D316 is critical for the enzymatic activity and HIV-1 restriction potential of human and rhesus APOBEC3B

Abstract

APOBEC3B is one of seven human APOBEC3 DNA cytosine deaminases that function to inhibit the replication and persistence of retroelements and retroviruses. Human APOBEC3B restricts the replication of HIV-1 in HEK293 cells, while our laboratory clone of rhesus macaque APOBEC3B did not. We mapped the restriction determinant to a single amino acid difference that alters enzymatic activity. Human APOBEC3B D316 is catalytically active and capable of restricting HIV-1 while rhesus APOBEC3B N316 is not; swapping these residues alters the activity and restriction phenotypes respectively. Genotyping of primate center rhesus macaques revealed uniform homozygosity for aspartate at position 316. Considering the C-to-T nature of the underlying mutation, we suspect that our rhesus APOBEC3B cDNA was inactivated by its own gene product during subcloning in Escherichia coli. This region has been previously characterized for its role in substrate specificity, but these data indicate it also has a fundamental role in deaminase activity.

Fig. 1

HIV-1 restriction potential maps to the human APOBEC3B C-terminal domain. (A) HIV-1 infectivity in the presence of increasing amounts of human APOBEC3B, rhesus APOBEC3B, or the human-rhesus/rhesus-human chimeras. A constant amount of Vif-deficient A200C HIV-1_IIIB molecular clone was co-transfected into HEK293T cells with an increasing gradient of the indicated APOBEC3B vector. Percent infectivity of HIV-1_IIIB is measured by infection of CEM-GFP in duplicate and flow cytometry, reported as the mean of the two technical replicates ± standard deviation. Representative immunoblots of HA-tagged APOBEC3B proteins (HA) in cell lysates and HIV-1 particles produced by those cells are shown with their respective tubulin (TUB) and p24 (CA) loading controls. (B) Schematic of human/rhesus APOBEC3B C-terminal domain chimeras and their HIV-1 restriction potential. HIV-1 single cycle assays were performed with each chimera as in (A) and found to be restrictive or non-restrictive depending on their similarity to the human or rhesus APOBEC3B parent, respectively. An alignment of human (top) and rhesus (bottom) APOBEC3B amino acid sequences to which the restriction determinant mapped is shown above. Differences between the human and rhesus sequences are highlighted in gray and the catalytic glutamate is highlighted in green. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

HIV-1 restriction potential tracks with the putative alpha 4 helix region of human APOBEC3B. HIV-1 infectivity in the presence of increasing amounts of human APOBEC3B, rhesus APOBEC3B, or various human-rhesus alpha helix chimeras in the C-terminal domain. A constant amount of Vif-deficient A200C HIV-1_IIIB molecular clone was co-transfected into HEK293T cells with an increasing gradient of the indicated APOBEC3B vector. Percent infectivity of HIV-1_IIIB is measured by infection of CEM-GFP in duplicate and flow cytometry, reported as the mean of the two technical replicates±standard deviation. Representative immunoblots of HA-tagged APOBEC3B proteins (HA) in cell lysates and HIV-1 particles produced by those cells are shown with their respective tubulin (TUB) and p24 (CA) loading controls.

Fig. 3

An aspartic acid at APOBEC3B residue 316 is crucial for HIV-1 restriction and deaminase activity. (A) HIV-1 infectivity in the presence of increasing amounts of human APOBEC3B, rhesus APOBEC3B, or various helix or single amino acid substitution constructs. A constant amount of Vif-deficient A200C HIV-1_IIIB molecular clone was co-transfected into HEK293T cells with an increasing gradient of the indicated APOBEC3B vector. Percent infectivity of HIV-1_IIIB is measured by infection of CEM-GFP in duplicate and flow cytometry, reported as the mean of the two technical replicates ± standard deviation. Representative immunoblots of HA-tagged APOBEC3B proteins (HA) in cell lysates and HIV-1 particles produced by those cells are shown with their respective tubulin (TUB) and p24 (CA) loading controls. (B) Deaminase activity of human APOBEC3B, rhesus APOBEC3B, or various helix or single amino acid substitution constructs in cell lysates as measured by an oligo cleavage assay. A constant amount of each indicated APOBEC3 expression construct was transfected into HEK293T cells and lysates collected for oligo cleavage (top) or immunoblotting (bottom). Deaminase activity is indicated by cleavage of the oligo substrate to a smaller product. Expression of each deaminase was verified by immunoblotting for each of the HA-tagged APOBEC3 proteins as shown below.

Fig. 4

Conservation and predicted structural context of APOBEC3B D316. (A) Amino acid sequence alignment of the predicted loop 7 regions of human and rhesus APOBEC3B and their related family members. Blue residues indicate perfect conservation. APOBEC3B residue 316 and its predicted homologs are bolded in black (aspartic acid) or red (asparagine). (B) Predicted structure of human APOBEC3B C-terminal domain based on the human APOBEC3G C-terminal domain crystal structure (PDB: 3IR2). D316 is highlighted in red in close proximity to the catalytic E255 in green. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Naturally occurring APOBEC3B variants in rhesus macaques. An amino acid alignment displaying a portion of rhesus APOBEC3B near residue 316 and polymorphisms observed among 45 individual rhesus macaques. A dash ‘–’ indicates that all animals surveyed were homozygous for alleles encoding the residue in the above consensus. At positions in red, some individuals were homozygous for alleles encoding alternate amino acids, as indicated. An ‘X’ indicates that the individual is heterozygous for alleles encoding the two possible amino acids observed at this site. The numbers of individuals found to have each of the indicated genotypes are indicated to the right. A schematic of the predicted structural motifs based on the human APOBEC3G C-terminal domain crystal structure is depicted above (PDB: 3IR2). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)