The Localization of APOBEC3H Variants in HIV-1 Virions Determines Their Antiviral Activity

Abstract

Several members of the human APOBEC3 family of cytidine deaminases can potently restrict retroviruses such as HIV-1. The single-domain APOBEC3H (A3H) is encoded by four haplotypes, of which only A3H haplotype II-RDD (hapII-RDD) restricts HIV-1 efficiently. The goal of this study was to elucidate the mechanisms underlying the differences in antiviral activity among A3H haplotypes. The naturally occurring A3H hapI-GKE and hapII-RDD variants differ at three amino acid positions. A panel of six site-directed mutants containing combinations of the three variable residues was used to determine A3H protein expression, requirements of A3H virion incorporation, and A3H-Gag interactions. The catalytic activity of each A3H protein was assessed directly by using an Escherichia coli mutator assay. We found that the incorporation efficiencies of A3H variants into HIV-1 virions were comparable despite major differences in cellular expression. An assessment of the enzymes' catalytic activities showed that the deaminase activity of each A3H variant correlated with protein expression, suggesting similar enzymatic efficiencies. Surprisingly, virion incorporation experiments using Gag deletion mutants demonstrated that A3H haplotypes interacted with different Gag regions. A3H hapII-RDD associated with nucleocapsid in an RNA-dependent manner, whereas A3H hapI-GKE associated with the C-terminal part of matrix and the N-terminal capsid domain. Our results show that the A3H hapII-RDD interaction with nucleocapsid is critical for its antiviral activity and that the inability of A3H hapI-GKE to interact with nucleocapsid underlies its limited antiviral potential. Thus, the antiviral activity of A3H haplotypes is determined by its incorporation into the viral core, in proximity to the reverse transcription complex.

Analysis of Human APOBEC3H Haplotypes and Anti-Human Immunodeficiency Virus Type 1 Activity

“…4B). Thus, we conclude that A3H binds HIV-1 NC in an RNA-dependent manner, which is consistent with a recent study (29). Next, we determined whether A3H contains a similar YYFW motif that serves as the packaging signal.…”

“…Recently, Ooms et al reported that although A3H hap I protein was poorly expressed in cells, it could be still packaged efficiently into HIV-1 virions. They also presented evidence that A3H hap I specifically interacted with MA-CA other than NC for virion packaging (29). We found that A3H hap I was poorly packaged into virions because it was not expressed in the cell, and, therefore, we cannot confirm this observation.…”

“…We have studied the function of A3H CDD and found that it is required for A3H anti-HIV-1 activity, but it is not required for virion packaging. Since the deaminase activity of A3H has been demonstrated in Escherichia coli (28,29), we believe it is most likely that the deaminase activity is required for A3H anti-HIV-1 activity. Although its virion packaging is independent of its CDD, it is dependent on an YYXW motif, which was identified as a packaging signal for A3G.…”

Determinants of Efficient Degradation of APOBEC3 Restriction Factors by HIV-1 Vif

“…A 63 K-linked poly-Ub chain on Gag or a cellular protein in the vicinity of the emerging viral bud is necessary to stimulate virus release (73,74). HIV may take advantage of 63 K-linked poly-Ub A3H, which can associate with Gag through an RNA intermediate or directly (75), to promote virus release. This may aid in virus budding and titrate out the effect of the A3H during viral adaptation akin to how Vif mutants unable to degrade A3G can acquire mutations that enable the production of more virus particles to minimize A3G packaging (76,77).…”

Suppression of HIV-1 Infection by APOBEC3 Proteins in Primary Human CD4 + T Cells Is Associated with Inhibition of Processive Reverse Transcription as Well as Excessive Cytidine Deamination

“…The first family member to be recognized for this activity, APOBEC3G (A3G), was identified on account of being the cellular target for the human immunodeficiency virus type 1 (HIV-1) regulatory protein Vif (5), a feature that is also shared by APOBEC3D (A3D) (6,7), APOBEC3F (A3F) (8)(9)(10)(11) and haplotypes II, V, and VII of APOBEC3H (A3H) (12)(13)(14). In the context of HIV-1 infection, the current weight of evidence indicates that A3G may exert the more potent antiviral effect, with A3D, A3F, and A3H eliciting less pronounced effects, though this continues to be debated.…”