Extracellular vesicles and viruses: Are they close relatives?

Esther Nolte-‘t Hoen^a, Tom Cremer^a, Robert C. Gallo^b,1, and Leonid B. Margolis^c 

Edited by Peter K. Vogt, The Scripps Research Institute, La Jolla, CA, and approved June 27, 2016 (received for review April 4, 2016) 

Extracellular vesicles (EVs) released by various cells are small phospholipid membrane-enclosed entities that can carry miRNA. They are now central to research in many fields of biology because they seem to constitute a new system of cell–cell communication. Physical and chemical characteristics of many EVs, as well as their biogenesis pathways, resemble those of retroviruses. Moreover, EVs generated by virus-infected cells can incorporate viral proteins and fragments of viral RNA, being thus indistinguishable from defective (noninfectious) retroviruses. EVs, depending on the proteins and genetic material incorporated in them, play a significant role in viral infection, both facilitating and suppressing it. Deciphering the mechanisms of EV-cell interactions may facilitate the design of EVs that inhibit viral infection and can be used as vehicles for targeted drug delivery. 

extracellular vesicles | exosomes | viruses | defective viruses | infection 

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 simi larity 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 biogen esis 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 Dictio nary 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 dis tanced itself from this strict definition of virus by its wide use of the terms noninfectious and defective virus. There fore, EVs generated by retrovirus-infected cells that carry viral proteins and even fragments of viral genomes essen tially fall under the definition of noninfectious viruses. 

Based on current knowledge, there are many aspects in which EVs resemble viruses, in particular

^aDepartment of Biochemistry and Cell Biology, Faculty Veterinary Medicine, Utrecht University, 3584 CM Utrecht, The Netherlands; ^bInstitute of Human Virology, University of Maryland, Baltimore, MD 21201; and ^cSection of Intercellular Interactions, Eunice Kennedy-Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 

Author contributions: E.N.-t.H., T.C., R.C.G., and L.B.M. analyzed data and wrote the paper. 

The authors declare no conflict of interest. 

This article is a PNAS Direct Submission. 

Freely available online through the PNAS open access option. 

See Core Concepts on page 9126. 

¹To whom correspondence should be addressed. Email: rgallo@ihv.umaryland.edu. 

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).