Stabilized Human TRIM5α Protects Human T Cells From HIV-1 Infection

Abstract

Rhesus (rh) but not human (hu) TRIM5α potently restricts human immunodeficiency virus (HIV)-1 infection. It is not clear why huTRIM5α fails to effectively block HIV infection, but it is thought to have a lower affinity for the viral core. Using primary human CD4 T cells, we investigated the ability of huTRIM5α, rhTRIM5α, and the huTRIM5αR323-332 B30.2/SPRY patch-mutant to form cytoplasmic bodies, postulated as key components of the HIV-1 restriction apparatus. Both rhTRIM5α and huTRIM5αR323-332 formed pronounced cytoplasmic bodies, whereas cytoplasmic bodies in T cells overexpressing huTRIM5α were present but more difficult to detect. As expression of all three TRIM5α orthologs was similar at the RNA level, we next investigated the role of protein stability in conferring TRIM5α-mediated HIV-1 restriction. Both steady-state and pulse-chase experiments revealed that the huTRIM5α protein was much less stable than rhTRIM5α, and this difference correlated with higher self-ubiquitination activity. Using a stabilized form of huTRIM5α in which the steady-state expression level was more similar to rhTRIM5α, we observed comparable HIV-1 restriction activity in multi-round HIV-1 challenge assays. Lastly, primary human CD4 T cells expressing a stabilized huTRIM5α were protected from HIV-1-mediated destruction in vivo, indicating that efforts to stabilize huTRIM5α should have significant long-term therapeutic value.

Preclinical Assessment of Mutant Human TRIM5α as an Anti-HIV-1 Transgene

“…In addition, mutations in this PRYSPRY region have been shown to modulate protein stability. 63 In conclusion, transgenic TRIM5a was moderately overexpressed, consistent with predominantly single integration events in our experimental system, with the R332G-R335G variant possibly being expressed at slightly lower steadystate levels.…”

Potent and Broad Inhibition of HIV-1 by a Peptide from the gp41 Heptad Repeat-2 Domain Conjugated to the CXCR4 Amino Terminus

“…To evaluate the ability of C34-conjugated coreceptors to protect CD4 T cells in vivo , we employed the NSG mouse model that has been used extensively to evaluate the ability of a wide range of antiviral agents to protect T cells from HIV-1 infection in vivo [23, 48–51]. We infused 10 million untransduced, GFP-transduced, or C34-CXCR4 transduced T cells into cohorts of mice.…”

Supraphysiologic control over HIV-1 replication mediated by CD8 T cells expressing a re-engineered CD4-based chimeric antigen receptor

“…Three weeks later mice were tail vein injected with 15ng HIV Bal mixed 1:1 with PBS. Peripheral blood was obtained by retro-orbital bleeding, and human CD4 and CAR + CD8 lymphocyte counts were enumerated using BD lysis buffer and BD TruCount tubes as previously described [73], staining with mouse anti human CD45 PerCp Cy5.5 (BD Biosciences), mouse anti human CD4 BV421 (Biolegend), and mouse anti human CD8α BV711 (Biolegend).…”