Haplotype II (HapII-RDD, with RDD standing for the substitutions at position 105, 121, and 178, respectively) was seen in 3 of the 12 donors (Fig. APOBEC3H transcripts encoding a cluster of three substitutions, G105R, K121D, and E178D, were found in 6 of the 12 donors (Fig. APOBEC3H alleles encoding GKE at position 105, 121, and 178, respectively, were amplified from 10 of 12 donors, indicating that haplotype 1 is common (Fig. 1A displays the exon location of the mutations the rs numbers are listed in Materials and Methods section). We detected six SNPs in our data set at previously published polymorphic positions (Fig. The frequency of G-to-A mutations and the dinucleotide context of the mutations were analyzed with the Hypermut program ( 29).
Serum serial number splice software#
Reverse transcriptase (RT) sequences (600 bp) were manually edited and aligned using DNAStar and Bioedit software packages. DNA sequencing was performed by Agencourt Biosciences using BigDye Terminator v3.1 reagents. To assess the frequency of mutations in the proviral genome, a 1,905-nucleotide-long region of pol (HXB2, nucleotides 2928 to 4833) was amplified by PCR and cloned using a StrataClone kit as previously described ( 24). At 12 h postinfection, the cells were extensively washed with phosphate-buffered saline and genomic DNA was extracted using a DNeasy DNA isolation kit (Qiagen). TZM-bl cells were infected in 24-well tissue culture plates with DNase I (Invitrogen)-treated viral stocks. Culture medium was replaced the next day and supernatants were harvested 36 h later. Viral stocks were generated by transfecting NL4-3 WT (500 ng) and pTR600-FLAG-APOBEC3H variants (50 ng), pTR600-FLAG-APOBEC3G (50 ng), or pTR600 (50 ng) in HEK 293T cells. Moreover, APOBEC3H has been reported to cause hypermutation in both the hepatitis B virus ( 19) and in human papillomavirus genomes ( 33), suggesting the presence of enzymatic activity in mammalian systems. Indeed, human APOBEC3H displayed cytidine deaminase activity comparable to its rhesus homologue in a bacterial mutator assay ( 28). Protein expression levels of human APOBEC3H and that of the rhesus homologue differ greatly upon transfection into mammalian cells ( 9, 28), suggesting that the lack of potency of human APOBEC3H is a reflection of insufficient expression and/or protein stability in the producer cell, rather than a lack of enzymatic activity.
In contrast to the strong Vif-independent HIV-1 restriction exerted by the rhesus macaque APOBEC3H, the human protein seems to be limited in its antiretroviral activity ( 9, 28). APOBEC3H lacks the cytidine deaminase domain (CDA) that mediates RNA binding, homodimerization, and virion encapsidation of APOBEC3G ( 13, 26). Since prevalence of hapII-RDD is high in populations of African descent, these findings raise the possibility that some individuals may harbor effective as well as HIV-1 Vif-resistant intracellular antiviral defense mechanisms.ĪPOBEC3H mRNA has been detected in several human tissues (e.g., peripheral blood mononuclear cells, liver, skin, ovary, and testis) ( 19, 28). Thus, the anti-HIV activity of APOBEC3H seems to be regulated by a combination of genomic variation and alternative splicing. HIV-1 mutagenesis as a mode of action for APOBEC3H is suggested by the decrease of restriction observed with a cytidine deaminase domain mutant and the inverse correlation between G-to-A mutations and infectivity.
APOBEC3H variants were catalytically active and, similarly to APOBEC3F, favored a GA dinucleotide context. Site-directed mutagenesis identified G105R in hapI-GKE and D121K in hapII-RDD as critical substitutions leading to an average additional 10-fold increase in antiviral activity. Splice variants of hapI-GKE displayed a wide range of antiviral activities, whereas similar splicing events in hapII-RDD resulted in proteins that uniformly and efficiently restricted viral infectivity (>20-fold). Alternative splicing of APOBEC3H was common and resulted in variants with distinct C-terminal regions and variable antiretroviral activities. All APOBEC3H variants tested were resistant to HIV-1 Vif, the viral protein that efficiently counteracts APOBEC3G/3F activity.
We found that APOBEC3H variants encoding a SNP cluster (G105R, K121D and E178D, hapII-RDD) restricted human immunodeficiency virus type 1 (HIV-1) more efficiently than wild-type APOBEC3H (hapI-GKE). In this study, we show that APOBEC3H transcripts derived from human peripheral blood mononuclear cells are polymorphic in sequence and subject to alternative splicing. The impact of single nucleotide polymorphisms (SNP) and alternative splicing on the antiretroviral activity of human APOBEC3H is currently unknown. Human APOBEC3H belongs to the APOBEC3 family of cytidine deaminases that potently inhibit exogenous and endogenous retroviruses.