A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors.

Authors:
Park RJ, Wang T, Koundakjian D, Hultquist JF, Lamothe-Molina P, Monel B, Schumann K, Yu H, Krupzcak KM, Garcia-Beltran W, Piechocka-Trocha , Krogan NJ, Marson A, Sabatini DM, Lander ES, Hacohen N, Walker BD.
In:
Source: Nat Genet
Publication Date: (2017)
Issue: 49(2): 193-203
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
CD4+, human
Species: human
Tissue Origin: blood
Culture Media:
Platform:
4D-Nucleofector™ X-Unit
Experiment

Donor 1 and 2: primary CD4+ T cells were activated using 10 µl of ImmunoCult Human CD3–CD28 T cell activator (StemCell Technologies) per milliliter of medium. After 3 d, 1 million cells were electroporated with 3 µl of Cas9–RNPs using Nucleocuvette strips in an Amaxa 4D-Nucleofector System X unit (P3 solution, program EO-115). Six days later, cells were re-activated using 2.5 µl of ImmunoCult reagent per milliliter of medium and cultured for three additional days prior to use in downstream assays including HIV challenge; For donors 3 and 4, primary CD4+ T cells were activated for 2 d with 5 µg/ml soluble anti-CD28 (Tonbo Biosceiences) on plates that were coated overnight with 10 µg/ml anti-CD3 (Tonbo Biosciences). Cells were electroporated with Cas9–RNPs as described above, except with 300,000 cells per condition.

Abstract

Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors, but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors, including the HIV co-receptors CD4 and CCR5, that are required for HIV infection yet are dispensable for cellular proliferation and viability. Tyrosylprotein sulfotransferase 2 (TPST2) and solute carrier family 35 member B2 (SLC35B2) function in a common pathway to sulfate CCR5 on extracellular tyrosine residues, facilitating CCR5 recognition by the HIV envelope. Activated leukocyte cell adhesion molecule (ALCAM) mediates cell aggregation, which is required for cell-to-cell HIV transmission. We validated these pathways in primary human CD4+ T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest that these pathways can be studied for therapeutic intervention