ABSTRACT

The earth hath bubbles as the water has…

William Shakespeare, Macbeth

Act I, Scene 3

Cells in vivo and ex vivo release membrane vesicles. These extracellular vesicles (EVs) are 50- to 100-nm-sized lipid bilayer-enclosed entities containing proteins and RNA. Not long ago, EVs were considered to be “cellular dust” or garbage and did not attract much attention. However, it has recently been found that EVs can have important biological functions and that in both structural and functional aspects they resemble viruses. This resemblance becomes even more evident with EVs produced by cells productively infected with viruses. Such EVs contain viral proteins and parts of viral genetic material. In this article, we emphasize the similarity between EVs and viruses, in particular retroviruses. Moreover, we emphasize that in the specific case of virus-infected cells, it is almost impossible to distinguish EVs from (noninfectious) viruses and to separate them.

Let us start with definitions. Although EVs were discovered decades ago, EV research emerged as a separate field relatively recently and currently lacks sufficient practical nomenclature. In full analogy with viral biogenesis, some of these vesicles are generated inside cells and on release into the extracellular milieu are called “exosomes,” whereas others pinch off from the plasma membrane and are generally referred to as “microvesicles” (1). Most commonly, the general term EVs is used to refer to any membrane vesicle of a type that is released into the extracellular space. However, use of this general term not only masks the fact that EVs are highly heterogeneous in size, structure, and biogenesis but may also lead to apparent controversies when different studies deal with different entities but call them by the same name. The diversity of EVs may also underlie the large variety of roles ascribed to them in normal cell function and in pathologies (2).

In contrast to EVs, the definition of viruses developed by 20th century virologists was quite precise: both the Encyclopedia Britannica and the Oxford English Dictionary define virus as “an infectious agent of small size that can multiply only in living cells.” EVs do not fall under this definition, because despite their resemblance to viruses in many aspects, they are fundamentally different, as they do not replicate. However, contemporary virology has distanced itself from this strict definition of virus by its wide use of the terms noninfectious and defective virus. Therefore, EVs generated by retrovirus-infected cells that carry viral proteins and even fragments of viral genomes essentially fall under the definition of noninfectious viruses.

Based on current knowledge, there are many aspects in which EVs resemble viruses, in particular retroviruses. First, although some EVs may be up to a micrometer in size, the majority of EVs are <300 nm, the size of a typical RNA virus. Like enveloped viruses, EVs are surrounded by a lipid membrane that also contains cell membrane proteins. Like many viruses, EVs are formed in the endosomal system or at the plasma membrane via defined biogenesis pathways, for example, involving the endosomal sorting complexes required for transport (ESCRT) machinery (1). Like viruses, EVs can bind to the plasma membranes of other cells, enter them either through fusion or endocytosis, and trigger specific reactions from these recipient cells (1). Finally, EVs carry genetic material, and this genetic material can change functions of the recipient cells (2, 3). Especially in the case of retroviruses, EVs generated in infected cells contain selected molecules of viral origin (4) and can be so similar to noninfectious defective viruses that have lost their ability to replicate that the difference between them becomes blurred. In other cases, EVs provide an “envelope” to nonenveloped viruses, e.g., hepatitis A, and these EV-encapsulated viruses can infect cells (5). Similarly, EV released by hepatitis C-infected cells can carry fully infectious viral genomes that in target cells generate new infectious viral particles (6).

In this Perspective, we suggest that in retrovirus infections a variety of diverse vesicles is released, such that on one extreme there are EVs consisting entirely of host cell components and on the other replication-capable viruses. In between these extremes are nonreplicating particles that can be considered both as defective viruses and as EVs containing various amounts of virus-specific molecules (Fig. 1).

Obviously, unlike true viruses, EVs that contain viral proteins and fragments of viral genomes do not cause outbreaks and epidemics. However, EVs can either directly interact with retroviruses or modulate host cells, thereby affecting the infection. Studies on other virus infections in which EVs were shown to affect antiviral immune responses [e.g., human herpesviruses, in particular Epstein-Barr virus (EBV)] or in which EVs were shown to entrap nonenveloped viruses (like hepatitis A virus and hepatitis E virus) have been reviewed elsewhere (7, 8).

EVS AND VIRUSES CROSS PATHS IN BIOGENESIS

Early discussions on relationships between EVs and viruses (9, 10) were largely based on the fact that both EVs and retroviruses use the cellular vesiculation machinery, which explained striking similarities between EVs and retroviruses in lipid composition (high cholesterol and glycosphingolipids) and protein content (tetraspanins, GPI proteins, and cytoplasmic proteins). Moreover, it was hypothesized that retroviruses exploit preexisting pathways for intracellular vesicle trafficking (The Trojan exosome hypothesis) (9) and could be regarded as “modified or mutated exosomes.” Others disputed the idea, because in contrast to retroviruses, there was little evidence for an active role of EVs in functional modification of target cells via transport of bioactive proteins, lipids, and genetic material (10). Later, it was found that EVs do contain genetic material, mainly in the form of small RNAs (3,11,12). Besides the involvement of molecular mechanisms for sorting of specific proteins into EVs (13), numerous studies also indicate that the RNA content of EVs doesn’t simply reflect the RNA content of the EV-producing cell. Although some RNAs may passively diffuse into EVs in the course of their biogenesis, active sorting of specific RNAs has been shown to depend on defined RNA-binding proteins (14). Moreover, EV-associated miRNAs and mRNAs have been found to be enriched in certain sorting motifs (14–16). Recent scientific breakthroughs have shown that EV-associated proteins, lipids, and genetic material can be functionally transferred to target cells (13, 17–19), strongly implying that EVs and (retro)viruses have in common not only structural but also some functional aspects. This similarity is a reflection of the similarity in biogenesis of EVs and viruses (Fig. 2).

“MISTER POSTMAN”: WHAT DO EVS AND VIRUSES DELIVER

Published data indicate that EVs share with viruses an important function that played a critical role in evolution, namely delivering bioactive material from one cell to another (7, 8, 20, 21). Specific combinations of lipids and proteins, in particular, tetraspanins (22), in the EV membrane can mediate specific targeting of vesicles to recipient cells and may determine the ability of vesicles to fuse with cellular membranes. These molecules, as well as genetic material and proteins enclosed in EVs (e.g., transcription factors and cytokines), constitute molecular signals that can affect the function of recipient cells. It is exactly this trait of being multicomponent transport units that EVs share with enveloped viruses. Below, we discuss further characteristics shared by EVs and viruses.

As suggested from the above, like viral envelope proteins, EV surface proteins can determine adhesion to the plasma membrane of specific target cells. The intercellular adhesion molecule 1 (ICAM 1), present in dendritic cell (DC)-derived EVs, for example, mediates EV recruitment by other DCs and activated T cells (23, 24). Interestingly, the combination of integrin proteins on tumor cell EVs was recently shown to determine their delivery to specific target organs, where these EVs prepare the site of metastasis (25). A number of cellular proteins are incorporated both in EVs and in virions. Tetraspanins, for example, are associated with EVs and have also been reported to be incorporated into retroviruses, in which these host proteins can play a role in infectivity (26). Other EV membrane proteins can act as ligands for receptors on the target cell plasma membrane. MHC class II–peptide complexes on DC-derived EVs, for example, can bind or target T-cell receptors (27). Besides proteins associated with the EV surface, lipids too can mediate signaling to target cells. Examples of EV-associated bioactive lipids include prostaglandin E2, which can play a role in tumor evasion and immune suppression, and lysophosphatidylcholine, which also affects membrane fusion and induces immune cell activation and chemotaxis (28, 29). EV surface proteins and lipids may also determine the ability of vesicles to fuse with cellular membranes, as they do in the case of viruses (30). Fusion of EVs with target cells allows EV-entrapped signaling molecules to exert effects on target cell functioning. These molecules include cytosolic proteins such as transcription factors and also cytokines such as IL-1β that lack an N-terminal signal peptide and that are released via alternative secretion routes (31). Moreover, EVs can carry specific enzymes, such as metalloproteinases and leukotriene-synthesis enzymes (32). Interestingly, DNA polymerase can also be transported by EVs. Whereas early studies reported the association of a DNA polymerase that catalyzed ribonuclease-sensitive DNA synthesis (thus resembling viral reverse transcriptase but not proving its identity) with particulate structures in the cytoplasm (27), more recent data show that tumor EVs can display endogenous reverse transcriptase activity (28). This suggests that under certain conditions, reverse transcriptase can be incorporated into EVs.

Some data indicate that EVs, although less effectively than virions themselves, can transfer cytosolic proteins involved in antiviral responses, such as APOBEC3G and cGAMP (33–36), to recipient cells. However, the relative efficiency of virions and EVs in transferring these proteins may be dependent on cell type and environmental conditions.

In some cases, EVs can also deliver genetic material into target cells. After the initial discovery that EVs carry protein-encoding mRNAs and small noncoding RNAs involved in regulation of gene expression [microRNA (miRNA)] (3), several groups demonstrated alterations in target cell gene expression due to the transfer of such RNAs via EVs (2). Besides miRNAs, EVs also contain a large variety of other small noncoding RNAs, such as fragments of protein-coding regions and repeat sequences, which could also act as regulatory RNAs by influencing gene expression (11). Although the most of genetic material enclosed in virions encodes for viral proteins that are essential for virus replication, viruses and EVs unite in their capacity to transfer RNAs that can trigger pathogen recognition receptors (PRRs) in target cells. Fragments of the viral genome, as well as virus-encoded small RNAs, such as those encoded by EBV, and certain host cell miRNAs, have been shown to trigger target cell PRRs. Although triggering of the PRR system results in complex responses, in some cases it may induce an increased activation status of these cells (37–39). Most of the described EV-mediated effects on the function of other cells are restricted to in vitro systems or occur within the same organism. Whereas viruses transfer between organisms as well as from cell to cell within an organism, the functional transfer of EVs from one individual to the other, as has been suggested for semen- or mother’s milk-derived EVs, has not been proven (12).

MISSION (ALMOST) IMPOSSIBLE: SEPARATION OF VIRIONS FROM EVS

Because EVs are produced by virtually all cells, probably every viral preparation is in fact a mixture of virions and EVs. To study their respective functions, it is necessary to separate EVs and virions. This is very difficult with some viruses, such as retroviruses, because both EVs and retroviruses are comparable in size (EVs ranging from 50 to 100 nm, virions being ∼100 nm) and buoyant density (EVs: 1.13–1.18 g/L; most retroviruses: 1.16–1.18 g/L). Other membrane-derived materials may also have similar characteristics. Therefore, density gradients, which are often used to separate EVs from contaminating protein aggregates on the basis of differences in buoyant densities (40), are not always reliable for separation of EVs from viral particles. Similar technical hurdles were also experienced at the early stages of retrovirus research, when there were long-lasting disagreements and controversies regarding replication-incompetent oncoviral particles causing cancer and their dependence on competent helper viruses for propagation (41). In those early days, electron microscopists observed that ultracentrifuged viruses copelleted with other 100-nm-sized membrane-enclosed particles. In the case of mouse erythroleukemia, pseudorabies, and polio virus these particles were termed “defective interfering particles” (42). Such particles were found to be functionally active, e.g., in repressing virus infection or oncogenic transformation (43), and would nowadays perhaps be classified as “virus-induced EVs.” At this time, it was discovered that these noninfectious viruses could be separated from their infectious counterparts (helper virus) on the basis of their slower migration in density gradients (42). Interestingly, a similar method has more recently been reported for separation of EVs from HIV virions that are produced in HEK 293 T cells or present in the plasma of HIV-1–positive individuals (44, 45). Virus particles and EVs were separated on the basis of migration in velocity gradients and distinguished on the basis of the presence of p24 in virus particles and, for example, acetylcholinesterase and CD45 in EVs but not in HIV. Although it has been reported that in contrast to EVs, HIV particles do not incorporate CD45 (46) or acetylcholinesterase (44), it is not clear if this is universal, and of course, these markers may not be carried by all of EVs. The very criteria for purity of the isolated preparations become murky with the realization that the border between retrovirus virions, like HIV, and EVs is blurry (Fig. 1). This is obviously different in the case of EV-enclosed nonenveloped viruses, such as hepatitis A, which can be distinguished from nonenveloped virions using neutralizing antibodies. This approach cannot be applied to enveloped viruses, because viral envelope proteins to which neutralizing antibodies are formed can be incorporated into EVs. Unless more specifically defined, it is currently virtually impossible to specifically separate and identify EVs that carry viral proteins, host proteins, and viral genomic elements from enveloped viral particles that carry the same molecules. Nevertheless, high-throughput methods to analyze individual nano-sized particles may facilitate discrimination of different particles in the EV–virus continuum in the future. For example, recent developments in flow cytometry-based techniques have opened up the possibility to quantify and characterize particles 50–200 nm in size. Such developments include not only hardware adaptations in high-end flow cytometers to improve signal-to-noise ratios, but also optimized staining protocols for general labeling of EVs and the use of magnetic nanoparticles to screen the surface antigenic composition of EVs (47, 48).

TO BE OR NOT TO BE INFECTED: EVS IN PRO- AND ANTIVIRAL STRATEGIES

In vivo, EVs can interact with viruses and with each other either directly or via modulation of host responses, thus participating in a “War and Peace” between viruses and host (49, 50). Some viruses induce the infected cells to release modified EVs that facilitate infection by increasing the pool of susceptible target cells (e.g., by increasing the number of activated cells) or their susceptibility to viral infection or by serving as decoys that absorb antiviral antibodies, thereby compromising antiviral immunity. In contrast, EVs carrying viral proteins can also be beneficial to the host, for example, by providing dendritic cells with viral antigens to facilitate the initiation of adaptive immune responses. Hypothetically, the capacity of EVs to regulate the lifespan of permissive cells and to modify antiviral immune responses may give additional flexibility to the host in responding to viral infection. Thus, EVs formed during viral infection may play either pro- or counter-viral roles (Fig. 3). It is currently unknown whether the diverse functions ascribed to virus-induced EV may in part be explained by differences in the purity of EV populations used in various studies. A general understanding of parameters that determine the net effect of EVs on viral infections is therefore still lacking.

CONCLUSIONS: PROSPECTS FOR EV THERAPY

A growing body of evidence indicates that cells infected with enveloped or nonenveloped viruses release EVs that contain viral components. Here, we aimed to create awareness that virus preparations may never be pure but rather are contaminated with diverse subpopulations of EVs, and some of these EVs may be either indistinguishable from or very similar to so-called defective viruses. Because of their common biogenesis paths, viruses and EVs may be close relatives, although only the former can replicate in cells. Importantly, EVs generated by infected cells are not neutral, as they can either facilitate virus propagation or enhance the antiviral response. Understanding of the structure of EVs produced by infected cells, determining their cargo, and deciphering the fine mechanisms by which they affect viral infection are required not only for basic virology but also for translation into therapy. Below, we present three examples of potential utilizations of EVs in immunotherapy, vaccine development, and drug delivery:

(i) EVs with viral proteins can serve as decoys for antiviral antibodies by binding them, leaving infectious virions partially undetected. Eliminating these EVs (e.g., with immunoadsorption based on their nonviral markers) may enhance antiviral immune responses. (ii) Understanding the roles of EVs in antiviral immune reactions may guide engineering of EVs that have strong antiviral properties. (iii) Knowledge of phenotypes and functions of EVs generated in response to viral inoculation can in the future be applied to improve virus vaccines by eliminating or adding defined subsets of EVs.

Targeted drug delivery is one of the most important and unresolved problems in pharmacology. By contrast, viruses are highly targeted: in the course of evolution they have acquired high specificity toward their cellular targets by incorporating specific binding proteins. Incorporation of such viral proteins onto the EV membrane may facilitate EV-mediated delivery of drugs to specific cells (63).

However, to achieve these goals several important questions need to be answered regarding the role of EVs in intercellular communication in the steady state and during viral infections:

• i)
  What are the exact mechanisms by which EVs affect viral infection at both cellular and systemic levels?
• ii)
  Can we use new technologies, some of which are described in this report, to obtain viral preparations free of contaminating EVs and, reciprocally, EV preparations produced by infected cells and free of contaminating viruses? Only after we can obtain clean populations, can question # 1 be addressed experimentally.
• iii)
  How can we predict either in vitro or in vivo net biological activity when viruses and EVs are mixed?
• iv)
  Can we obtain EVs with specific (viral) surface proteins to target vesicles to particular cells and organs?
• v)
  Can we efficiently scale up the production of EVs so that we have sufficient quantities to test their in vivo effects and even perform clinical trials in the future?
• vi)
  Can we design and engineer EVs that block newly evolving viruses? Can we, for example, use EVs to block Zika viral infection developing in fetuses or to enhance antiviral activity to new influenza strains?

Answers to these questions will show whether the newly emergent field of extracellular vesicle research will become important for understanding fundamental mechanisms of virus infections and be translated into anti-viral therapeutic strategies.

ACKNOWLEDGMENTS

FOOTNOTES

REFERENCES