Sequences within RNA coding for HIV‐1 Gag p17 are efficiently targeted to exosomes

HIV budding requires the interaction with cell factors involved in the biogenesis of exosomes. This implies the possibility that viral products undergo exosome incorporation. While this has been already described for both Gag and Nef HIV‐1 proteins, no conclusive results on HIV genome have been produced so far. Here, we report that unspliced, but not single or double spliced, HIV‐1 RNA species are incorporated in exosomes. Deletion mutant analysis indicated that the presence of a stretch of sequences within the 5′ end of the Gag p17 open reading frame is sufficient for HIV‐1 RNA exosome incorporation. These sequences were found associating with exosomes also out of the HIV‐1 context, thus indicating that the diversion towards the vesicular compartment can occur without need of additional HIV‐1 sequences. Finally, the incorporation of genomic HIV‐1 RNA in exosomes significantly increased when producer cells express HIV‐1 defective for viral genome packaging. Manipulating infected cells to favour the selective incorporation in exosomes of genomic HIV‐1 RNA might have therapeutic implications.     

Viral miRNAs exploiting the endosomal-exosomal pathway for intercellular cross-talk and immune evasion

The class of persistent gamma-herpesviruses has developed a variety of strategies that exploit host-cell regulatory pathways to ensure a long-lasting, well-balanced infection of their host. However when these pathways are deregulated, an otherwise harmless infection can lead to disease including cancer. We recently demonstrated that the human herpes virus 4 (HHV4) also known as Epstein-Barr virus (EBV), encodes for small regulatory non-coding microRNAs (miRNAs) that can be transferred from an infected cell to uninfected neighboring cells. Upon arrival these miRNAs are functional in the recipient cell, in that they are able to down regulate specific target genes. These secreted miRNAs are transported to recipient cells via small nano-sized vesicles (known as exosomes) that are of endosomal origin, formed as intraluminal vesicles (ILV) inside multivesicular bodies (MVB). One question that needs to be addressed is how viral miRNAs are sorted into these exosomes. Mature miRNAs, including those of viral origin, are loaded into RNA-induced silencing complexes (RISC) for gene silencing via blocking mRNA translation and/or initiating mRNA decay. Recent insights indicate that cytoplasmic RNA granules rich in RISC complexes are closely associated with endosomes. In fact, selective components of RISC, including GW182 and Argonaut proteins, miRNAs and mRNAs are present in exosomes. Thus miRNA function, mRNA stability and exosome-mediated intercellular communication converge at the level of endosomes. Since endosomes can be considered as key intracellular cross-roads that regulate communication of cells with their exterior, including neighboring cells, it is perhaps not surprising that viruses have found means to exploit this pathway to their benefit. Little is known however, how and if (micro) RNA species are specifically sorted into ILVs and what (micro)RNA-binding proteins are involved. Here we discuss recent developments relating to intracellular trafficking and function of miRNA-containing protein complexes that EBV may exploit for promoting or restricting miRNAs sorting into exosomes for intercellular regulatory functions. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.     

Exosomes Released from Rabies Virus-Infected Cells May be Involved in the Infection Process

Exosomes are cell-derived vesicles that are secreted by many eukaryotic cells. It has recently attracted attention as vehicles of intercellular communication. Virus-infected cells release exosomes, which contain viral proteins, RNA, and pathogenic molecules. However, the role of exosomes in virus infection process remains unclear and needs to be further investigated.In this study, we aimed to evaluate the effects of exosomes on rabies virus infection. OptiPrep~(TM) density gradient centrifugation was used to isolate exosomes from rabies virus-infected cell culture supernatants. A rabies virus G protein enzyme-linked immunosorbent assay and acetylcholinesterase activity assays were performed to verify the centrifugation fractions. Exosomes were then characterized using transmission electron microscopy and Western blotting. Our results showed that rabies virus infection increased the release of exosomes. Treatment with GW4869 and si-Rab27 a, two exosomal secretion inhibitors, inhibited exosome release. Furthermore, the inhibitors reduced the levels of extracellular and intracellular viral RNA. These data indicated that exosomes may participate in the viral infection process. Moreover, our results establish a basis for future research into the roles of exosomes in rabies virus infection and as potential targets for developing new antiviral strategies.     

Cell to Cell Signalling via Exosomes Through esRNA

Exosomes are small vesicles of endosomal origin that can be released by many different cells to the microenvironment. Exosomes have been shown to participate in the immune system, by mediating antigen presentation. We have recently shown the presence of both mRNA and microRNA in exosomes, specifically in exosomes derived from mast cells. This RNA can be transferred between one mast cell to another, most likely through fusion of the exosome to the recipient cell membrane. The delivered RNA is functional, as the mRNA can lead to translation of new proteins in a recipient cell. The RNA shuttled between cells via exosomes is called esRNA. We propose that several types of exosomes may exist, and that an additional function of exosomes is to communicate to neighboring cells through delivery of RNA-signals.     

Exosomes Derived from HIV-1-infected Cells Contain Trans-activation Response Element RNA

Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 10⁴–10⁶ copies/ml TAR RNA in exosomes derived from infected culture supernatants and 10³ copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS.     

Cell to Cell Signalling via Exosomes Through esRNA

Exosomes are small vesicles of endosomal origin that can be released by many different cells to the microenvironment. Exosomes have been shown to participate in the immune system, by mediating antigen presentation. We have recently shown the presence of both mRNA and microRNA in exosomes, specifically in exosomes derived from mast cells. This RNA can be transferred between one mast cell to another, most likely through fusion of the exosome to the recipient cell membrane. The delivered RNA is functional, as the mRNA can lead to translation of new proteins in a recipient cell. The RNA shuttled between cells via exosomes is called esRNA. We propose that several types of exosomes may exist, and that an additional function of exosomes is to communicate to neighbouring cells through delivery of RNA-signals.Key words: esRNA, exosomes, microRNA, mRNA, cell communication, signalling     

Identification of an inhibitory budding signal that blocks the release of HIV particles and exosome/microvesicle proteins

Animal cells bud exosomes and microvesicles (EMVs) from endosome and plasma membranes. The combination of higher-order oligomerization and plasma membrane binding is a positive budding signal that targets diverse proteins into EMVs and retrovirus particles. Here we describe an inhibitory budding signal (IBS) from the human immunodeficiency virus (HIV) Gag protein. This IBS was identified in the spacer peptide 2 (SP2) domain of Gag, is activated by C-terminal exposure of SP2, and mediates the severe budding defect of p6-deficient and PTAP-deficient strains of HIV. This IBS also impairs the budding of CD63 and several other viral and nonviral EMV proteins. The IBS does not prevent cargo delivery to the plasma membrane, a major site of EMV and virus budding. However, the IBS does inhibit an interaction between EMV cargo proteins and VPS4B, a component of the endosomal sorting complexes required for transport (ESCRT) machinery. Taken together, these results demonstrate that inhibitory signals can block protein and virus budding, raise the possibility that the ESCRT machinery plays a role in EMV biogenesis, and shed new light on the role of the p6 domain and PTAP motif in the biogenesis of HIV particles.     

Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells

Exosomes are vesicles of endocytic origin released by many cells. These vesicles can mediate communication between cells, facilitating processes such as antigen presentation. Here, we show that exosomes from a mouse and a human mast cell line (MC/9 and HMC-1, respectively), as well as primary bone marrow-derived mouse mast cells, contain RNA. Microarray assessments revealed the presence of mRNA from approximately 1300 genes, many of which are not present in the cytoplasm of the donor cell. In vitro translation proved that the exosome mRNAs were functional. Quality control RNA analysis of total RNA derived from exosomes also revealed presence of small RNAs, including microRNAs. The RNA from mast cell exosomes is transferable to other mouse and human mast cells. After transfer of mouse exosomal RNA to human mast cells, new mouse proteins were found in the recipient cells, indicating that transferred exosomal mRNA can be translated after entering another cell. In summary, we show that exosomes contain both mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location. We propose that this RNA is called "exosomal shuttle RNA" (esRNA).     

Integrating Protein Engineering and Bioorthogonal Click Conjugation for Extracellular Vesicle Modulation and Intracellular Delivery

Exosomes are small, cell-secreted vesicles that transfer proteins and genetic information between cells. This intercellular transmission regulates many physiological and pathological processes. Therefore, exosomes have emerged as novel biomarkers for disease diagnosis and as nanocarriers for drug delivery. Here, we report an easy-to-adapt and highly versatile methodology to modulate exosome composition and conjugate exosomes for intracellular delivery. Our strategy combines the metabolic labeling of newly synthesized proteins or glycan/glycoproteins of exosome-secreting cells with active azides and bioorthogonal click conjugation to modify and functionalize the exosomes. The azide-integrated can be conjugated to a variety of small molecules and proteins and can efficiently deliver conjugates into cells. The metabolic engineering of exosomes diversifies the chemistry of exosomes and expands the functions that can be introduced into exosomes, providing novel, powerful tools to study the roles of exosomes in biology and expand the biomedical potential of exosomes.     

Virion-Independent Transfer of Replication-Competent Hepatitis C Virus RNA between Permissive Cells

In this study, we show that replication-competent subgenomic hepatitis C virus (HCV) RNA can be transferred to permissive Huh7 cells, leading to the establishment of viral RNA replication. Further, we show that these events are mediated by exosomes rather than infectious virus particles. If similar events occur in vivo, this could represent a novel, albeit inefficient, mechanism of viral spread and immune escape.     

Disruption of Type I Interferon Induction by HIV Infection of T Cells

Our main objective of this study was to determine how Human Immunodeficiency Virus (HIV) avoids induction of the antiviral Type I Interferon (IFN) system. To limit viral infection, the innate immune system produces important antiviral cytokines such as the IFN. IFN set up a critical roadblock to virus infection by limiting further replication of a virus. Usually, IFN production is induced by the recognition of viral nucleic acids by innate immune receptors and subsequent downstream signaling. However, the importance of IFN in the defense against viruses has lead most pathogenic viruses to evolve strategies to inhibit host IFN induction or responses allowing for increased pathogenicity and persistence of the virus. While the adaptive immune responses to HIV infection have been extensively studied, less is known about the balance between induction and inhibition of innate immune defenses, including the antiviral IFN response, by HIV infection. Here we show that HIV infection of T cells does not induce significant IFN production even IFN I Interferon production. To explain this paradox, we screened HIV proteins and found that two HIV encoded proteins, Vpu and Nef, strongly antagonize IFN induction, with expression of these proteins leading to loss of expression of the innate immune viral RNA sensing adaptor protein, IPS-1 (IFN-β promoter stimulator-1). We hypothesize that with lower levels of IPS-1 present, infected cells are defective in mounting antiviral responses allowing HIV to replicate without the normal antiviral actions of the host IFN response. Using cell lines as well as primary human derived cells, we show that HIV targeting of IPS-1 is key to limiting IFN induction. These findings describe how HIV infection modulates IFN induction providing insight into the mechanisms by which HIV establishes infection and persistence in a host.     

Role of HIV-1 Gag domains in viral assembly

After entry of the human immunodeficiency virus type 1 (HIV-1) into T cells and the subsequent synthesis of viral products, viral proteins and RNA must somehow find each other in the host cells and assemble on the plasma membrane to form the budding viral particle. In this general review of HIV-1 assembly, we present a brief overview of the HIV life cycle and then discuss assembly of the HIV Gag polyprotein on RNA and membrane substrates from a biochemical perspective. The role of the domains of Gag in targeting to the plasma membrane and the role of the cellular host protein cyclophilin are also reviewed.     

Role of HIV-1 Gag domains in viral assembly

After entry of the human immunodeficiency virus type 1 (HIV-1) into T cells and the subsequent synthesis of viral products, viral proteins and RNA must somehow find each other in the host cells and assemble on the plasma membrane to form the budding viral particle. In this general review of HIV-1 assembly, we present a brief overview of the HIV life cycle and then discuss assembly of the HIV Gag polyprotein on RNA and membrane substrates from a biochemical perspective. The role of the domains of Gag in targeting to the plasma membrane and the role of the cellular host protein cyclophilin are also reviewed.     

Biochemical and virological analysis of the 18-residue C-terminal tail of HIV-1 integrase

Background

Mouse mammary tumor virus (MMTV) encodes the Rem protein, an HIV Rev-like protein that enhances nuclear export of unspliced viral RNA in rodent cells. We have shown that Rem is expressed from a doubly spliced RNA, typical of complex retroviruses. Several recent reports indicate that MMTV can infect human cells, suggesting that MMTV might interact with human retroviruses, such as human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), and human endogenous retrovirus type K (HERV-K). In this report, we test whether the export/regulatory proteins of human complex retroviruses will increase expression from vectors containing the Rem-responsive element (RmRE).

Results

MMTV Rem, HIV Rev, and HTLV Rex proteins, but not HERV-K Rec, enhanced expression from an MMTV-based reporter plasmid in human T cells, and this activity was dependent on the RmRE. No RmRE-dependent reporter gene expression was detectable using Rev, Rex, or Rec in HC11 mouse mammary cells. Cell fractionation and RNA quantitation experiments suggested that the regulatory proteins did not affect RNA stability or nuclear export in the MMTV reporter system. Rem had no demonstrable activity on export elements from HIV, HTLV, or HERV-K. Similar to the Rem-specific activity in rodent cells, the RmRE-dependent functions of Rem, Rev, or Rex in human cells were inhibited by a dominant-negative truncated nucleoporin that acts in the Crm1 pathway of RNA and protein export.

Conclusion

These data argue that many retroviral regulatory proteins recognize similar complex RNA structures, which may depend on the presence of cell-type specific proteins. Retroviral protein activity on the RmRE appears to affect a post-export function of the reporter RNA. Our results provide additional evidence that MMTV is a complex retrovirus with the potential for viral interactions in human cells.     

Multifunctional role of microRNAs in mesenchymal stem cell-derived exosomes in treatment of diseases

Mesenchymal stem cells can be replaced by exosomes for the treatment of inflammatory diseases, injury repair, degenerative diseases, and tumors. Exosomes are small vesicles rich in a variety of nucleic acids [including messenger RNA, Long non-coding RNA, microRNA (miRNA), and circular RNA], proteins, and lipids. Exosomes can be secreted by most cells in the human body and are known to play a key role in the communication of information and material transport between cells. Like exosomes, miRNAs were neglected before their role in various activities of organisms was discovered. Several studies have confirmed that miRNAs play a vital role within exosomes. This review focuses on the specific role of miRNAs in MSC-derived exosomes (MSC-exosomes) and the methods commonly used by researchers to study miRNAs in exosomes. Taken together, miRNAs from MSC-exosomes display immense potential and practical value, both in basic medicine and future clinical applications, in treating several diseases.     

The use of ribozyme gene therapy for the inhibition of HIV replication and its pathogenic sequelae

Human immunodeficiency virus (HIV) is a lentivirus, a separate genus of the Retroviridae which are RNA viruses that integrate as DNA copies into the genomes of host cells and replicate intracellularly through various RNA intermediates. Several of these RNA molecules can be targeted by ribozymes and a number of investigators, including our group, have demonstrated the ability of ribozymes to suppress HIV replication in cultured cells. It is argued that the use of this ribozyme gene therapy approach for the treatment of HIV infection may act as an adjunct to chemotherapeutic drugs and may affect not just viral suppression, but also immune restoration. This approach can be tested in Clinical Trials, several of which are currently under way.