Abstract
Extracellular vesicles (EVs) are involved in intercellular communication and affect processes including immune and antiviral responses. Blood serum, a common cell culture medium component, is replete with EVs and must be depleted prior to EV-related experiments. The extent to which depletion processes deplete non-EV particles is incompletely understood, but depleted serum is associated with reduced viability and growth in cell culture. Here, we examined whether serum depleted by two methods affected HIV-1 replication. In cell lines, including HIV-1 latency models, increased HIV-1 production was observed, along with changes in cell behavior and viability. Add-back of ultracentrifuge pellets (enriched in EVs but possibly other particles) rescued baseline HIV-1 production. Primary cells were less sensitive to serum depletion processes. Virus produced under processed serum conditions was more infectious. Finally, changes in cellular metabolism, surface markers, and gene expression, but not miRNA profiles, were associated with depleted serum culture. In conclusion, depleted serum conditions have a substantial effect on HIV-1 production and infectivity. Dependence of cell cultures on “whole serum” must be examined carefully along with other experimental variables, keeping in mind that the effects of EVs may be accompanied by or confused with those of closely associated or physically similar particles.
Highlights
Extracellular vesicles (EVs) are a diverse group of bilayer-membraned particles that include so-called “exosomes” (canonically defined as budding into multivesicular bodies (MVBs) and being released upon MVB fusion with the plasma membrane) and “microvesicles”[1, 2]
We show here an impact of serum processing protocols—which were designed and have been widely implemented for EV depletion—on HIV-1 production by susceptible cells
HIV-1 latency models undergo a degree of viral activation under depleted serum conditions
Summary
Extracellular vesicles (EVs) are a diverse group of bilayer-membraned particles that include so-called “exosomes” (canonically defined as budding into multivesicular bodies (MVBs) and being released upon MVB fusion with the plasma membrane) and “microvesicles” (often described as blebbing directly from the plasma membrane)[1, 2]. We observed a slight but significant decline in replication and viability in EV-depleted conditions[26] The magnitude of these effects was variable, and, notably, a primary glioblastoma cell line (U87) did not appear to be affected. We used serum depleted by two methods and examined the effects of media prepared with these sera on the growth and behavior of cells that are susceptible to or infected with HIV-1, including immortalized cell lines, HIV-1 latency models, and primary T-cells and monocyte-derived macrophages. We conclude that two distinct serum depletion protocols—meant to remove EVs—have a profound effect on HIV-1 replication and infection in cultures of some cells, and that bulk EVs in serum, and/or their closely associated or co-depleted factors, tend to exert a virus-suppressive effect
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