The rare antibodies selected functioned by inhibiting the virus-encoded protease that is necessary for viral maturation.
Defenders against Death
A review of literature describing how researchers used intracellular combinatorial libraries to select antibodies that protected cells from death.
The infective antibody library platform developed by the Lerner team approximately 2 years ago, in which lentiviral infection of a mammalian cell host by an antibody library effectively links antibody genotype and cell phenotype, has been used in several successful screens, delivering antibodies with unique properties such as those having the ability to transdifferentiate cells without the need for transitional induced pluripotency . Here the team uses the platform to find antibody tools to dissect a fundamental process of cell death brought about by an acute viral infection.
An iterative selection was devised in which antibody genes recovered from surviving cells were used for each new round. A 108-member naive antibody lentivirus library was used to induce cytoplasmic expression of antibodies in HeLa cells prior to infecting them with rhinovirus. Cells expressing functional antibodies were expected to be protected from death while other cells were killed. The antibody-coding information from the surviving cells was fed into a secondary lentiviral library that served as the starting point for the next round of selection (see Figure). After five rounds of such selection, the team obtained antibodies that when expressed in the cytoplasm as distinct agents were shown to offer protection to mammalian cells from rhinovirus-induced death. Mechanism of action studies implicated the viral 3C protease, responsible for virus maturation, as the target to which the antibodies bound and whose blockage rescued the mammalian host cell from virus-induced cell death.
Figure. Scheme of the phenotype selection-based information enrichment. The selection starts with a naive intrabody lentiviral library. HeLa cells were sequentially infected with the antibody library followed by infection with human rhinovirus. Cells expressing functional antibodies were protected from death while other cells were killed. The integrated antibody fragments (the information) from the surviving cells were recovered and converted into a secondary lentiviral library that was the starting point for the next round of selection. The selection was carried out for five rounds. The number of surviving cells (green circles) relative to killed cells (red circle) increased in each round. The percentage of functional antibody sequences (red and black patches) also enriched after each round of selection.
The present work is a fine example of a selection strategy that carries an additional kinetic-based component, as if the virus infection and host cell replication enter a race of sorts during the initial stages of which any rare rescue antibody clones remain invisible and are only revealed if they are truly effective and after several successive rounds of enrichment. It is anticipated that future uses of this approach will incorporate earlier markers for cell death, such as onset of apoptosis, autophagy, or cell cycle arrest. In turn, knowing the factors that preferentially protect cells from death should inform the efforts directed at finding ways to selectively kill unwanted cells, such as rapidly proliferating cancers.
One of the most important phenotypes in biology is cell death. One way to probe the mechanism(s) of cell death is to select molecules that prevent it and learn how this was accomplished. Here, intracellular combinatorial antibody libraries were used to select antibodies that protected cells from killing by rhinovirus infection. These rare antibodies functioned by inhibiting the virus-encoded protease that is necessary for viral maturation. Snapshots of the selection process after each round could be obtained by deep sequencing the ever-enriching populations. This detailed analysis of the enrichment process allowed an interesting look at a “test tube” selection process that pitted two replicating systems against each other. Thus, initially a minority of cells containing protective antibodies must compete against a majority of unprotected cells that continue to produce large amounts of virus.