HCV transmission by hepatic exosomes establishes a productive infection

Efficient entry into cells is key for virus propagation. Unsurprisingly, it is antagonized by the anti-viral armaments, such as neutralizing antibodies that can block viral entry at different steps or such as innate response sensors that decode structural features of viral particles and trigger anti-viral responses in host cells. Several cell entry routes and programs exist that seem to almost invariably involve the initial attachment of viral particles to one or several cellular receptors and/or entry factors. Explicitly, the canonical entry process of enveloped viruses depends on viral surface (glyco-)proteins for attachment to cells, and merging of the viral membrane with cellular membranes by specialized fusion proteins []. Yet, recent unveiling of virus seizure of specific cell membrane structures [] and lipids [] has highlighted novel and highly original entry pathways.

Because the host defense arsenal efficiently targets various entry steps of cell-free viral particles, viruses have evolved a plethora of strategies to counteract or escape host immune mechanisms. For example, apart from conventional cell-free transmission by viral particles, hepatitis C virus (HCV), a prominent liver disease pathogen, may use alternative itineraries for cell invasion such as antibody-resistant cell-cell transmission routes [] and subterfuges such as camouflage with lipoprotein components, as lipo-viro-particles, which also impairs antibody neutralization []. Strikingly, a recent study by Ramakrishnaiah and colleagues [] inferred that hepatic exosomes transmit productive HCV infection in vitro in a process that is partly resistant to antibody neutralization and to antisera raised against some HCV entry receptors (i.e., CD81, scavenger receptor class B member 1 (SR-BI), and Claudin 1).

Exosomes are extracellular microvesicles of 30–150 nm in size that are likely formed within multivesicular bodies (MVBs) – a type of late endosome – and that are secreted in body fluids under normal and pathological conditions []. Exosomes contribute to many cell-cell communication processes, including transfer of proteins, of mRNAs and microRNAs, or, within infected organisms, to the transfer of pathogen-derived antigens and virulence factors []. Previous studies reported the presence of viral RNA in exosomes isolated from plasma of HCV-infected patients []. A recent study demonstrated that full length HCV RNAs are encapsulated within hepatocyte-derived exosomes and can be transferred to plasmacytoid dendritic cells (pDC), where they trigger IFN-α production []. The report by Ramakrishnaiah et al. [] is the first to demonstrate an exosomal route of HCV transmission between hepatocytes.

Interestingly, the mass spectrometric analysis of exosomes purified from both naïve or HCV-infected HuH7.5.1 hepatoma cells [] revealed that they display both expected exosomal markers and microRNAs but also ApoE and ApoB apolipoproteins that, coincidently, are also usually present on bona fide infectious HCV particles [], for which they mediate virion attachment to cells independently of virus-encoded surface glycoproteins []. Importantly, exosomes purified from both infected HuH7.5.1 cells and HCV subgenomic replicon (SGR) cells – that are devoid of viral structural proteins and hence, do not assemble infectious viral particles – contained complete HCV subgenomes that could transmit HCV RNA and establish replication and/or infection in HCV permissive naïve HuH7.5.1 cells. Exosomes isolated from HCV SGR cells could induce viral replication in target cells less efficiently than those produced from infected cells, likely owing to reduced levels of viral RNAs in exosome-producer SGR cells. Yet, this part of the study was essential since it allowed firm demonstration of exosome-mediated HCV genome transmission without the confounding effect of infectious viral particles that may contaminate exosome preparations. Indeed, technically, it is very challenging to totally exclude viral particles from isolated exosome samples because density of the latter vesicles (1.10–1.19 g/ml) partially overlaps with that of HCV particles (from <1.06 to >1.16 g/ml in the blood of infected patients and in the supernatants of infected cells []). This potential limitation, as well as the possibility that exosomes may randomly pick up HCV surface glycoproteins [, ], may explain why the exosomal transfer process of HCV was not completely resistant to neutralizing antibodies. However, it is clear that a significant part of HCV exosomal transmission escapes neutralization since a high proportion of sera from patients with chronic HCV exhibited limited inhibition of exosome-mediated infection, as compared to HCV infectious particles, and since immunoglobulins purified from patient’ sera not only did not neutralize exosomes isolated from HCV SGR cells but, instead, could enhance their transmission. Additionally, since neutralizing antibodies are typically directed against virus surface glycoproteins, these results directly suggest that different processes are involved in HCV transmission by genuine HCV particles vs. exosomes.

A recent study demonstrating that a non-enveloped virus, hepatitis A virus, may conceal into exosomes to penetrate cells and escape humoral immunity [] further spices up the results of Ramakrishnaiah et al. []. Exciting research addressing, e.g., the usurpation of cellular factors associated to MVB formation, i.e., the endosomal sorting machineries for encapsidation and/or transfer of viral elements [, ] or the discovery of cell surface molecule(s) involved in uptake and membrane fusion of exosomes will unravel the mechanisms of exosomal transmission of HCV regarding the biogenesis of these fascinating vesicles and their entry process into target cells. Additionally, these questions will be important for understanding the contribution of exosomes to the known immune escape properties of HCV and to its dissemination or, conversely, to their capacity to stimulate immune cells []. Conceivably, this may also lead to overcome the unsuccessfulness of prophylactic neutralizing antibodies and agents targeting the entry of HCV into cells.