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). In this paper, 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 the 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. Fusion chimeras with haplotype II allowed for stabilization, cytoplasmic retention, and packaging of the N15del-containing haplotype III, but the haplotype III component of these chimeras was unable to restrict HIV-1 on its own. Thus, the evolutionary loss of A3H activity in many humans involves functional deficiencies independent of protein stability.

Single-nucleotide polymorphism of the DNA cytosine deaminase APOBEC3H haplotype I leads to enzyme destabilization and correlates with lung cancer

“…Computational models of the G105R system exhibit dynamic motion and HBond patterns similar to the WT, indicating that this mutation does not negatively affect the structural stability of the protein (see Supplementary Figures S6–S8 for G105R computational data). The G105R may simply increase the propensity of the protein to become polyubiquitinated and degraded ( 35 ), in contrast to K121E that destabilizes the structural integrity of A3H Hap I (Figures 2 and 3 ). To test whether 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 Sf 9 insect cells to obtain a quantitative measurement.…”

“…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 ( 35 ). Thus, the SNP studied in this work for A3H Hap I is unique since it appears to destabilize the enzyme by disrupting an HBond network.…”

“…The other A3H haplotypes are primarily in the cytoplasm ( 34 ). A3H has several determinants for stability, such as the propensity to become ubiquitinated and ability to bind cellular RNA ( 12 , 35 ). A3H forms a dimer that has no protein–protein contacts and is mediated by binding cellular RNA ( 8 , 12 , 36,37 ).…”

APOBEC1 cytosine deaminase activity on single-stranded DNA is suppressed by replication protein A

“…A3H Hap VII is a proxy for A3H Hap I. The A3H Hap VII differs only by a G105R polymorphism and is not rapidly ubiquitinated and degraded in cells, in contrast to A3H Hap I ( 58 , 59 ). APOBECs access genomic DNA during times when it is transiently single-stranded.…”

Retroviral Restriction Factors and Their Viral Targets: Restriction Strategies and Evolutionary Adaptations

“…In the case of A3C, a single haplotype found in African populations is the only known variant capable of blocking Vif-deficient HIV-1 [ 132 , 148 ]. Moreover, Both A3F and A3H are highly polymorphic in humans with several variants showing anti-HIV-1 activity [ 133 , 146 , 147 , 152 , 153 , 154 , 155 , 156 ].…”