Biology Reference
In-Depth Information
transduction of a naive cell population, concluded by a new round of selection.
This process can be repeated sequentially for an unlimited number of selection/
enrichment steps.
For an additional level of selection, different challenge virus genomes
can be used, each encoding a different drug-selectable marker (e.g., puromycin
(PuroR) or zeocin (ZeoR) resistance), in self-inactivating retroviruses. Thus,
after challenge and selection of a library-transduced population with one pseu-
doparticle-packaged selection cassette (e.g., PuroR), the population can be
pooled and rechallenged with the second-selectable pseudovirus (e.g., ZeoR).
Then, during CPR, only the full-length retroviral transcripts from the non-self-
inactivating provirus that encodes the library but not the self-inactivating
challenge virus genomes are repackaged and transferred to the naive cell popu-
lation. This enables researchers to perform multiple rounds of selection, thereby
overcoming the background of nonspecific pseudoparticle uptake. Moreover,
using this scheme, underrepresented cDNA will be enriched. Once the final
round of selection of pseudotype-susceptible cells has been achieved, genomic
DNA can be prepared from selected clones and used as a template in a PCR
across the provirus cDNA-cloning site.
The nonpermissive target cell line for the cDNA screen adheres to
several stringent criteria. As stated above, the primary requirement is that (1) to
minimize nonspecific background, cell lines with minimal uptake of pseudopar-
ticle of interest and “no envelope” pseudoparticles were preferred. (2) To ensure
that nonpermissiveness was a phenotype due to the lack of a pseudoparticle-
specific entry factor(s) rather than poor infection by pseudotypes in general,
chosen cell lines should be highly permissive to an unrelated pseudoparticle
(VSVGpp, rhabdovirus) infection. In addition, this also ensures that the target
cell line would be easily transduced with the cDNA library. (3) For selection,
candidate cell lines also had to be susceptible to the desired drug selections.
(4) To perform multiple rounds of screening involving CPR, the ideal cell line
had to demonstrate this method well and be highly transfectable. (5) Finally, to
facilitate the screen, the chosen cell line needed to be relatively fast growing and
clone efficiently.
C. Determining the endocytosis pathway of entry and the different
cell proteins involved in entry by RNA interference screens
To gain insights into virus entry, it is necessary to examine several inhibitors of
pathway-mediated endocytosis in terms of their role in blocking infection
mediated by pseudotypes with different EnvGP. An advantage of pseudoparticles
is that the entry process of pathogens of BSL4 can be studied in BSL2 conditions.
For example, to gain insights into Ebola virus entry, inhibitors against different
endocytosis pathways have been examined for their ability to block infection
