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Updated: Apr 18, 2026
Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells
Published on: January 30, 2019
Sequence and structural determinants of human APOBEC3H deaminase and anti-HIV-1 activities
Insights
Human APOBEC3H (A3H) exhibits potent antiviral activity against HIV-1, functioning similarly to double-domain A3 proteins. A3H utilizes both deaminase-dependent and -independent pathways to inhibit viral replication.
Area of Science:
- Immunology
- Virology
- Molecular Biology
Background:
- Human APOBEC3H (A3H) is a cytidine deaminase and a host restriction factor.
- A3H has seven haplotypes with diverse phenotypes and a unique Z3 deaminase domain.
- A3H HapII shows potent activity against HIV-1, distinct from A3A and A3C.
Purpose of the Study:
- To identify determinants of A3H HapII deaminase and antiviral activities.
- To elucidate the molecular mechanisms underlying A3H's restriction of HIV-1.
Main Methods:
- Homology modeling of A3H HapII structure.
- Site-directed mutagenesis and sequence/structure-guided modifications.
- Cell-based, biochemical, and HIV-1 infectivity assays.
Main Results:
- A homology model revealed basic residues crucial for nucleic acid binding.
- RNase A treatment is required for A3H deaminase activity, indicating RNA inhibition.
- Mutagenesis of nucleic acid binding sites reduced or abolished enzymatic activity.
- A3H mutants, even catalytically defective ones, retained antiviral activity against HIV-1.
Conclusions:
- A3H's molecular and biological activities resemble double-domain A3 proteins.
- A3H employs both deaminase-dependent and -independent mechanisms to restrict HIV-1.
- A3H targets reverse transcription to inhibit HIV-1 replication.
Background:
Human APOBEC3H (A3H) belongs to the A3 family of host restriction factors, which are cytidine deaminases that catalyze conversion of deoxycytidine to deoxyuridine in single-stranded DNA. A3 proteins contain either one (A3A, A3C, A3H) or two (A3B, A3D, A3F, A3G) Zn-binding domains. A3H has seven haplotypes (I-VII) that exhibit diverse biological phenotypes and geographical distribution in the human population. Its single Zn-coordinating deaminase domain belongs to a phylogenetic cluster (Z3) that is different from the Z1- and Z2-type domains in other human A3 proteins. A3H HapII, unlike A3A or A3C, has potent activity against HIV-1. Here, we sought to identify the determinants of A3H HapII deaminase and antiviral activities, using site-directed sequence- and structure-guided mutagenesis together with cell-based, biochemical, and HIV-1 infectivity assays.
Results:
We have constructed a homology model of A3H HapII, which is similar to the known structures of other A3 proteins. The model revealed a large cluster of basic residues (not present in A3A or A3C) that are likely to be involved in nucleic acid binding. Indeed, RNase A pretreatment of 293T cell lysates expressing A3H was shown to be required for detection of deaminase activity, indicating that interaction with cellular RNAs inhibits A3H catalytic function. Similar observations have been made with A3G. Analysis of A3H deaminase substrate specificity demonstrated that a 5' T adjacent to the catalytic C is preferred. Changing the putative nucleic acid binding residues identified by the model resulted in reduction or abrogation of enzymatic activity, while substituting Z3-specific residues in A3H to the corresponding residues in other A3 proteins did not affect enzyme function. As shown for A3G and A3F, some A3H mutants were defective in catalysis, but retained antiviral activity against HIV-1vif (-) virions. Furthermore, endogenous reverse transcription assays demonstrated that the E56A catalytic mutant inhibits HIV-1 DNA synthesis, although not as efficiently as wild type.
Conclusions:
The molecular and biological activities of A3H are more similar to those of the double-domain A3 proteins than to those of A3A or A3C. Importantly, A3H appears to use both deaminase-dependent and -independent mechanisms to target reverse transcription and restrict HIV-1 replication.
