Editing of the human TRIM5 gene to introduce mutations with the potential to inhibit HIV-1

Abstract

The type I interferon (IFN-I)-inducible human restriction factor TRIM5α inhibits the infection of human cells by specific nonhuman retroviruses, such as N-MLV and EIAV, but does not generally target HIV-1. However, the introduction of two aminoacid substitutions, R332G and R355G, in the human TRIM5α (huTRIM5α) domain responsible for retroviral capsid recognition leads to efficient HIV-1 restriction upon stable over-expression. CRISPR-Cas-based approaches to precisely edit DNA could be employed to modify TRIM5 in human cells. Toward this aim, we used a DNA transfection-based CRISPR-Cas9 genome editing protocol to successfully mutate TRIM5 to its potentially HIV-1-restrictive version by homology-directed repair (HDR) in HEK293T cells. Nine clones bearing at least one HDR-edited TRIM5 allele containing both mutations were isolated (5.6% overall efficiency), whereas another one contained only the R332G mutation. Of concern, several of these HDR-edited clones contained on-target undesired mutations, and none had all the alleles corrected. Our study demonstrates the feasibility of editing the TRIM5 gene in human cells and identifies the main challenges to be addressed in order to use this approach to confer protection from HIV-1.

CRISPR/Cas9-Based Antiviral Strategy: Current Status and the Potential Challenge

“…This CRISPR/Cas9d-based viral promoter-activating antiviral approach showed promising antiviral efficacy [28,29,30,31,32]. On the other hand, induction of host restriction factors such as interferons and interferon-stimulated genes (ISG) by CRISPR/Cas9-mediated transcriptional activation was envisaged as another plausible approach for this host-targeting antiviral strategy [33,34]. In addition, a number of anti-HIV applications of CRISPR/Cas9 involving the enhanced expression of host restriction factors against HIV infection such as apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC3G), and tripartite motif-containing protein 5 alpha (TRIM5α) genes through Cas9d-mediated transcriptional activation were also proposed and tested [33,34].…”

“…On the other hand, induction of host restriction factors such as interferons and interferon-stimulated genes (ISG) by CRISPR/Cas9-mediated transcriptional activation was envisaged as another plausible approach for this host-targeting antiviral strategy [33,34]. In addition, a number of anti-HIV applications of CRISPR/Cas9 involving the enhanced expression of host restriction factors against HIV infection such as apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC3G), and tripartite motif-containing protein 5 alpha (TRIM5α) genes through Cas9d-mediated transcriptional activation were also proposed and tested [33,34]. In the following chapter, different kinds of CRISPR/Cas9-based anti-HIV strategies such as direct disruption of an HIV genome, induction of latency reversal, disruption of a host dependency factor, and induction of a host restriction factor will be discussed in detail.…”

Do Human Endogenous Retroviruses Contribute to Multiple Sclerosis, and if So, How?

“…This is particularly intriguing given that SNPs in the TRIM5 locus within intron 1 have been found to be inversely associated with MS; intron 1 is immediately adjacent to the exon coding for a RING (really interesting new gene) finger domain [ 40 ]. There is a considerable body of evidence demonstrating the influence of SNPs on TRIM5 activity [ 202 ]; hence, the SNPs in question could plausibly reduce the strength of the immune response to HERV RNA and explain the reduction in risk of MS development associated with these polymorphisms.…”

Restriction Factors: From Intrinsic Viral Restriction to Shaping Cellular Immunity Against HIV-1

“…This combination anti-HIV-1 lentiviral vector is currently in a phase I/II clinical trial (ID: NCT02797470). The exploitation of the novel CRISPR-Cas9 technology could also be a suitable tool to precisely manipulate hTRIM5 α gene to increase the affinity of hTRIM5α for the incoming retroviral capsid and enhance antiviral potency (262, 263).…”