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.
Highlights
Viruses are obligate parasites whose success at infecting a host cell typically requires evasion from antiviral factors
These results suggest that human embryonic kidney 293T cells (HEK293T) cells provide a suboptimal environment for HIV-1 restriction by over-expressed TRIM5α
To investigate whether this observation extended to the endogenous TRIM5α in HEK293T, we infected the parental cells with increasing amounts of N-MLVGFP or with the huTRIM5α-insensitive B-MLVGFP
Summary
Viruses are obligate parasites whose success at infecting a host cell typically requires evasion from antiviral factors. Many cellular antiviral factors that can potentially interfere with the progression of viral infections have been identified These factors can often act without external stimulation, but their expression and activity are enhanced by type I interferons (IFN-I) [1]. At its C-terminus, a domain called SPRY (PRYSPRY, B30.2) determines the retrovirus targeting specificity This domain comprises hyper-variable loops that directly interact with the N-terminal domain of capsid proteins early after entry of the retrovirus into the host cell membrane [4]. When such interactions occurs, the retrovirus is inhibited (“restricted”) through mechanisms that include destabilization of the capsid core [5], proteasomal degradation of some core components [6] and sequestration of the viral particle in TRIM5α
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