The Enzymatic Activity of CEM15/Apobec-3G Is Essential for the Regulation of the Infectivity of HIV-1 Virion but Not a Sole Determinant of Its Antiviral Activity

Abstract

Human immunodeficiency virus, type 1 (HIV-1) Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion. Vif functions to counteract an anti-HIV-1 cellular factor in non-permissive cells, CEM15/Apobec-3G, which shares a cytidine deaminase motif. CEM15/Apobec-3G deaminates dC to dU in the minus strand DNA of HIV-1, resulting in G to A hypermutation in the plus strand DNA. In this study, we have done the mutagenesis analysis on two cytidine deaminase motifs in CEM15/Apobec-3G and examined their antiviral functions as well as the DNA editing activity. Point mutations in the C-terminal active site such as E259Q and C291A almost completely abrogated the antiviral function, while those in the N-terminal active site such as E67Q and C100A retained this activity to a lesser extent as compared with that of the wild type. The DNA editing activities of E67Q and E259Q mutants were both retained but impaired to the same extent. This indicates that the enzymatic activity of this protein is essential but not a sole determinant of the antiviral activity. Furthermore, all the deletion mutants tested in this study lost the antiviral activity because of the loss of the activity for dimerization, suggesting that the entire protein structure is necessary for the antiviral function.

The Retroviral Hypermutation Specificity of APOBEC3F and APOBEC3G Is Governed by the C-terminal DNA Cytosine Deaminase Domain

“…Both studies reported that mutations in either deaminase domain rendered APOBEC3G unable to restrict infection, and it was therefore not possible to define the source of the deaminase activity causing retroviral hypermutation or whether other mechanistic steps were disrupted. In contrast, a third study indicated that the N-terminal glutamate was dispensable, whereas the C-terminal glutamate of APOBEC3G was essential for restricting HIV-1 infection (29). Although this initially appears consistent with our data, both mutants still showed moderate levels of G3 A mutation suggesting that either domain can mediate catalysis (29).…”

“…In contrast, a third study indicated that the N-terminal glutamate was dispensable, whereas the C-terminal glutamate of APOBEC3G was essential for restricting HIV-1 infection (29). Although this initially appears consistent with our data, both mutants still showed moderate levels of G3 A mutation suggesting that either domain can mediate catalysis (29). This apparent conundrum may be attributable to reverse transcriptase error and/or to the APOBEC3G mutants retaining a low level of deaminase activity.…”

“…The apoB mRNA editing protein APOBEC1 and the antibody gene deaminase AID are examples of single domain cytosine deaminases, and their activity is abolished by the mutation of the conserved deaminase domain residues (8, 26 -28). In contrast, APOBEC3F and -3G have two deaminase domains, both of which appear to be essential for retroviral restriction (1,3,29). However, it is not presently clear whether one or both of these domains participates directly in the deamination reaction itself.…”

Enzymatically Active APOBEC3G Is Required for Efficient Inhibition of Human Immunodeficiency Virus Type 1

“…Some of these effects do not require catalytically active APO3G (19,22), and several reports suggested that deaminase-defective APO3G and APO3F have antiviral activity when transiently coexpressed with HIV-1 in 293T cells (3,12,28,35). Our own data concerning the antiviral properties of the deaminase-defective APO3G C288S/C291A mutant supported these conclusions (30).…”

“…In the absence of Vif, APO3G is efficiently packaged into HIV virions and inhibits virus replication. A number of studies reported that the presence of APO3G in the virus can result in hypermutation of the viral minus-strand cDNA during reverse transcription (11,18,23,24,42,45), inhibition of reverse transcription (9), tRNA annealing or tRNA processing (10, 26), DNA strand transfer (19, 26), or integration (22, 26).Some of these effects do not require catalytically active APO3G (19,22), and several reports suggested that deaminase-defective APO3G and APO3F have antiviral activity when transiently coexpressed with HIV-1 in 293T cells (3,12,28,35). Our own data concerning the antiviral properties of the deaminase-defective APO3G C288S/C291A mutant supported these conclusions (30).…”