Key role of exportin 6 in exosome-mediated viral transmission from insect vectors to plants.

Exosomes play a key role in virus exocytosis and transmission. The exportin family is usually responsible for cargo nucleocytoplasmic trafficking, and they are frequently found in exosomes. However, the function of exportins sorted in exosomes remains unknown. Here, we successfully isolated "cup holder"-like exosomes from the saliva of ∼30,000 small brown planthoppers, which are vectors of rice stripe virus (RSV). RSV virions were packed in comparatively large exosomes. Four viral genomic RNAs at a certain ratio were identified in the saliva exosomes. The virions contained in the saliva exosomes were capable of replicating and causing disease in rice plants. Interference with each phase of the insect exosome system affected the transmission of RSV from the insect vectors to rice plants. Fragmented exportin 6 was coimmunoprecipitated with viral nucleocapsid protein in saliva and sorted to exosomes via interactions with the cargo sorting protein VPS37a. When the expression of exportin 6 was knocked down, the amounts of RSV secreted in saliva and rice plants were reduced by 60% and 74%, respectively. These results showed that exportin 6 acted as a vehicle for transporting RSV into exosomes to overcome the barrier of insect salivary glands for horizontal transmission. Exportin 6 would represent an ideal target that could be manipulated to control the outbreak of insect-borne viruses in the future.

PPRV-Induced Autophagy Facilitates Infectious Virus Transmission by the Exosomal Pathway.

Peste des petits ruminants virus (PPRV) is an important pathogen that seriously influences the productivity of small ruminants worldwide. We showed previously that PPRV induced sustained autophagy for their replication in host cells. Many studies have shown that exosomes released from virus-infected cells contain a variety of viral and host cellular factors that are able to modulate the recipient's cellular response and result in productive infection of the recipient host. Here, we show that PPRV infection results in packaging of the viral genomic RNA and partial viral proteins into exosomes of Vero cells and upregulates exosome secretion. We provide evidence showing that the exosomal viral cargo can be transferred to and establish productive infection in a new target cell. Importantly, our study reveals that PPRV-induced autophagy enhances exosome secretion and exosome-mediated virus transmission. Additionally, our data show that TSG101 may be involved in the sorting of the infectious PPRV RNA into exosomes to facilitate the release of PPRV through the exosomal pathway. Taken together, our results suggest a novel mechanism involving autophagy and exosome-mediated PPRV intercellular transmission. IMPORTANCE Autophagy plays an important role in PPRV pathogenesis. The role of exosomes in viral infections is beginning to be appreciated. The present study examined the role of autophagy in secretion of infectious PPRV from Vero cells. Our data provided the first direct evidence that ATG7-mediated autophagy enhances exosome secretion and exosome-mediated PPRV transmission. TSG101 may be involved in the sorting of the infectious PPRV RNA genomes into exosomes to facilitate the release of PPRV through the exosomal pathway. Inhibition of PPRV-induced autophagy or TSG101 expression could be used as a strategy to block exosome-mediated virus transmission.

Density Analysis of Enterovirus D68 Shows Viral Particles Can Associate with Exosomes.

Enterovirus D68 (EV-D68) is an emerging pathogen which causes respiratory disease and is associated with an acute flaccid myelitis that predominately affects children. EV-D68 can infect motor neurons, causing cell death and a loss of motor control leading to flaccid paralysis. However, it remains unknown how viral particles gain entry into the central nervous system (CNS). Here, we show that three distinct densities of EV-D68 particle can be isolated from infected muscle and neural cell lines (RD and SH-SY5Y) using high-speed density centrifugation to separate cell supernatant. The lowest-density peak is composed of viral particles, which have adhered to the exterior surface of a small extracellular vesicle called an exosome. Analysis of prototypic (historic) and contemporary EV-D68 strains suggests that binding to exosomes is a ubiquitous characteristic of EV-D68. We further show that interaction with exosomes increases viral infectivity in a neural cell line. Analysis of the two higher-density peaks, which are not associated with exosomes, revealed that a significant amount of viral titer in the modern (2014) EV-D68 strains is found at 1.20 g/cm3, whereas this density has a very low viral titer in the prototypic Fermon strain. IMPORTANCE Despite the strong causal link between enterovirus D68 (EV-D68) and acute flaccid myelitis (AFM), it remains unclear how EV-D68 gains entry into the central nervous system and what receptors enable it to infect motor neurons. We show that EV-D68 particles can adhere to exosomes, placing EV-D68 among a handful of other picornaviruses which are known to interact with extracellular vesicles. Uptake and infection of permissive cells by virally contaminated exosomes would have major implications in the search for the EV-D68 receptor, as well as providing a possible route for viral entry into motor neurons. This work identifies a novel cellular entry route for EV-D68 and may facilitate the identification of genetic risk factors for development of AFM.

Transcriptome analysis of cervical cancer exosomes and detection of HPVE6*I transcripts in exosomal RNA.

BACKGROUND: Exosomes play a key role in cell-to-cell communication and are integral component of the tumor microenvironment. Recent observations suggest transfer of RNA through tumor-derived exosomes that can potentially translate into regulatory proteins in the recipient cells. Role of cervical cancer-derived exosomes and their transcript cargo is poorly understood. MATERIALS AND METHODS: The total RNA of exosomes from HPV-positive (SiHa and HeLa) and HPV-negative (C33a) cervical cancer cell lines were extracted and the transcripts were estimated using Illumina HiSeq X. Further, validation of HPV transcripts were performed using RT-PCR. RESULTS: 3099 transcripts were found to be differentially-exported in HPV-positive vs. HPV-negative exosomes (p value <0.05). Analysis of top 10 GO terms and KEGG pathways showed enrichment of transcripts belonging to axon guidance and tumor innervation in HPV-positive exosomes. Among top 20 overexpressed transcripts, EVC2, LUZP1 and ANKS1B were the most notable due to their involvement in Hh signaling, cellular migration and invasion, respectively. Further, low levels of HPV-specific reads were detected. RT-PCR validation revealed presence of E6*I splice variant of HPV18 in exosomal RNA of HeLa cells. The E6*I transcripts were consistently retained in exosomes obtained from HeLa cells undergoing 5-FU and cisplatin-induced oxidative stress. CONCLUSION: Our data suggests the enrichment of poly-A RNA transcripts in the exosomal cargo of cervical cancer cells, which includes pro-tumorigenic cellular RNA and viral transcripts such as HPV E6, which may have clinical utility as potential exosomal biomarkers of cervical cancer.

New insights into Epstein­Barr virus­associated tumors: Exosomes (Review).

Epstein­Barr virus (EBV) is endemic worldwide and is associated with a number of human tumors. EBV­associated tumors have unique mechanisms of tumorigenesis. EBV encodes multiple oncogenic molecules that can be loaded into exosomes released by EBV+ tumor cells to mediate intercellular communication. Moreover, different EBV+ tumor cells secrete exosomes that act on various target cells with various biological functions. In addition to oncogenicity, EBV+ exosomes have potential immunosuppressive effects. Investigating EBV+ exosomes could identify the role of EBV in tumorigenesis and progression. The present review summarized advances in studies focusing on exosomes and the functions of EBV+ exosomes derived from different EBV­associated tumors. EBV+ exosomes are expected to become a new biomarker for disease diagnosis and prognosis. Therefore, exosome­targeted therapy displays potential.

Ubiquitinated Hepatitis D Antigen-Loaded Microvesicles Induce a Potent Specific Cellular Immune Response to Inhibit HDV Replication in Vivo.

Hepatitis D is the most severe form of human viral hepatitis and currently lacks an efficient therapy. Dendritic cell-derived exosomes (Dexs) have been found to induce immune responses capable of eliminating viruses. However, the therapeutic potential of antigen-loaded exosomes in hepatitis D is still unknown. Recently, we designed exosomes loaded with ubiquitinated hepatitis delta virus (HDV) small delta antigen (Ub-S-HDAg) and then treated mice bearing replicating HDV with these exosomes to explore their antiviral effect and mechanism. Mature dendritic cell-derived exosomes (mDexs) were loaded with Ub-S-HDAg and their antivirus function was evaluated in mice with HDV viremia. Furthermore, the proportion of CD8+ cells, the ratio of Th1/Th2 cells, the postimmunization levels of cytokines were explored, and the Janus kinases (JAK)/signal transducer and activator of transcription (STAT) pathway was evaluated with a JAK2 inhibitor AG490. In Ub-S-HDAg-Dexs group, the HDV RNA viral load was significantly decreased compared with other groups by CD8+ cell enrichment and an increase Th1/Th2 cell ratio. Furthermore, lymphocyte infiltration was increased, while the HDAg level was decreased in mouse liver tissue. However, there were no significant differences in HBV surface antigen (HBsAg), alanine aminotransferase (ALT), or aspartate aminotransferase (AST) levels among the groups. Moreover, p-JAK2, p-STAT1, p-STAT4, STAT1, and STAT4 expression was increased in Ub-S-HDAg-Dexs group. In conclusion, Ub-S-HDAg-Dexs might be a potential immunotherapeutic agent for eradicating HDV by inducing specific cellular immune response via the JAK/STAT pathway. IMPORTANCE Hepatitis D is the most severe viral hepatitis with accelerating the process of liver cirrhosis and increasing the risk of hepatocellular carcinoma. However, there are no effective antiviral drugs. Exosomes derived from mature dendritic cells are used not only as immunomodulators, but also as biological carriers to deliver antigens to induce robust immune response. Based on these properties, exosomes could be used as a biological immunotherapy by enhancing adaptive immune response to inhibit hepatitis D virus replication. Our research may provide a new therapeutic strategy to eradicate HDV in the future.

Semen-Derived Exosomes Mediate Immune Escape and Transmission of Reticuloendotheliosis Virus.

Reticuloendotheliosis virus (REV) causes immune-suppression disease in poultry, leading to a significant economic burden worldwide. Recent evidence demonstrated that the REV can enter the semen and then induce artificial insemination, but how the virus gets into semen was little known. Accumulating studies indicated that exosomes serve as vehicles for virus transmission, but the role of exosomes in viral shedding through the semen remains unclear. In this study, exosomes purified from the REV-positive semen were shown with reverse transcription-PCR and mass spectrometry to contain viral genomic RNA and viral proteins, which could also establish productive infections both in vivo and in vitro and escape from the REV-specific neutralizing antibodies. More importantly, compared with the infection caused by free virions, the exosome is more efficient for the virus to ensure effective infection and replication, which can also help the REV compromise the efficacy of the host immune response. In summary, this study demonstrated that semen-derived exosomes can medicate the transmission and immune escape of REV, implicating a novel mechanism for REV entering the semen and leading to vertical transmission.

Decreased inhibition of exosomal miRNAs on SARS-CoV-2 replication underlies poor outcomes in elderly people and diabetic patients.

Elderly people and patients with comorbidities are at higher risk of COVID-19 infection, resulting in severe complications and high mortality. However, the underlying mechanisms are unclear. In this study, we investigate whether miRNAs in serum exosomes can exert antiviral functions and affect the response to COVID-19 in the elderly and people with diabetes. First, we identified four miRNAs (miR-7-5p, miR-24-3p, miR-145-5p and miR-223-3p) through high-throughput sequencing and quantitative real-time PCR analysis, that are remarkably decreased in the elderly and diabetic groups. We further demonstrated that these miRNAs, either in the exosome or in the free form, can directly inhibit S protein expression and SARS-CoV-2 replication. Serum exosomes from young people can inhibit SARS-CoV-2 replication and S protein expression, while the inhibitory effect is markedly decreased in the elderly and diabetic patients. Moreover, three out of the four circulating miRNAs are significantly increased in the serum of healthy volunteers after 8-weeks' continuous physical exercise. Serum exosomes isolated from these volunteers also showed stronger inhibitory effects on S protein expression and SARS-CoV-2 replication. Our study demonstrates for the first time that circulating exosomal miRNAs can directly inhibit SARS-CoV-2 replication and may provide a possible explanation for the difference in response to COVID-19 between young people and the elderly or people with comorbidities.

Proteomic Alterations in Salivary Exosomes Derived from Human Papillomavirus-Driven Oropharyngeal Cancer.

BACKGROUND: Increasing evidence supports the notion that human papillomavirus (HPV) DNA integration onto the human genome can influence and alter the molecular cargo in the exosomes derived from head and neck cancer cells. However, the molecular cargo of salivary exosomes derived from HPV-driven oropharyngeal cancer (HPV-driven OPC) remains unelucidated. METHODS AND MATERIALS: Salivary exosomes morphology and molecular characterizations were examined using the nanoparticle tracking (NTA), western blot analysis, transmission electron microscopy (TEM) and mass spectrometry analysis. RESULTS: We report that HPV16 DNA was detected (80%) in isolated salivary exosomes of HPV-driven OPC patients. Importantly, we demonstrate elevated protein levels of six main glycolytic enzymes [i.e., aldolase (ALDOA), glyceraldehye-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase A/B (LDHA and LDHB), phosphoglycerate kinase 1 (PGK1) and pyruvate kinase M1/2 (PKM)] in isolated salivary exosomes of HPV-driven OPC patients, suggesting a novel mechanism underlying the potential role of salivary exosomes in mediating the reciprocal interplay between glucose metabolism and HPV-driven OPC. CONCLUSION: Our data demonstrate the potential diagnostic value of HPV16 DNA and glycolytic enzymes in salivary exosomes in discriminating healthy controls from HPV-driven OPC patients, thereby opening new avenues in the future for clinical translation studies.

The interferon-stimulated exosomal hACE2 potently inhibits SARS-CoV-2 replication through competitively blocking the virus entry.

Since the outbreak of coronavirus disease 2019 (COVID-19), it has become a global pandemic. The spike (S) protein of etiologic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specifically recognizes human angiotensin-converting enzyme 2 (hACE2) as its receptor, which is recently identified as an interferon (IFN)-stimulated gene. Here, we find that hACE2 exists on the surface of exosomes released by different cell types, and the expression of exosomal hACE2 is increased by IFNα/ß treatment. In particular, exosomal hACE2 can specifically block the cell entry of SARS-CoV-2, subsequently inhibit the replication of SARS-CoV-2 in vitro and ex vivo. Our findings have indicated that IFN is able to upregulate a viral receptor on the exosomes which competitively block the virus entry, exhibiting a potential antiviral strategy.

Proteomic analysis of the exosomes secreted from Ctenopharyngodon idellus kidney cells infected with grass carp reovirus reveals their involvement in the cellular responses to viral infection.

Exosomes are small membrane-enclosed vesicles secreted by various types of cells. Exosomes not only participate in different physiological processes in cells, but also involve in the cellular responses to viral infection. Grass carp reovirus (GCRV) is a non-enveloped virus with segmented, double-stranded RNA genome. Nowadays, the exact role of exosomes in regulating the life cycle of GCRV infection is still unclear. In this study, the exosomes secreted from Ctenopharyngodon idellus kidney (CIK) cells infected or uninfected with GCRV were isolated, and the differential protein expression profiles were analyzed by proteomic technologies. A total of 1297 proteins were identified in the isolated exosomes. The differentially abundant proteins were further analyzed with functional categories, and numerous important pathways were regulated by exosomes in GCRV-infected CIK cells. These exosomal proteins were estimated to interact with the genes (proteins) of the top 10 most enriched signaling pathways. Furthermore, GW4869 exosome inhibitor suppressed the expression level of VP7 in GCRV-infected cells, suggesting that exosomes play a crucial role in the life cycle of GCRV infection. These findings could shed new lights on understanding the functional roles of exosomes in the cellular responses to GCRV infection.

Proteomic and phospholipidomic characterization of extracellular vesicles inducing tumor microenvironment in Epstein-Barr virus-associated lymphomas.

Epstein-Barr virus (EBV) causes malignant carcinomas including B cell lymphomas accompanied by the systemic inflammation. Previously, we observed that phosphatidylserine (PS)-exposing subset of extracellular vesicles (EVs) secreted from an EBV strain Akata-transformed lymphoma (Akata EVs) convert surrounding phagocytes into tumor-associated macrophages (TAMs) via induction of inflammatory response, which is in part mediated by EBV-derived micro RNAs. However, it is still unclear about EV-carried other potential inflammatory factors associated with TAM formation in EBV lymphomas. To this end, we sought to explore proteomic and phospholipidomic profiles of PS-exposing EVs derived from EBV-transformed lymphomas. Mass spectrometric analysis revealed that several immunomodulatory proteins including integrin αLß2 and fibroblast growth factor 2 (FGF2) were highly expressed in PS-exposing Akata EVs compared with another EBV strain B95-8-transformed lymphoma-derived counterparts which significantly lack TAM-inducing ability. Pharmacological inhibition of either integrin αLß2 or FGF2 hampered cytokine induction in monocytic cultured cells elicited by PS-exposing Akata EVs, suggesting the involvement of these proteins in EV-mediated TAM induction in EBV lymphomas. In addition, phospholipids containing precursors of immunomodulatory lipid mediators were also enriched in PS-exposing Akata EVs compared with B95-8 counterparts. Phospholipidomic analysis of fractionated Akata EVs by density gradient centrifugation further demonstrated that PS-exposing Akata EVs might be identical to certain Akata EVs in low density fractions containing exosomes. Therefore, we concluded that a variety of immunomodulatory cargo molecules in a certain EV subtype are presumably conducive to the development of EBV lymphomas.

Syntenin-knock out reduces exosome turnover and viral transduction.

Exosomal transfers represent an important mode of intercellular communication. Syntenin is a small scaffold protein that, when binding ALIX, can direct endocytosed syndecans and syndecan cargo to budding endosomal membranes, supporting the formation of intraluminal vesicles that compose the source of a major class of exosomes. Syntenin, however, can also support the recycling of these same components to the cell surface. Here, by studying mice and cells with syntenin-knock out, we identify syntenin as part of dedicated machinery that integrates both the production and the uptake of secreted vesicles, supporting viral/exosomal exchanges. This study significantly extends the emerging role of heparan sulfate proteoglycans and syntenin as key components for macromolecular cargo internalization into cells.

HCV-Associated Exosomes Upregulate RUNXOR and RUNX1 Expressions to Promote MDSC Expansion and Suppressive Functions through STAT3-miR124 Axis.

RUNX1 overlapping RNA (RUNXOR) is a long non-coding RNA and plays a pivotal role in the differentiation of myeloid cells via targeting runt-related transcription factor 1 (RUNX1). We and others have previously reported that myeloid-derived suppressor cells (MDSCs) expand and inhibit host immune responses during chronic viral infections; however, the mechanisms responsible for MDSC differentiation and suppressive functions, in particular the role of RUNXOR-RUNX1, remain unclear. Here, we demonstrated that RUNXOR and RUNX1 expressions are significantly upregulated and associated with elevated levels of immunosuppressive molecules, such as arginase 1 (Arg1), inducible nitric oxide synthase (iNOS), signal transducer and activator of transcription 3 (STAT3), and reactive oxygen species (ROS) in MDSCs during chronic hepatitis C virus (HCV) infection. Mechanistically, we discovered that HCV-associated exosomes (HCV-Exo) can induce the expressions of RUNXOR and RUNX1, which in turn regulates miR-124 expression via STAT3 signaling, thereby promoting MDSC differentiation and suppressive functions. Importantly, overexpression of RUNXOR in healthy CD33+ myeloid cells promoted differentiation and suppressive functions of MDSCs. Conversely, silencing RUNXOR or RUNX1 expression in HCV-derived CD33+ myeloid cells significantly inhibited their differentiation and expressions of suppressive molecules and improved the function of co-cultured autologous CD4 T cells. Taken together, these results indicate that the RUNXOR-RUNX1-STAT3-miR124 axis enhances the differentiation and suppressive functions of MDSCs and could be a potential target for immunomodulation in conjunction with antiviral therapy during chronic HCV infection.

Exosomes in Hepatitis B Virus Transmission and Related Immune Response.

The chronicity of Hepatitis B virus (HBV) infection relates to both viral factors and host factors. HBV could result in persistent infection and even serious liver disease, including chronic hepatitis B (CHB), cirrhosis and hepatocellular carcinoma (HCC). Although the HBV vaccine can effectively prevent HBV infection, chronic HBV infection still endangers human health and results in a large social burden. Moreover, the mechanisms underlying the HBV-mediated imbalance of the immune response and persistent infection are not fully understood. Exosomes are extracellular vesicles (EVs) 40-160 nm in size that are released from many cells and transfer specific functional RNAs, proteins, lipids and viral components from donor to recipient cells. These exosome nanovesicles are associated with various biological processes, such as cellular homeostasis, immune response and cancer progression. Besides, previous studies on exosomes have shown that they take part in viral pathogenicity due to the similarity in structure and function between exosomes and enveloped viruses. Moreover, exosome as a novel immunomodulatory carrier plays a significant role in viral immunology. In this review, we focus on the latest progress in understanding the role of exosomes in HBV transmission as well as their vital roles in immune regulation during HBV infection. Furthermore, we discuss the potential clinical applications of exosomes in hepatitis B infection, including the use of exosomes in the auxiliary diagnosis and treatment of hepatitis B.

Mathematical modeling of hepatitis C RNA replication, exosome secretion and virus release.

Hepatitis C virus (HCV) causes acute hepatitis C and can lead to life-threatening complications if it becomes chronic. The HCV genome is a single plus strand of RNA. Its intracellular replication is a spatiotemporally coordinated process of RNA translation upon cell infection, RNA synthesis within a replication compartment, and virus particle production. While HCV is mainly transmitted via mature infectious virus particles, it has also been suggested that HCV-infected cells can secrete HCV RNA carrying exosomes that can infect cells in a receptor independent manner. In order to gain insight into these two routes of transmission, we developed a series of intracellular HCV replication models that include HCV RNA secretion and/or virus assembly and release. Fitting our models to in vitro data, in which cells were infected with HCV, suggests that initially most secreted HCV RNA derives from intracellular cytosolic plus-strand RNA, but subsequently secreted HCV RNA derives equally from the cytoplasm and the replication compartments. Furthermore, our model fits to the data suggest that the rate of virus assembly and release is limited by host cell resources. Including the effects of direct acting antivirals in our models, we found that in spite of decreasing intracellular HCV RNA and extracellular virus concentration, low level HCV RNA secretion may continue as long as intracellular RNA is available. This may possibly explain the presence of detectable levels of plasma HCV RNA at the end of treatment even in patients that ultimately attain a sustained virologic response.

DEFA1B inhibits ZIKV replication and retards cell cycle progression through interaction with ORC1.

AIMS: Zika virus (ZIKV) infection causes a public health concern because of its potential association with the development of microcephaly. During viral infections, the host innate immune response is mounted quickly to produce some endogenous functional molecules to limit virus replication and spread. Exosomes contain molecules from their cell of origin following virus infection and can enter recipient cells for intercellular communication. Here, we aim to clarify whether ZIKV-induced exosomes can regulate viral pathogenicity by transferring specific RNAs. MAIN METHODS: In this study, exosomes were isolated from the supernatants of A549 cells with or without ZIKV infection. Human transcriptome array (HTA) was performed to analyze the profiling of RNAs wrapped in exosomes. Then qPCR, western blotting and ELISA were used to determine ZIKV replication. CCK-8 and flow cytometry were used to test the cell proliferation and cell cycles. Co-culture assay was used to analyze the effect of exosomes on the cell cycles of recipient cells. KEY FINDINGS: Through human transcriptome array (HTA) we found the defensin alpha 1B (DEFA1B) expression was significantly increased within exosomes isolated from ZIKV infected A549 cells. Additionally, we found that the extracellular DEFA1B exerts significant anti-ZIKV activity, mainly before ZIKV entering host cells. Interestingly, up-regulated DEFA1B retards the cell cycle of host cells. Further studies demonstrated that DEFA1B interacted with the origin recognition complex 1 (ORC1) which is required to initiate DNA replication during the cell cycle and increased DEFA1B expression decreased the ORC1 level in the cell nuclei. Accordingly, DEFA1B-containing exosomes can be internalized by the recipient cells to retard their cell cycles. SIGNIFICANCE: Together, our results demonstrated that the anti-ZIKV activity of DEFA1B can be mediated by exosomes, and DEFA1B interacts with ORC1 to retard cell cycles. Our study provides a novel concept that DEFA1B not only acts as an antiviral molecule during ZIKV infection but also may correlate with cell proliferation by retarding the progression of cell cycles.

Reticulon-3 modulates the incorporation of replication competent hepatitis C virus molecules for release inside infectious exosomes.

BACKGROUND: Cell released microvesicles specifically, exosomes, play an important role in mediating immunologic escape, treatment resistance, and disease persistence of Hepatitis C virus (HCV) infection. Reports on the molecular compositions of exosomes released by cells under diverse conditions, especially during viral infections, suggest that their cargo contents are not randomly loaded. However, the precise molecular mechanisms directing the selective cargo sorting and loading inside infectious viral exosomes remains elusive. AIM: To decipher the role of Reticulon 3 (RTN3) in the selective molecular cargo sorting and loading inside infectious viral exosomes during HCV infection. METHODS: We used Huh7 cells-JFH1 HCV infection and HCV Full-Length (FL) replicon systems. Additionally, we analyzed human liver and serum exosome samples from healthy and treatment naïve HCV infected individuals. Our experiments made use of molecular biology and immunology techniques. RESULTS: HCV infection (JFH1-Huh7 or HCV-FL replicon cells) was associated with increased RTN3L&S isoforms expression in cells and cell released exosomes. Accordingly, increased expression of RTN3L&S was observed in liver and serum exosome samples of HCV infected individuals compared to healthy controls. RNA-ChIP analysis revealed that RTN3L&S interacted with dsHCV RNA. Lentiviral CRISPR/Cas9 mediated knockdown (KD) of RTN3 and plasmid overexpression (OE) of wild type, C- and N-terminal deletion mutants of RTN3L&S in HCV- infected Huh7 cells differentially impacted the cellular release of infectious viral exosomes. RTN3L&S KD significantly decreased, while RTN3S OE significantly increased the number of Huh7 cell-released infectious exosomes. The C-terminal domain of RTN3 interacted with and modulated the loading of dsHCV RNA inside infectious exosomes. Antiviral treatment of HCV infected Huh7 cells reduced virus-induced RTN3L&S expression and attenuated the release of infectious exosomes. CONCLUSION: RTN3 constitutes a novel regulator and a potential therapeutic target that mediates the specific loading of infectious viral exosomes.

Intercellular transmission of Seneca Valley virus mediated by exosomes.

Seneca Valley virus (SVV) is a non-encapsulated single-stranded positive-strand RNA virus whose transmission routes have not yet been fully elucidated. Exosomes have been implicated in the intercellular transport of a variety of materials, such as proteins, RNA, and liposomes. However, whether exosomes can mediate SVV intercellular transmission remains unknown. In this study, we extracted exosomes from SVV-infected IBRS-2 cells to investigate intercellular transmission. Our results suggest that the intercellular transmission of SVV is mediated by exosomes. The results of co-localization and RT-qPCR studies showed that exosomes harbor SVV and enable the virus to proliferate in both susceptible and non-susceptible cells. Furthermore, the replication of SVV was inhibited when IBRS-2 cells were treated with interfering RNA Rab27a and exosome inhibitor GW4869. Finally, neutralization experiments were performed to further verify whether the virus was encapsulated by the exosomes that mediated transmission between cells. It was found that exosome-mediated intercellular transmission was not blocked by SVV-specific neutralizing antibodies. This study reveals a new transmission route of SVV and provides clear evidence regarding the pathogenesis of SVV, information which can also be useful for identifying therapeutic interventions.

Extracellular Vesicles in Viral Infections of the Nervous System.

Almost all types of cells release extracellular vesicles (EVs) into the extracellular space. EVs such as exosomes and microvesicles are membrane-bound vesicles ranging in size from 30 to 1000 nm in diameter. Under normal conditions, EVs mediate cell to cell as well as inter-organ communication via the shuttling of their cargoes which include RNA, DNA and proteins. Under pathological conditions, however, the number, size and content of EVs are found to be altered and have been shown to play crucial roles in disease progression. Emerging studies have demonstrated that EVs are involved in many aspects of viral infection-mediated neurodegenerative diseases. In the current review, we will describe the interactions between EV biogenesis and the release of virus particles while also reviewing the role of EVs in various viral infections, such as HIV-1, HTLV, Zika, CMV, EBV, Hepatitis B and C, JCV, and HSV-1. We will also discuss the potential uses of EVs and their cargoes as biomarkers and therapeutic vehicles for viral infections.