Extracellular vesicles from HTLV-1 infected cells modulate target cells and viral spread

Abstract

Background The Human T-cell Lymphotropic Virus Type-1 (HTLV-1) is a blood-borne pathogen and etiological agent of Adult T-cell Leukemia/Lymphoma (ATLL) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). HTLV-1 has currently infected up to 10 million globally with highly endemic areas in Japan, Africa, the Caribbean and South America. We have previously shown that Extracellular Vesicles (EVs) enhance HTLV-1 transmission by promoting cell–cell contact. Results Here, we separated EVs into subpopulations using differential ultracentrifugation (DUC) at speeds of 2 k (2000×g), 10 k (10,000×g), and 100 k (100,000×g) from infected cell supernatants. Proteomic analysis revealed that EVs contain the highest viral/host protein abundance in the 2 k subpopulation (2 k > 10 k > 100 k). The 2 k and 10 k populations contained viral proteins (i.e., p19 and Tax), and autophagy proteins (i.e., LC3 and p62) suggesting presence of autophagosomes as well as core histones. Interestingly, the use of 2 k EVs in an angiogenesis assay (mesenchymal stem cells + endothelial cells) caused deterioration of vascular-like-tubules. Cells commonly associated with the neurovascular unit (i.e., astrocytes, neurons, and macrophages) in the blood–brain barrier (BBB) showed that HTLV-1 EVs may induce expression of cytokines involved in migration (i.e., IL-8; 100 k > 2 k > 10 k) from astrocytes and monocyte-derived macrophages (i.e., IL-8; 2 k > 10 k). Finally, we found that EVs were able to promote cell–cell contact and viral transmission in monocytic cell-derived dendritic cell. The EVs from both 2 k and 10 k increased HTLV-1 spread in a humanized mouse model, as evidenced by an increase in proviral DNA and RNA in the Blood, Lymph Node, and Spleen. Conclusions Altogether, these data suggest that various EV subpopulations induce cytokine expression, tissue damage, and viral spread.

Extracellular Vesicles from Infected Cells Are Released Prior to Virion Release

“…Lastly, we observed that the HTLV-1infected cells contained higher intracellular env RNA compared to secreted env RNA (EV-bound) at each timepoint (Figure 5A,B). This is consistent with observations that HTLV-1 primarily exists intracellularly, and that infectious virion release occurs sporadically in infected cells [14,18,56,58]. Collectively, these data indicate that HTLV-1 RNA synthesis is increased post-release/induction and that viral RNA can be found in the extracellular environment.…”

“…The infectivity of the 24-h samples but not of the 6-h samples could be augmented by the presence of EVs. This is in line with data from Figure 1 (lane 10) that demonstrate that the concentration of EVs peaks at 24 h. Collectively, these results imply that virions released at 24 h are indeed infectious, and the presence of EVs may contribute to their infectivity, since EVs from virally infected cells have been found to contain viral proteins and RNA that can increase the pool of surrounding cells susceptible to infection [14,[18][19][20]32,51,52].…”

“…These early EVs contain viral proteins and RNAs that can activate the cell cycle and render naïve recipient T cells and monocytes more vulnerable to subsequent viral infection. Altogether, these data could allow further understanding of the effects of EVs on naïve recipient cells [18,69] and contribute to knowledge about the effects of EVs in enhancing susceptibility to infection. More importantly, these findings show the importance of EVs during viral infection, since they are secreted before the virions and contain several viral proteins with the potential to induce inflammation and disease.…”

“…These changes in EV marker expression over time could potentially be due to the increase of EV release at different timepoints (Figure S1). Furthermore, this differential tetraspanin expression could also be attributed to the concept that cells produce different EV populations that are heterogenous in their protein markers [18,30] and that tetraspanins such as CD9 could potentially enhance viral spread and EV biogenesis [45]. Moreover, viral capsid protein p24 was found to interact with CD81 and colocalize in tetraspanin-enriched microdomains composed of CD63, CD9, and CD81, which is crucial for virion assembly [46,47].…”

“…EVs carry functional cargoes including viral proteins, lipids, cytokines, RNA and, in some cases, the virion itself (i.e., hepatitis B) [12,13]. As such, EVs are able to modulate disease pathogenesis in several types of infections such as HIV-1, HTLV-1, Ebola virus, and Rift Valley fever by promoting cell-to-cell spread, immune evasion, persistent inflammation, and cell cycle regulation in recipient cells [14][15][16][17][18]. Recently, we found that EVs from HIV-1-infected cells transport TAR RNA to naïve cells, compromising their resistance to HIV-1 through downregulation of apoptosis by depleting CDK9 levels [19].…”