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HIV restriction by APOBEC3 in humanized mice.

ABSTRACT: Innate immune restriction factors represent important specialized barriers to zoonotic transmission of viruses. Significant consideration has been given to their possible use for therapeutic benefit. The apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 (APOBEC3) family of cytidine deaminases are potent immune defense molecules capable of efficiently restricting endogenous retroelements as well as a broad range of viruses including Human Immunodeficiency virus (HIV), Hepatitis B virus (HBV), Human Papilloma virus (HPV), and Human T Cell Leukemia virus (HTLV). The best characterized members of this family are APOBEC3G (A3G) and APOBEC3F (A3F) and their restriction of HIV. HIV has evolved to counteract these powerful restriction factors by encoding an accessory gene designated viral infectivity factor (vif). Here we demonstrate that APOBEC3 efficiently restricts CCR5-tropic HIV in the absence of Vif. However, our results also show that CXCR4-tropic HIV can escape from APOBEC3 restriction and replicate in vivo independent of Vif. Molecular analysis identified thymocytes as cells with reduced A3G and A3F expression. Direct injection of vif-defective HIV into the thymus resulted in viral replication and dissemination detected by plasma viral load analysis; however, vif-defective viruses remained sensitive to APOBEC3 restriction as extensive G to A mutation was observed in proviral DNA recovered from other organs. Remarkably, HIV replication persisted despite the inability of HIV to develop resistance to APOBEC3 in the absence of Vif. Our results provide novel insight into a highly specific subset of cells that potentially circumvent the action of APOBEC3; however our results also demonstrate the massive inactivation of CCR5-tropic HIV in the absence of Vif.

SUBMITTER: Krisko JF

PROVIDER: S-EPMC3610649 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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HIV restriction by APOBEC3 in humanized mice.

Krisko John F JF Martinez-Torres Francisco F Foster John L JL Garcia J Victor JV

PLoS pathogens 20130328 3

Innate immune restriction factors represent important specialized barriers to zoonotic transmission of viruses. Significant consideration has been given to their possible use for therapeutic benefit. The apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 (APOBEC3) family of cytidine deaminases are potent immune defense molecules capable of efficiently restricting endogenous retroelements as well as a broad range of viruses including Human Immunodeficiency virus (HIV), Hepatitis B virus ...[more]

PMID: 23555255

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Project description:BackgroundFeline immunodeficiency virus (FIV) is a global pathogen of Felidae species and a model system for Human immunodeficiency virus (HIV)-induced AIDS. In felids such as the domestic cat (Felis catus), APOBEC3 (A3) genes encode for single-domain A3Z2s, A3Z3 and double-domain A3Z2Z3 anti-viral cytidine deaminases. The feline A3Z2Z3 is expressed following read-through transcription and alternative splicing, introducing a previously untranslated exon in frame, encoding a domain insertion called linker. Only A3Z3 and A3Z2Z3 inhibit Vif-deficient FIV. Feline A3s also are restriction factors for HIV and Simian immunodeficiency viruses (SIV). Surprisingly, HIV-2/SIV Vifs can counteract feline A3Z2Z3.ResultsTo identify residues in feline A3s that Vifs need for interaction and degradation, chimeric human-feline A3s were tested. Here we describe the molecular direct interaction of feline A3s with Vif proteins from cat FIV and present the first structural A3 model locating these interaction regions. In the Z3 domain we have identified residues involved in binding of FIV Vif, and their mutation blocked Vif-induced A3Z3 degradation. We further identified additional essential residues for FIV Vif interaction in the A3Z2 domain, allowing the generation of FIV Vif resistant A3Z2Z3. Mutated feline A3s also showed resistance to the Vif of a lion-specific FIV, indicating an evolutionary conserved Vif-A3 binding. Comparative modelling of feline A3Z2Z3 suggests that the residues interacting with FIV Vif have, unlike Vif-interacting residues in human A3s, a unique location at the domain interface of Z2 and Z3 and that the linker forms a homeobox-like domain protruding of the Z2Z3 core. HIV-2/SIV Vifs efficiently degrade feline A3Z2Z3 by possible targeting the linker stretch connecting both Z-domains.ConclusionsHere we identified in feline A3s residues important for binding of FIV Vif and a unique protein domain insertion (linker). To understand Vif evolution, a structural model of the feline A3 was developed. Our results show that HIV Vif binds human A3s differently than FIV Vif feline A3s. The linker insertion is suggested to form a homeo-box domain, which is unique to A3s of cats and related species, and not found in human and mouse A3s. Together, these findings indicate a specific and different A3 evolution in cats and human.

Project description:UnlabelledThe APOBEC3 deoxycytidine deaminases can restrict the replication of HIV-1 in cell culture to differing degrees. The effects of APOBEC3 enzymes are largely suppressed by HIV-1 Vif that interacts with host proteins to form a Cullin5-Ring E3 ubiquitin ligase that induces (48)K-linked polyubiquitination (poly-Ub) and proteasomal degradation of APOBEC3 enzymes. Vif variants have differing abilities to induce degradation of APOBEC3 enzymes and the underlying biochemical mechanisms for these differences is not fully understood. We hypothesized that by characterizing the interaction of multiple APOBEC3 enzymes and Vif variants we could identify common features that resulted in Vif-mediated degradation and further define the determinants required for efficient Vif-mediated degradation of APOBEC3 enzymes. We used Vifs from HIV-1 NL4-3 (IIIB) and HXB2 to characterize their induced degradation of and interaction with APOBEC3G, APOBEC3G D128K, APOBEC3H, and APOBEC3B in 293T cells. We quantified the APOBEC3G-Vif and APOBEC3H-Vif interaction strengths in vitro using rotational anisotropy. Our biochemical and cellular analyses of the interactions support a model in which the degradation efficiency of VifIIIB and VifHXB2 correlated with both the binding strength of the APOBEC3-Vif interaction and the APOBEC3-Vif interface, which differs for APOBEC3G and APOBEC3H. Notably, Vif bound to APOBEC3H and APOBEC3B in the natural absence of Vif-induced degradation and the interaction resulted in (63)K-linked poly-Ub of APOBEC3H and APOBEC3B, demonstrating additional functionality of the APOBEC3-Vif interaction apart from induction of proteasomal degradation.ImportanceAPOBEC3 enzymes can potently restrict the replication of HIV-1 in the absence of HIV-1 Vif. Vif suppresses APOBEC3 action by inducing their degradation through a direct interaction with APOBEC3 enzymes and other host proteins. Vif variants from different HIV-1 strains have different effects on APOBEC3 enzymes. We used differing Vif degradation capacities of two Vif variants and various APOBEC3 enzymes with differential sensitivities to Vif to delineate determinants of the APOBEC3-Vif interaction that are required for inducing efficient degradation. Using a combined biochemical and cellular approach we identified that the strength of the APOBEC3-Vif binding interaction and the APOBEC3-Vif interface are determinants for degradation efficiency. Our results highlight the importance of using Vif variants with different degradation potential when delineating mechanisms of Vif-induced APOBEC3 degradation and identify features important for consideration in the development of HIV-1 therapies that disrupt the APOBEC3-Vif interaction.

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HIV restriction by APOBEC3 in humanized mice.

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HIV restriction by APOBEC3 in humanized mice.

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