NEW YORK (GenomeWeb) – A CRISPR/Cas9-based genome-wide screen has found five potential drug targets for HIV infection.
Researchers from more than a dozen institutions in the Boston and San Francisco Bay areas collaborated to interrogate host factors necessary for HIV infection. They identified five factors — including the canonical HIV co-receptors CD4 and CCR5 — required for HIV infection, but seemingly unnecessary for cellular proliferation and viability. Tryosylprotein sulfotransferse 2 (TPST2), solute carrier family 35 member B2 (SLC35B2), and activated leukocyte cell adhesion molecule (ALCAM) were newly identified proteins. They published their results in Nature Genetics.
"Current anti-HIV medications overwhelmingly target viral proteins," Ragon Institute and Broad Institute researcher, and study co-author Ryan Park said in a statement. "Because HIV mutates so rapidly, drug-resistant strains frequently emerge, particularly when patients miss doses of their medication. Developing new drugs to target human genes required for HIV infection is a promising approach to HIV therapy, with potentially fewer opportunities for the development of resistance."
While RNA interference screening has identified hundreds of possible candidate host factors "there is little agreement among studies and few factors have been validated," the authors wrote in their paper. They suggested that RNAi screens had chosen cell types, such as HeLA cells, that were good experimental models because they were efficiently transfected; however, they were not representative of cells naturally targeted by HIV.
The authors surmised that pooled loss-of-function screening using CRISPR/Cas9-based knockout, using primary T cells, would allow for specific, unbiased results. Their screening library consisted of 187,536 guide RNAs, targeting 18,543 protein-coding genes. For each of the five genes identified by the screen, at least five sgRNAs were enriched — in some cases, it was up to 10. The researchers then validated the hits and ran experiments to uncover the mechanisms through which the gene knockouts helped cells resist infection.
TPST2 and SLC35B2 help HIV gain entry to cells through their critical role in sulfating the CCR5 receptor, catalyzing the O-sulfation of tyrosines on plasma membrane proteins and transporting the activated sulfate donor, respectively.
ALCAM knockout appeared to disrupt cell-to-cell HIV transmission. as gene-gene interactions help mediate infected cells aggregate with each other and disrupting primary T cell aggregation hinders the spread of HIV.
"ALCAM is necessary for cell-to-cell adhesion in our cell line, allowing more efficient viral transfer from one cell to the next," Park said. "In fact, we found that artificially inducing the aggregation of cells lacking ALCAM restored the cell-to-cell transmission of HIV."
He also noted that while further studies are needed to investigate whether targeting these genes would be toxic to humans, targeting these pathways could provide an opportunity to strengthen the arsenal of drugs to fight HIV infection.
"Global use of combination antiretroviral therapy for HIV has saved tens of millions of lives and slowed the AIDS epidemic, but it has failed to prevent the emergence and spread of drug-resistant strains," the authors wrote. "Anti-HIV therapies that target host genes required by HIV may raise the barrier to drug resistance and potentially offer new interventional or curative strategies through gene therapy."
Co-author Tim Wang of the Broad and Whitehead Institutes added that the method could be used to identify therapeutic targets for other viral pathogens and that it could "pinpoint a novel class of potential therapeutic targets that have previously been underexplored for the treatment of infectious disease."