidic organelles. However, JEV entry was not affected by chlorpromazine, overexpression of a dominant-negative form of EPS 15 protein, or silencing of the clathrin heavy chain by small interfering RNA (siRNA). These results suggested that JEV entry depended on the acidic intracellular pH but was independent of clathrin. We found that endocytosis of JEV was dependent on membrane cholesterol and was inhibited by inactivation of caveolin-1 with siRNA or dominant-negative mutants. It was also shown, by using the inhibitor dynasore, the K44A mutant, and specific siRNA, that dynamin was required for JEV entry. Phagocytosis or macropinocytosis did not play a role in JEV internalization. In addition, we showed that JEV entry into the neuroblastoma cells is not virus strain specific by assessing the effect of the pharmacological inhibitors on the internalization of JEV belonging to different genotypes. Taken together, our results demonstrate that JEV enters B104 cells through a dynamin-dependent caveola-mediated uptake with a pH-dependent step, which is distinct from the clathrin-mediated endocytosis used by most flaviviruses.

HCV infection has a complex and dynamic process which involves a large number of viral and host factors. Long non-coding RNA GAS5 inhibits liver fibrosis and liver tumor migration and invasion. However, the contribution of GAS5 on HCV infection remains unknown. In this study, GAS5 was gradually upregulated during HCV infection in Huh7 cells. In addition, GAS5 attenuated virus replication with its 5' end sequences, as confirmed by different GAS5 truncations. Moreover, this 5' end sequences showed RNA-protein interaction with HCV NS3 protein that could act as a decoy to inhibit its functions, which contributed to the suppression of HCV replication. Finally, the innate immune responses remained low in HCV infected Huh7 cells, ruling out the possibility of GAS5 to modulate innate immunity. Thus, HCV stimulated endogenous GAS5 can suppress HCV infection by acting as HCV NS3 protein decoy, providing a potential role of GAS5 as a diagnostic or therapeutic target.

Hepatitis C virus (HCV) infection is a global health problem, with an estimated 170 million people being chronically infected. HCV cell entry is a complex multi-step process, involving several cellular factors that trigger virus uptake into the hepatocytes. The high- density lipoprotein receptor scavenger receptor class B type I, tetraspanin CD81, tight junction protein claudin-1, and occludin are the main receptors that mediate the initial step of HCV infection. In addition, the virus uses cell receptor tyrosine kinases as entry regulators, such as epidermal growth factor receptor and ephrin receptor A2. This review summarizes the current understanding about how cell surface molecules are involved in HCV attachment, internalization, and membrane fusion, and how host cell kinases regulate virus entry. The advances of the potential antiviral agents targeting this process are introduced.

This work provides a detailed picture of the entry route and intricate cellular events following the entry of JEV into human neuronal cells, and promotes a better understanding of JEV entry.

Hepatitis C virus (HCV) infection affects approximately 3% of the world's population and causes chronic liver diseases, including liver fibrosis, cirrhosis, and hepatocellular carcinoma. Although current antiviral therapy comprising direct-acting antivirals (DAAs) can achieve a quite satisfying sustained virological response (SVR) rate, it is still limited by viral resistance, long treatment duration, combined adverse reactions, and high costs. Moreover, the currently marketed antivirals fail to prevent graft reinfections in HCV patients who receive liver transplantations, probably due to the cell-to-cell transmission of the virus, which is also one of the main reasons behind treatment failure. HCV entry is a highly orchestrated process involving initial attachment and binding, post-binding interactions with host cell factors, internalization, and fusion between the virion and the host cell membrane. Together, these processes provide multiple novel and promising targets for antiviral therapy. Most entry inhibitors target host cell components with high genetic barriers and eliminate viral infection from the very beginning of the viral life cycle. In future, the addition of entry inhibitors to a combination of treatment regimens might optimize and widen the prevention and treatment of HCV infection. This review summarizes the molecular mechanisms and prospects of the current preclinical and clinical development of antiviral agents targeting HCV entry.

Hepatitis C virus (HCV) infection involves a variety of viral and host factors, some of which promote the infection process. A small nucleolar RNA, C/D box 126 (SNORD126), was previously shown to be associated with hepatocellular carcinoma (HCC). However, the role of SNORD126 in HCV infection, which is one of the primary reasons for HCC development, has not been elucidated. In the present study, using small nucleolar RNA profiling, we observed that SNORD126 was significantly downregulated during HCV infection in both Huh7 and Huh7.5.1 cells. In addition, overexpression of SNORD126 enhanced HCV entry into host cells, whereas SNORD126 knockdown showed the opposite effect, suggesting that SNORD126 promotes HCV infection, especially through viral entry. Further functional analysis revealed that SNORD126 could enhance the expression level of claudin-1 (CLDN1), a key HCV entry factor, by increasing the levels of phosphorylated AKT. Additionally, the function of SNORD126 in HCV infection was associated with ribonucleoprotein (RNP) complexes. In summary, our findings demonstrate that oncogenic SNORD126 levels are decreased during HCV infection probably due to the host defense reaction, and SNORD126 may be important to promote viral entry by increasing CLDN1 expression through activation of the PI3K-AKT pathway, the mechanism of which is partly associated with SNORD126mediated snoRNA RNP (snoRNP) function. Our work here provides initial evidence that endogenous snoRNA takes part in HCV infection and shows potential as a diagnostic or antiviral agent.