Polymorphisms in human APOBEC3H differentially regulate ubiquitination and antiviral activity

Abstract

The APOBEC3 family of cytidine deaminases are an important part of the host innate immune defense against endogenous retroelements and retroviruses like human immunodeficiency virus (HIV).APOBEC3H (A3H) is the most polymorphic of the human APOBEC3 genes, with four major haplotypes circulating in the population. Haplotype II is the only antivirally-active variant of A3H, while the majority of the population possess independently destabilizing polymorphisms present in haplotype I (R105G) and haplotypes III and IV (N15del). Here, we show that instability introduced by either polymorphism is positively correlated with degradative ubiquitination, while haplotype II is protected from this modification.Inhibiting ubiquitination by mutating all of the A3H lysines increased expression of haplotypes III and IV, but these stabilized forms of haplotype III and IV had a strict nuclear localization, and did not incorporate into virions, nor exhibit antiviral activity, thus separating stabilization from function. On the other hand, the instability and functional deficiencies of haplotype III could be rescued by fusion to haplotype II, supporting a model by which antiviral A3H is actively stabilized through a cytoplasmic retention mechanism. Thus, the evolutionary loss of A3H activity in many humans involves functional deficiencies independent of protein stability.

Human APOBEC3 Variations and Viral Infection

“…The haplotypes II (NRRDD), V (NRRDE), and VII (NRRKE) show strong antiviral activities [ 32 , 83 , 85 , 146 , 147 ]. Variants with a deletion in position 15 and/or G in position 105 are unstable and have little/no antiviral activity, likely due to ubiquitination [ 148 ]. Differential interactions with HIV-1 Gag have also been suggested to be responsible for the significantly reduced antiviral activity of HapI, which has a G in position 105 [ 149 ].…”

Stability evolution as a major mechanism of human protein adaptation in response to viruses

“…We further validate Thermonet estimates in multiple ways. First, Thermonet correctly identifies a known destabilizing genetic variant (R105G) in antiviral VIP APOBEC3H as strongly destabilizing (stability change ∆∆G = 0.71 kcal/mol) (Chesarino and Emerman, 2020). Second and most importantly, we find strong, highly significant evidence of compensatory evolution of protein stability in non-immune proviral VIPs with multiple amino acid changes, where it is the most expected (see below).…”

Induced mutagenesis by the DNA cytosine deaminase APOBEC3H Haplotype I protects against lung cancer

“…The antiviral properties of these enzymes are likely why they are maintained despite the negative effect of their off-target activity. The destabilizing SNPs for A3H other than the one studied here, are not thought to destabilize protein structure, but to promote A3H ubiquitination and proteosomal degradation 42 . Thus, the SNP studied in this work for A3H Hap I is unique since it appears to destabilize the enzyme by disrupting a hydrogen bonding network.…”

“…The G105R may simply increase the propensity of the protein to become polyubiquitinated and degraded 42 , in contrast to K121E that destabilizes the structural integrity of A3H Hap I (Figure 2). To test if the longer steady state half-life of A3H K117E and K117E/K121E in cells correlates with increased catalytic activity similar to A3H Hap VII, we conducted an in vitro deamination assay using protein purified from Sf9 insect cells to obtain a quantitative measurement.…”