Efficient transduction tools are a hallmark for both research and therapy development. Here, we introduce new insights into the generation of lentiviral vectors with improved performance by utilizing producer cells with increased production rates of extracellular vesicles through CD9 overexpression. Most human cells secrete small vesicles from their surface (microvesicles) or intraluminal endosome-derived membranes (exosomes). In particular, enhanced levels of the tetraspanin CD9 result in significantly increased numbers of extracellular vesicles with exosome-like features that were secreted from four different human cell lines. Intriguingly, exosomes and their biogenesis route display similarities to lentivirus and we examined the impact of CD9 expression on release and infectivity of recombinant lentiviral vectors. Although the titers of released viral particles were not increased upon production in high CD9 cells, we observed improved performance in terms of both speed and efficiency of lentiviral gene delivery into numerous human cell lines, including HEK293, HeLa, SH-SY5Y, as well as B and T lymphocytes. Here, we demonstrate that enhanced CD9 enables lentiviral transduction in the absence of any pseudotyping viral glycoprotein or fusogenic molecule. Our findings indicate an important role of CD9 for lentiviral vector and exosome biogenesis and point out a remarkable function of this tetraspanin in membrane fusion, viral infectivity, and exosome-mediated horizontal information transfer.

These findings enable a better understanding of horizontal cell-to-cell transfer of molecules and information via exosomes and, at the same time, indicate a path for more efficient manufacturing of recombinant LVs.

Recently, extracellular vesicles (EVs), in particular, microvesicles and exosomes, have gained interest as a research objective in horizontal information exchange as biomarkers and in infection biology. In contrast to membrane-shedding microvesicles, exosomes are small (30–100 nm) extracellular vesicles of intraluminal or endosomal origin from intraluminal membranes of many, if not all, cell types.These EVs were shown to be involved in routes of cell-cell communication beyond peptides or small molecules and with both endocrine and paracrine effects and are thus capable of a horizontal transfer of information.After the first description in 1983 by Stahl and Johnstone, a key finding in interpreting the role of exosomes was made in 2007 when Valadi et al. discovered microRNAs (miRNAs) and mRNAs as a cargo of the cell-free vesicles.Besides their utilization as biomarkers in the diagnostic of certain cancers, their potential to deliver functional RNAs implies putative clinical relevance, e.g., in gene therapy.Interestingly, the production of LVs in various adherent growing cell types parallels to some extent the biogenesis route of exosomes.The characterization of exosomes and microvesicles during HIV infection revealed that viral particles are found within fractions of isolated exosomes and microvesicles. A potential interplay of the secretome with LVs is also reflected by the fact that HIV proteins co-localize with exosomal markers on the subcellular level. HIV-infected macrophages displayed increased production rates of extracellular vesicles and were able to infect cells in a CD4-independent manner.Here, we studied relations of the amounts and size distributions of extracellular vesicles and LV production during constitutive expression of three exosomal marker proteins, i.e., CD9, TSG101, and Alix, in various human cell lines to explore the possibilities of increased exosome production. An increased rate of exosome production was expected to impact the outcome of LVs produced under such conditions. Our positively selected candidate CD9 belongs to the tetraspanin superfamily, which contains several differentiation antigens, including CD53, CD63, and CD81.TSG101 and Alix are soluble cytoplasmic components of the endosomal sorting complexes required for transport (ESCRT) machinery, which is required for multivesicular body (MVB) formation and cargo sorting into intraluminal vesicles (ILVs).Tetraspanins are characterized by four transmembrane regions and they expose two extracellular loops.CD9 is involved in cell motility, adhesion, and fusion,and the loss of CD9 expression leads to tumor progression and metastasis in several types of cancer.Furthermore, CD9, CD63, and CD81 are expressed on the mammalian oocyte surface and an essential role of CD9 for sperm egg fusion was identified.CD81 and CD9 play a role in HIV-1 infection that is controversially discussed because the respective functions during late replication and early infection remain elusive. However, a function via the extracellular domains of tetraspanins is very likely to be involved in HIV-1 infection.Also, CD81, CD53, and CD9 have been shown to be present in intraluminal membrane domains during HIV-1 assembly in macrophages and to inhibit direct cell-to-cell transmission of HIV-1.In brief, we observed that the tetraspanin CD9 has a positive influence on the rate of exosome production and release, whereas the exosomal proteins Alix and TSG101 decreased the extracellular vesicle concentration. Therefore, we decided to use the positive effects of CD9 on exosome biogenesis to investigate the potential of LVs generated in high CD9 conditions.

In macrophages, HIV-1 assembles into an intracellular plasma membrane domain containing the tetraspanins CD81, CD9, and CD53.

Recombinant extracellular domains of tetraspanin proteins are potent inhibitors of the infection of macrophages by human immunodeficiency virus type 1.

Progression of cervical carcinomas is associated with down-regulation of CD9 but strong local re-expression at sites of transendothelial invasion.

Expression of the neuroglandular antigen and analogues in melanoma. CD9 expression appears inversely related to metastatic potential of melanoma.

Identification of a highly specific surface marker of T-cell acute lymphoblastic leukemia and neuroblastoma as a new member of the transmembrane 4 superfamily.

In macrophages, HIV-1 assembles into an intracellular plasma membrane domain containing the tetraspanins CD81, CD9, and CD53.

In macrophages, HIV-1 assembles into an intracellular plasma membrane domain containing the tetraspanins CD81, CD9, and CD53.

Exosomes and their role in the life cycle and pathogenesis of RNA viruses.

Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor.

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

During the past two decades, lentiviral vectors (LVs) for gene delivery in research and therapy were improved and optimized with respect to efficiency, cell or tissue targeting, and safety. LVs belong to the retroviridae family and have a diploid, positive strand RNA genome, which is reversely transcribed and integrated into the host genome.The viral genome can integrate in both dividing and non-dividing cells; in particular, HIV-1-based LVs have become a widespread tool for gene delivery applications.The recent third generation of vectors utilizes replication-deficient viruses that only require three HIV-1 genes from the original genome (i.e., gag, pol, and rev).One particularly useful feature of LVs is the possibility of pseudotyping or retargeting, i.e., a manipulation of the exposed viral surface that enables adaptations of the natural tropism by expressing foreign viral glycoproteins on its envelope. One prominent example is the vesicular stomatitis virus glycoprotein (VSVG), which facilitates LV transduction in a wide range of cells from many different vertebrate species. In particular, HIV-1-based LVs with VSVG instead of the original HIV-GP120/GP41 have been used extensively in vitro and in vivo.Immense efforts have successfully been devoted to create LVs with a high selectivity for specific target cells, e.g., by engineering specific ligands, peptides, or single chain antibodies (scFv) that, by co-expression during production of the LV, are integrated into the envelope and in turn enable recognition of the addressed target-cell markers.This, in combination with high loading capacities of genes of interest (GOIs) of up to 10 kbp and options, including integrating or non-integrating LV vectors and the high in vitro and in vivo expression levels, make LVs an attractive tool for research and gene therapy.

Modifications in the binding domain of avian retrovirus envelope protein to redirect the host range of retroviral vectors.

Generation of targeted retroviral vectors by using single-chain variable fragment: an approach to in vivo gene delivery.

Characterization of membrane components of the erythrocyte involved in vesicular stomatitis virus attachment and fusion at acidic pH.

Functional characterization of fractions 8–11 of both viruses revealed that fractions 8 and 11 of LV-VSVG and LV-VSVG-CD9have only minor differences in their low transduction efficiencies. However, fractions 9 and 10 displayed the drastically increased lentiviral infectivity at comparable MOIs for LV-VSVG-CD9 Figure 6 D). These findings implicate that the direct incorporation of transgenic CD9 is directly responsible for increased lentiviral infectivity.

Furthermore, lentiviral supernatants were subjected to density gradient centrifugation in iodixanol (Optiprep) to separate exosomes and virions. As was previously described, exosomes are recovered in fractions 3–6, whereas virions should be collected in fractions 8–11 Figure 6 A). Fractions were analyzed for exosome markers, transgenic CD9, the physical titers of recombinant virus, and its transduction efficiency ( Figure 6 ). The secreted and exosome enriched protein Alixwas detected in western blots of fractions 6–11, with the strongest both in gradients of culture supernatants for VSVG and CD9virus, whereas GFP was detectable in all fractions ( Figure 6 B). qPCR analysis displayed viral genomes of LV-VSVG and LV-VSVG-CD9mainly in fraction 10, with far weaker levels being seen in fractions 8–11, but not in the exosome fractions ( Figure 6 C).

(A) Lentiviral supernatant was fractionated by centrifugation through a gradient of iodixanol to separate lentiviral particles and exosomes. (B) Western blot analysis revealed high concentrations of CD9-GFP and GFP in fraction 6+7 and 10+11, whereas Alix showed an equal distribution, indicating a successful separation of exosomes (fraction 6+7) and lentiviruses (fraction 10+11). (C) qPCR titer measurements clarified the presence of lentiviral particles in fractions 9–11. (D) Comparative analysis of LV-VSVG and LV-VSVG-CD9 with similar MOIs displayed no enhanced infectivity for fractions 8 and 11, whereas in fractions 9 and 11, increased transduction rates were observed for LV-VSVG-CD9 lentiviral constructs on HEK293 cells. Error bars represent SD.

Because positive effects of exosomes on viral transduction are described in the literature,we co-incubated cells with isolated exosomes (CD9and WT exosomes) together with lentiviruses (LV-VSVG-CD9and LV-VSVG) and monitored the lentiviral transduction efficiency. This was performed to investigate the two alternatives of either having envelope-embedded CD9 on the LV or working with mixtures of exosomes and viral particles. LV-VSVG and LV-VSVG-CD9without or in combination with purified exosomes were supplemented to HEK293 and subsequently analyzed with respect to transduction efficiencies after 48 hr. The addition of extracellular vesicles reduced the transduction efficiency for both viruses and the effect was independent of the origin of exosomes, i.e., HEK293 and HEK293-CD9 Table S2 ).

Aiming to evaluate the effects of CD9 on LV production and subsequent fusion events, we utilized LV-CD9(a lentivirus without viral surface glycoproteins), together with a broad range of controls lacking viral core components ( Figures 5 B and S2 B). We observed successful transduction events for LV-CD9 Figure 5 A). The possibility of observing cell line artifacts of CD9 fusion was excluded by testing the CD9virus and relevant controls also on primary cells. We utilized CNS tissue of rats (E18 Wistar) to generate dissociated cortex cultures ( Figure S5 ). All three viruses (physical titer TU = 10viral particles) were added to fresh isolated neuronal rat cells, and transduction events were seen for all the tested virus strains (LV-VSVG, LV-CD9-VSVG, and LV-CD9).

(A) HEK293 cells were transduced with CD9, VSVG, and VSVG-CD9 LVs. All tested LVs show expression after 72 hr. The highest efficiency was observed with LV-VSVG-CD9 GFP , followed by LV-VSVG, but the virus without any viral glycoprotein (LV-CD9 GFP ) also successfully infected a minor proportion of cells. (B) Negative control without viral envelope proteins or CD9 GFP shows no transduction. Scale bars, 100 μm (A) and 200 μm (B). Exposure time: 1,000 ms.

To allocate the LV-driven GFP-reporter expression from the transfer of enveloped CD9, we replicated the experiments with viruses coding for an RFP reporter. No GFP was detectable in fluorescence microscopy analysis after LV-VSVG-CD9treatment ( Figure S2 B). Thus, we can exclude any detectable transfer of CD9protein to the target cell. Overall, LV-VSVG-CD9revealed a faster and more efficient transduction in all tested cell lines ( Figure 4 ). Summarized transduction efficiencies are listed in Tables 1 and S3

Moreover, following the production of LVs in HEK293FT-CD9cells, we noticed a change of CD9localization from the plasma membrane toward cytoplasmic speckles ( Figure S4 ). To examine the re-localization of CD9in HEK293FT-CD9cells during recombinant lentiviruses productions, we selected LV-producing cells by their RFP expression and checked the subcellular localization of the CD9-GFP signal. Cells without RFP expression, i.e., those not actively producing virus, showed in 86.5% CD9membrane location and 13.5% cytoplasmic diffuse localization, whereas 98% of producing cells displayed cytoplasmic or intraluminal localization of the CD9

In brief, LV-VSVG-CD9 GFP showed remarkably higher transduction rates and transgene expression, suggesting that CD9 GFP located in the viral envelope generates more potent recombinant lentivirus without changing the overall production rate of virions.

Flow cytometry analysis of GFP expression revealed an increased number of transduced cells for LV-VSVG-CD9compared to LV-VSVG when comparable amounts of virus are used ( Figures 4 E, 4F, S2 , and S3 ). Relative outcomes of FACS and microscopy analyses are summarized in Table 1

For microscopy analysis, equal amounts of viruses (physical titer of 1*10lentivirus particles according to p24 ELISA titration) were used to infect equal numbers of human HeLa, HEK293, or SH-SY5Y cells. In summary, a significantly higher proportion of GFP-expressing cells were detected upon infection with LV-VSVG-CD9when compared to LV-VSVG ( Figures 4 C and 4D).

Because CD9 showed positive effects on EV secretion, some positive effects of exosomes on viral transduction were described in the literature,and an influence of CD9 in membrane fusion was previously reported,we investigated the impact of increased levels of tetraspanin CD9 on production and performance of recombinant LVs. A constitutively CD9-expressing lentiviral packaging cell line (HEK293FT-CD9) was used to compare virus titers and transduction efficiency with a standard 2-generation lentiviral packaging cell line HEK293FT ( Figure 3 A). We generated three LV variants: (1) a pseudotyped positive control that uses the common VSVG; (2) CD9combined with VSVG; and (3) only with CD9on its envelope ( Figure 3 B). All three LVs transduced DNA coding for either a red fluorescent protein (RFP) or GFP as the reporter gene. Comparisons of the physical and genomic titers of the three pseudotyped viruses were performed as outlined in the Materials and Methods section. No significant differences of titers, i.e., Cap24/mL or genome copies/mL were observed for the viruses ( Figures 4 A and 4B ). Transduction efficiencies of the recombinant LVs and controls lacking either viral capsid proteins (Cap) or viral envelope proteins (Env) were determined by fluorescence microscopy and fluorescence-activated cell sorting (FACS) analysis ( Figures 4 and S2 ).

(A and B) Comparative analysis of three lentiviral productions revealed only minor differences in titer concentrations for LV-VSVG, LV-VSVG-CD9 GFP , and LV-CD9 GFP demonstrated by ELISA (A) and qPCR (B). Comparison of transduction efficiency was evaluated with LV-VSVG as a standard control and LV-VSVG-CD9 GF on HEK293, HeLa, and SH-SY5Y cells. (C) Successful transduction was confirmed via fluorescence microscopy in regular intervals from 20 to 108 hr after transduction. (D) In fluorescence microscopy analysis, positive cells were quantified by manual counting and the transduction efficiency was calculated. (E) Comparing equal amounts of LV-VSVG and LV-VSVG-CD9 revealed an increased efficiency for LV-VSVG-CD9 over a broad MOI range (30–300 MOI). (F) Density plot representation of lentiviral efficiency at equal MOI (150) through FACS analysis. Scale bars, 200 μm. Error bars represent SD.

(A) HEK293FT or HEK293FT-CD9 cells were transfected with three different plasmids encoding for envelope glycoproteins (i.e., CD9 and/or VSVG), viral capsid proteins, and the gene of interest, here, an RFP. LVs are shed from the cell membrane, and LVs produced in HEK293FT-CD9 cells carry additional CD9-GFP and/or VSVG within their envelope. (B) The three different LVs used in this study, i.e., exposing VSVG, CD9, or both VSVG and CD9, are schematically shown.

The fusing ability of sperm is bestowed by CD9-containing vesicles released from eggs in mice.

Fluorescence microscopy analysis of the subcellular CD9localization ( Figure 2 ) revealed that CD9is mainly associated to the plasma membranes of adherent and suspension cells. Also, CD9expression did not cause any visible changes of the cell morphology and no alterations of mortality or growth rates. However, transgene expression of TSG101 and Alix in HEK293 cells was severely cytotoxic (data not shown).

Cell membrane localization of CD9-GFP was observed via fluorescence microscopy. Green fluorescence was detected after at least 2 weeks of Blasticidin selection (scale bar, 100 μm).

Focusing the analyses of secreted vesicles to the exosome size range (30–100 nm), we detected a similar trend of exosome enrichment in the majority of cell lines. HEK293FT showed an >3-fold increase of exosomes, whereas Raji-CD9and Jurkat-CD9doubled their exosome secretion. HeLa cells kept the exosome release constant but also displayed the lowest relative CD9expression rate ( Figures S1 A and S1D; Table S1 ).

The CD9 effects were evaluated in four additional stable CD9cell lines ( Figure S1 ). A 22-fold overexpression of CD9 was recorded in HEK293FT cells (commonly used for the LV production), a much higher expression gain was seen in the human B cell line Raji, with 920-fold CD9expression, and the human T cell line Jurkat reached a 55-fold increase compared to the corresponding wild-type cell line ( Figure S1 A). Although the EV general production was generally lower in suspension cells (Raji and Jurkat), the exogenous expression of CD9 increased the amount of EVs in all the cell types that were tested ( Figure S1 C). In response to overexpression of CD9, the mean diameters of secreted vesicles changed significantly in HEK cell types and HeLa cells. However, in human lymphocyte cell lines, we did not observe significant changes of the average sizes of EVs ( Figure S1 B).

In summary, only the tetraspanin CD9 led to increased exosome production and release, whereas the exosomal ESCRT proteins Alix and TSG101 decrease the extracellular vesicle load. Also, a shift toward enlarged EVs was a result of TSG101 or Alix overexpression, whereas exosome-sized vesicles were enriched upon an increase of cellular CD9 levels. These findings indicate that CD9 empowers an accelerated production and/or release of exosomes, whereas ESCRT components counteract this pathway. Therefore, we solely used CD9 overexpression to investigate the effects on LV generation.

Secreted particles of all cell lines, including the respective parental wild-type cells, were examined by Nanoparticle Tracking Analysis (NTA) and transgene expression in HEK293-CD9resulted in reduced average sizes of EVs. In contrast, HEK293-TSG101 and HEK293-Alix released fewer vesicles, with an increased average size ( Figures 1 B and 1C; Table S1 ). The HEK293-CD9cells produced significantly more extracellular vesicles when compared to HEK293 WT, whereas lower concentrations of secreted vesicles were observed in HEK293-TSG101 and HEK293-Alix ( Figure 1 C). Vesicles in the size range of exosomes (i.e., diameters of 30–100 nm)were detected, with a 2.5-fold increase of HEK293-CD9compared to HEK293 WT, whereas a reduced exosome concentration was seen for HEK293-TSG101 and HEK293-Alix ( Figure 1 D; Table S1 ).

Exosomes and their role in the life cycle and pathogenesis of RNA viruses.

Previous studies reported the positive influence of exosomes on the infectivity of enveloped viruses (e.g., HIV and HSV).To investigate a suitable candidate for evaluating the generation of LVs in high exosome conditions, we selected three functionally diverse exosome marker proteins, i.e., CD9, TSG101, and Alix. This was to explore if an elevated output of exosomes increases the efficiencies of our HIV-1-based LVs. The exosome output was studied in cell lines that were constitutively expressing high levels of the exosomal marker proteins TSG101, Alix, or CD9. We examined their influence on the size distribution and the secretion of vesicles to the medium. The impact of CD9, TSG101, and Alix on the amount and size distribution of EVs was first studied on human HEK293 cells. Stable cell lines expressing CD9, Alix, or TSG101were generated by LVs, and gene expression was monitored by qPCR ( Figure 1 A). All cell lines displayed a significant overexpression of the respective GOI with a 12-fold increase of TSG101, 4-fold for Alix, and a 23-fold increase of the CD9 expression.

(A) qPCR analyses were performed and displayed a 4- to 22-fold higher expression of the respective transgenes. Nanoparticle tracking analysis of extracellular vesicles showed significantly enlarged particles for HEK293-TSG101 and HEK293-Alix compared to wild-type. (B) The overexpression of CD9 led to a decreased average size of secreted vesicles. (C) Total extracellular vesicle amount was decreased upon TSG101 and Alix overexpression, but the extracellular vesicle amount was significantly increased upon CD9 overexpression. (D) For vesicles within the size range of exosomes (30–100 nm), these trends were even more obvious. Error bars represent SD.

Overexpression of TSG101, Alix, or CD9 Alters the Amount and Mean Sizes of EVs

Discussion

52 Bassani S.

Cingolani L.A. Tetraspanins: interactions and interplay with integrins. Tetraspanins are small transmembrane proteins expressed on the surface of various cell types. By their interactions with transmembrane proteins, such as integrins, a role in cell adhesion, mobility, and cellular fusion is anticipated.

We observed a shift toward size-enlarged EVs in response to higher levels of TSG101 and Alix, but increased levels of exosome-sized EVs by upregulating CD9. These observations were confirmed in five human cell lines, i.e., HEK293, HeLa, SH-SY5Y neuroblastoma, B cells (Raji), and T cells (Jurkat). We report a drastically improved infectivity of LVs from producer cells (HEK293FT) with enhanced levels of CD9. The high-CD9 recombinant LVs were faster and more efficiently delivered their genetic cargos into all human cell types that were tested. Finally, we demonstrated that the presence of CD9 on the virus surface is sufficient to enable transduction of target cells in the absence of fusogenic molecules like VSVG. CD9-enriched LVs were capable of transducing human cell lines and also primary cells from a CNS specimen of rodent origin (rat).

In this study, we evaluated the influence of the tetraspanin CD9 on the production and efficiency of recombinant LVs in a manufactured producer cell line with an elevated CD9 level (HEK293FT-CD9 GFP ). As was expected from previous findings, we observed increased efficiencies of recombinant LVs when they were generated in CD9-enriched producer cells. The increase of efficiency was demonstrated in terms of the number of transduced cells, transgene expression, and onset of the expression. No substantial changes of lentiviral particle titers were observed, as was determined by p24 ELISA and qPCR of viral genomic copies.

GFP virus that delivered its genomic content into cells in the absence of any known receptor interaction and additional fusogen. Even though the proportion of transduced cells was minor, this observation indicated a direct contribution of CD9 to membrane fusion between the LV envelope and the target cell membrane. CD9-driven LV infections were confirmed in vitro on HEK293 cells as well as ex vivo with primary rat CNS tissue. Our data support findings of previous studies of Miyado et al., 38 Miyado K.

Yamada G.

Yamada S.

Hasuwa H.

Nakamura Y.

Ryu F.

Suzuki K.

Kosai K.

Inoue K.

Ogura A.

et al. Requirement of CD9 on the egg plasma membrane for fertilization. Unexpectedly, we detected transduction-competent LV-CD9virus that delivered its genomic content into cells in the absence of any known receptor interaction and additional fusogen. Even though the proportion of transduced cells was minor, this observation indicated a direct contribution of CD9 to membrane fusion between the LV envelope and the target cell membrane. CD9-driven LV infections were confirmed in vitro on HEK293 cells as well as ex vivo with primary rat CNS tissue. Our data support findings of previous studies of Miyado et al.,in which CD9 fusing capabilities facilitate sperm and egg fusion in vitro.

41 Krementsov D.N.

Weng J.

Lambelé M.

Roy N.H.

Thali M. Tetraspanins regulate cell-to-cell transmission of HIV-1. 53 Monk P.N.

Partridge L.J. Tetraspanins: gateways for infection. 40 Tippett E.

Cameron P.U.

Marsh M.

Crowe S.M. Characterization of tetraspanins CD9, CD53, CD63, and CD81 in monocytes and macrophages in HIV-1 infection. However, information about the influence of CD9 on viral infectivity is controversial. For example, Krementsow et al.discarded effects of CD9 overexpression on HIV-I release, whereas others give supporting evidence on tetraspanins as facilitators in uptake, trafficking, and spread of viruses.Importantly, the extracellular domains of tetraspanin (CD9, CD53, CD63, and CD81) are involved in HIV-1 infection.

54 Meckes Jr., D.G.

Raab-Traub N. Microvesicles and viral infection. , 55 Gould S.J.

Booth A.M.

Hildreth J.E. The Trojan exosome hypothesis. 27 Chahar H.S.

Bao X.

Casola A. Exosomes and their role in the life cycle and pathogenesis of RNA viruses. 56 Hudry E.

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Fitzpatrick Z.

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et al. Exosome-associated AAV vector as a robust and convenient neuroscience tool. , 57 Kadiu I.

Narayanasamy P.

Dash P.K.

Zhang W.

Gendelman H.E. Biochemical and biologic characterization of exosomes and microvesicles as facilitators of HIV-1 infection in macrophages. As mentioned earlier, CD9 is a well-known marker protein for extracellular vesicles and in particular for exosomes. The impact of extracellular vesicles (e.g., exosomes) on virus production and infection was discussed during the past decade and some evidence for a positive influence was postulated.However, the exact mechanism remains elusive and not all pathways and factors have been described in too much detail. For example, examinations of HIV infection suggested that EVs enhance both the infectivity and replication rates.Contributions of intraluminal vesicles and, in particular, exosomes to viral pathogenesis are currently unraveling and it has been convincingly demonstrated that a very active cellular vesiculation machinery is an enhancer for virus transduction, with positive effects on virus efficiency and stability.

In this study, we manipulated some key factors of the vesiculation machinery by constitutively overexpressing three exosomal marker proteins (i.e., CD9, TSG101, and Alix) and both qualitatively and quantitatively investigated the influence on the generation of extracellular vesicles.

58 Charrin S.

Jouannet S.

Boucheix C.

Rubinstein E. Tetraspanins at a glance. , 59 Andreu Z.

Yáñez-Mó M. Tetraspanins in extracellular vesicle formation and function. GFP expression for five different human cell lines, indicating a rather general and cell-type independent mode of action. Only overexpression of CD9 increased the overall level of microvesicles and, even more importantly, of exosomes as we repeatedly observed in NTA. This finding supported the previously proposed contribution of certain tetraspanins to intracellular vesicle biogenesis.Both increased levels of extracellular vesicles and the shift toward a higher proportion of exosomes were consistent for CD9expression for five different human cell lines, indicating a rather general and cell-type independent mode of action.

58 Charrin S.

Jouannet S.

Boucheix C.

Rubinstein E. Tetraspanins at a glance. , 59 Andreu Z.

Yáñez-Mó M. Tetraspanins in extracellular vesicle formation and function. The direct connection between CD9 overexpression and the production of extracellular vesicles was not described so far, although a general role of tetraspanins in microdomain formation and the assembly of ILVs has been discussed.

60 Wagner K.U.

Krempler A.

Qi Y.

Park K.

Henry M.D.

Triplett A.A.

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Rucker III, E.B.

Hennighausen L. Tsg101 is essential for cell growth, proliferation, and cell survival of embryonic and adult tissues. , 61 Oh K.B.

Stanton M.J.

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Wagner K.U. Tsg101 is upregulated in a subset of invasive human breast cancers and its targeted overexpression in transgenic mice reveals weak oncogenic properties for mammary cancer initiation. 62 Trioulier Y.

Torch S.

Blot B.

Cristina N.

Chatellard-Causse C.

Verna J.M.

Sadoul R. Alix, a protein regulating endosomal trafficking, is involved in neuronal death. 63 Dores M.R.

Paing M.M.

Lin H.

Montagne W.A.

Marchese A.

Trejo J. AP-3 regulates PAR1 ubiquitin-independent MVB/lysosomal sorting via an ALIX-mediated pathway. , 64 Metcalf D.

Isaacs A.M. The role of ESCRT proteins in fusion events involving lysosomes, endosomes and autophagosomes. , 65 Raiborg C.

Stenmark H. The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins. 66 Yi X.

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Abou-Samra A.B.

et al. Alix (AIP1) is a vasopressin receptor (V2R)-interacting protein that increases lysosomal degradation of the V2R. Contrasting the CD9 findings, TSG101 and Alix, two factors responsible for trafficking of the ESCRT machinery, led to a decreased amount of extracellular vesicles upon constitutive overexpression. In particular, exosomes were affected by increased expression of these marker proteins. TSG101 and Alix overexpression decreased the total vesicle amount and increased the average size of extracellular vesicles. However, in addition, we also observed toxic effects of high TSG101 levels. Its multiple roles in the regulation of cell growth, proliferation, and cell survival were previously reported, also providing some mechanistic insights to cytotoxic effects following deregulation.Increased levels of Alix were linked to neuronal cell death by activation of caspases, leading to apoptosis.Both Alix and TSG101 are components of the ESCRT machinery and are responsible for cargo sorting and decomposition of ubiquitin-tagged proteins,and increased lysosomal degradation of specific proteins were seen in response to overexpression.We think that the balance of cargo breakdown in lysosomes and cargo release via exosomes is shifted toward the lysosome pathway in the presence of high levels of these ESCRT proteins. However, opposite effects were observed for CD9. This well-known exosome marker protein is highly enriched in cell-free vesicles and was not described in the context of protein degradation.

47 Dettenhofer M.

Yu X.F. Highly purified human immunodeficiency virus type 1 reveals a virtual absence of Vif in virions. , 48 Cantin R.

Diou J.

Bélanger D.

Tremblay A.M.

Gilbert C. Discrimination between exosomes and HIV-1: purification of both vesicles from cell-free supernatants. GFP ) in all fractions, whereas we observed two intensity peaks around 12.0%–13.2% and 16.8%–18.0% iodixanol for both exosomal markers. These two peaks reflect the separation between virus and the exosome described previously. 48 Cantin R.

Diou J.

Bélanger D.

Tremblay A.M.

Gilbert C. Discrimination between exosomes and HIV-1: purification of both vesicles from cell-free supernatants. According to Cantin et al.,exosomes can be separated from LV particles by density gradient centrifugation, whereas exosomes accumulate among 8.4%–12.0% of iodixanol while LV particles are enriched at an iodixanol concentration between 14.4% and 16.8%. By qPCR and infection experiments, we confirmed that our gradient contains LVs between 14.4% and 18.0% iodixanol. We detected exosomal marker proteins (Alix and CD9) in all fractions, whereas we observed two intensity peaks around 12.0%–13.2% and 16.8%–18.0% iodixanol for both exosomal markers. These two peaks reflect the separation between virus and the exosome described previously.

To exclude an interference of extracellular vesicles on viral transduction events and strengthen the direct role of CD9, we tested the influence of extracellular vesicles on viral transduction efficiencies. External added exosomes (CD9 and WT), together with lentiviral particles, lead to a reduction of transduction events of LV-VSVG and LV-VSVG-CD9. These findings exclude positive effects of exosomes on LV efficiency, suggesting a direct effect of CD9 on viral transduction.

GFP upon virus production 24 hr after transfection with lentiviral plasmids, which is congruent with Deneka et al., 28 Deneka M.

Pelchen-Matthews A.

Byland R.

Ruiz-Mateos E.

Marsh M. In macrophages, HIV-1 assembles into an intracellular plasma membrane domain containing the tetraspanins CD81, CD9, and CD53. Moreover, we already observed a cytoplasmic re-localization of CD9upon virus production 24 hr after transfection with lentiviral plasmids, which is congruent with Deneka et al.,who observed HIV-1 assembly in intracellular vesicular structures containing tetraspanins like CD53, CD81, and CD9.

GFP exogenous expression increases the production of exosomes and that HIV-1 lentiviral-based vector produced on HEK293FT-CD9 GFP is more efficient compared to vectors derived from HEK293FT. Furthermore, we show that this effect is probably the results of three cooperative mechanisms: (1) GFP lentiviral particles. CD9-mediated membrane fusion was shown by Miyado and colleagues, 46 Miyado K. Yoshida K. Yamagata K. Sakakibara K. Okabe M. Wang X. Miyamoto K. Akutsu H. Kondo T. Takahashi Y. et al. The fusing ability of sperm is bestowed by CD9-containing vesicles released from eggs in mice. The interaction of CD9 with the target cell membrane was proven by transduction-competent LV-CD9lentiviral particles. CD9-mediated membrane fusion was shown by Miyado and colleagues,and our findings for CD9-driven transduction by recombinant LVs provide new insights into this subject.

(2) 34 Hemler M.E. Tetraspanin functions and associated microdomains. Because externally added exosomes decreased and did not increase the transduction efficiency of lentiviral particles, we propose a stabilizing effect of CD9 on extracellular membranes (e.g., exosomes and lentiviral particles). Tetraspanin tend to form microdomains,which could increase membrane stability and thereby escalate stability and transduction capacities of LV vectors.

(3) GFP interacts with viral components during virion formation on intraluminal membranes, which is reflected by CD9 relocation from the plasma membrane to the cytoplasm and internal membranes. This is supported by previous findings that HIV-1 assembly in macrophages occurred on intraluminal membrane domains containing CD9, 28 Deneka M. Pelchen-Matthews A. Byland R. Ruiz-Mateos E. Marsh M. In macrophages, HIV-1 assembles into an intracellular plasma membrane domain containing the tetraspanins CD81, CD9, and CD53. The CD9interacts with viral components during virion formation on intraluminal membranes, which is reflected by CD9 relocation from the plasma membrane to the cytoplasm and internal membranes. This is supported by previous findings that HIV-1 assembly in macrophages occurred on intraluminal membrane domains containing CD9,which matches our observations.

In brief, our results support that CD9exogenous expression increases the production of exosomes and that HIV-1 lentiviral-based vector produced on HEK293FT-CD9is more efficient compared to vectors derived from HEK293FT. Furthermore, we show that this effect is probably the results of three cooperative mechanisms:

Our findings provide a novel and, in part, unexpected insight into the influence of tetraspanins on virus production and performance and open new prospects of using this protein family in the context of future therapeutic interventions with LV-based delivery strategies.