mRNA expression pattern of APOBEC3H in human tissues. Both APOBEC3G and APOBEC3H were PCR amplified with specific primers from cDNAs made from a panel of different human tissues. Molecular weight markers are shown every ninth lane, and the tissue of origin is marked above each lane. Boxes indicate expression of human APOBEC3G and human APOBEC3H in PBLs and germ line cells (sites relevant to retroviral replication).

Steady-state expression levels of APOBEC3H proteins are drastically different. Lysates of 293T cells transiently transfected with APOBEC expression plasmids were analyzed by Western blotting and semiquantitative RT-PCR to determine relative levels of protein and mRNA, respectively. (A) APOBEC3H protein levels were detected in cell lysates 48 h after transfection. The predicted size of HA-tagged human APOBEC3H is ∼23 kDa, and that of macaque APOBEC3H is ∼26 kDa. Identical results were obtained with an alternate expression vector (data not shown), ruling out an effect specific to the pCS2HA construct used in these experiments. (B) Cytoplasmic mRNAs were reverse transcribed with an APOBEC3H-specific primer, and serial dilutions (1:2) were used as a template for PCR amplification. Amplification of APOBEC3H transcripts is equivalent for both the human and macaque APOBEC3H transfections. The control (no reverse transcriptase) was included to rule out the possibility of plasmid DNA contamination. (C) The proteasome inhibitor MG-132 was added to transient transfections of human APOBEC3H and macaque APOBEC3H, and lysates were used for Western blot analysis. Samples harvested at the 2- and 20-h time points revealed approximately fivefold stabilization of the human APOBEC3H protein, even at the earliest time point. In the bottom part, 10-fold less macaque APOBEC3H was transfected.

APOBEC3H induces G-to-A hypermutation of retroviral genomes. Retroviral hypermutation induced by APOBEC proteins was evaluated by sequencing HIVΔvif proviral genomes following infection of a target cell line, as described in Materials and Methods. (A) Sequences were obtained from infections with viral supernatants from transfections containing human APOBEC3G, macaque APOBEC3H, human APOBEC3H, or no APOBEC expression plasmid. G-to-A changes were the only mutations observed in ∼9,000 bp of the sequenced genomes, ruling out the likelihood of generalized reverse transcriptase errors or experimentally introduced mutations. (B) Hypermutated viral sequences were analyzed to determine the sequence context of the deaminated cytidine. The number of deamination target sites evaluated for each APOBEC is noted (huA3G = 21, macA3H = 20). Shown are percentages of each nucleotide found at the −2, −1, and +1 positions relative to the deaminated cytidine (C). Macaque APOBEC3H demonstrates a clear TC dinucleotide preference.

APOBEC3H is conserved in mammalian genomes and adaptively evolving in primates. APOBEC3 genes from humans, primates, and other mammals were used for an analysis of the evolutionary history and selective pressures affecting APOBEC3H throughout mammalian evolution. (A) A neighbor-joining tree was constructed in CLUSTAL_X based on a protein alignment of APOBEC sequences from humans and nonprimate eutherian mammals. The N-terminal and C-terminal domains of double-domain APOBEC proteins have been split and are designated -N and -C, respectively. APOBEC3 proteins from mammals in which more than one APOBEC3 was identified are numbered. An APOBEC3H-like domain (red lineages, Z2) has been conserved in a number of mammalian species, including mice, rats, dogs, horses, pigs, cows, and primates. This phylogeny is rooted with the most closely related activation-induced deaminase, cytidine deaminase, as an outgroup lineage; the placement of this root is shown by an arrow. Bootstrap support for the groupings is indicated by numbers next to the relevant branches; those nodes that were supported by a Bayesian maximum-likelihood analysis are indicated with an asterisk. (B) Schematic of APOBEC3 genes identified in representative mammalian genomes, including the mouse, cow, dog, and human genomes. Red denotes Z2 (APOBEC3H like), while blue and purple denote Z1A and Z1B, respectively (the nomenclature system used was suggested in reference 5). While rodents encode only one APOBEC3 gene (a Z1-Z2 fusion), the cow and pig genomes both contain at least two APOBEC3 genes, one whose domain structure mirrors that of the rodent Z1-Z2 fusion gene, as well as an additional Z1-type gene. The horse and dog genomes contain at least three single-domain APOBEC3 genes, two Z1-type genes, and a Z2-type gene; however, the dog genes have not been assigned to a genomic contig yet and hence the chromosomal assignment, and the order of the genes, is still tentative (this uncertainty is represented as a dotted line). (C) APOBEC3H is adaptively evolving in primates. dN/dS ratios were calculated and are indicated on the APOBEC3H phylogeny, which is completely congruent with the accepted primate phylogeny (24). We obtained APOBEC3H sequences from a panel of six hominoids, four OWMs, and three NWMs. dN/dS ratios were calculated along each branch of the phylogeny with the free-ratio model in the PAML package that allows the dN/dS ratio to vary along each branch. A dN/dS ratio of greater than 1 is indicative of positive selection. In some instances, zero synonymous substitutions lead to an apparent dN/dS ratio of infinity (shown with an asterisk). mya, million years ago.

Restriction of HIV and SIV by macaque but not human APOBEC3H. Single-round viral infectivity assays were used to assess the relative abilities of APOBEC3 proteins to inhibit retroviral replication, as described in Materials and Methods. (A) Effects of human APOBEC3G (huApo3G), macaque APOBEC3H (macApo3H), and human APOBEC3H (huApo3H) on HIV-1 infectivity. Black boxes are HIV-WT, and white boxes are HIVΔvif. Results are shown as percent infectivity of the control infection in which no APOBEC expression plasmid was included. Results from one representative experiment are shown (error bars reflect variation between triplicate infections within one experiment). (B) The same as panel A, except that APOBEC genes were assayed for activity against SIVagm, a related nonhuman primate lentivirus. Of note, the twofold decrease in SIVagmΔvif infectivity in the presence of human APOBEC3H was not reproducible in independent experiments. (C) Single-round viral infectivity assay of HIVΔvif produced in the presence of increasing amounts of APOBEC3H expression plasmids. Human APOBEC3H (open squares) plasmids decreased HIV-1 infectivity less than 2-fold at all of the concentrations of transfected plasmid used, while macaque APOBEC3H (solid circles, solid lines) decreased infectivity almost 50-fold. A catalytic-site mutant form of macaque APOBEC3H (macApo3H E56A; solid circles, dotted lines) was severely impaired in its ability to inhibit HIV-1 replication.

Cytidine deaminase activity is conserved in APOBEC3H homologs. (A) Macaque and human APOBEC3H homologs were expressed in a bacterial system. Whole-cell lysates were used for Western blot analysis to evaluate expression levels of APOBEC proteins in bacteria. Sizes of myc-tagged APOBEC proteins are ∼48 kDa for human APOBEC3G, ∼23 kDa for human APOBEC3H, and ∼26 kDa for macaque APOBEC3H. All of the APOBEC proteins were stably expressed in this system. (B) APOBEC proteins were evaluated for the ability to affect rates of acquired resistance to rifampin in a bacterial cytidine deamination assay. Results are shown as the number of rifampin-resistant (Rif^r) colonies normalized for cell number (OD₆₀₀). One representative experiment is shown in which eight independent cultures were evaluated. Of note, both the human APOBEC3H and macaque APOBEC3H mutation frequencies were higher than the control (Vector Only) in independent experiments (P < 0.05). However, the potency of the macaque and human APOBEC3H proteins relative to one another varied in independent experiments and should not be compared.