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The Role of RNA in HIV-1 Vif-Mediated Degradation of APOBEC3H.

ABSTRACT: As many as five members of the APOBEC3 family of DNA cytosine deaminases are capable of inhibiting HIV-1 replication by deaminating viral cDNA cytosines and interfering with reverse transcription. HIV-1 counteracts restriction with the virally encoded Vif protein, which forms a hybrid ubiquitin ligase complex that directly binds APOBEC3 enzymes and targets them for proteasomal degradation. APOBEC3H (A3H) is unique among family members by dimerization through cellular and viral duplex RNA species. RNA binding is required for localization of A3H to the cytoplasmic compartment, for efficient packaging into nascent HIV-1 particles and ultimately for effective virus restriction activity. Here we compared wild-type human A3H and RNA binding-defective mutants to ask whether RNA may be a factor in the functional interaction with HIV-1 Vif. We used structural modeling, immunoblotting, live cell imaging, and split green fluorescence protein (GFP) reconstitution approaches to assess the capability of HIV-1 Vif to promote the degradation of wild-type A3H in comparison to RNA binding-defective mutants. The results combined to show that RNA is not strictly required for Vif-mediated degradation of A3H, and that RNA and Vif are likely to bind this single-domain DNA cytosine deaminase on physically distinct surfaces. However, a subset of the results also indicated that the A3H degradation process may be affected by A3H protein structure, subcellular localization, and differences in the constellation of A3H interaction partners, suggesting additional factors may also influence the fate and functionality of this host-pathogen interaction.

SUBMITTER: Wang J

PROVIDER: S-EPMC6948013 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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The Role of RNA in HIV-1 Vif-Mediated Degradation of APOBEC3H.

Wang Jiayi J Becker Jordan T JT Shi Ke K Lauer Kate V KV Salamango Daniel J DJ Aihara Hideki H Shaban Nadine M NM Harris Reuben S RS

Journal of molecular biology 20191016 24

As many as five members of the APOBEC3 family of DNA cytosine deaminases are capable of inhibiting HIV-1 replication by deaminating viral cDNA cytosines and interfering with reverse transcription. HIV-1 counteracts restriction with the virally encoded Vif protein, which forms a hybrid ubiquitin ligase complex that directly binds APOBEC3 enzymes and targets them for proteasomal degradation. APOBEC3H (A3H) is unique among family members by dimerization through cellular and viral duplex RNA species ...[more]

PMID: 31628948

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Project description:Human APOBEC3H (A3H) is a cytidine deaminase that inhibits HIV-1 replication. To evade this restriction, the HIV-1 Vif protein binds A3H and mediates its proteasomal degradation. To date, little information on the Vif-A3H interface has been available. To decipher how both proteins interact, we first mapped the Vif-binding site on A3H by functionally testing a large set of A3H mutants in single-cycle infectivity and replication assays. Our data show that the two A3H ?-helixes ?3 and ?4 represent the Vif-binding site of A3H. We next used viral adaptation and a set of Vif mutants to identify novel, reciprocal Vif variants that rescued viral infectivity in the presence of two Vif-resistant A3H mutants. These A3H-Vif interaction points were used to generate the first A3H-Vif structure model, which revealed that the A3H helixes ?3 and ?4 interact with the Vif ?-sheet (?2-?5). This model is in good agreement with previously reported Vif and A3H amino acids important for interaction. Based on the predicted A3H-Vif interface, we tested additional points of contact, which validated our model. Moreover, these experiments showed that the A3H and A3G binding sites on HIV-1 Vif are largely distinct, with both host proteins interacting with Vif ?-strand 2. Taken together, this virus-host interface model explains previously reported data and will help to identify novel drug targets to combat HIV-1 infection.IMPORTANCE HIV-1 needs to overcome several intracellular restriction factors in order to replicate efficiently. The human APOBEC3 locus encodes seven proteins, of which A3D, A3F, A3G, and A3H restrict HIV-1. HIV encodes the Vif protein, which binds to the APOBEC3 proteins and leads to their proteasomal degradation. No HIV-1 Vif-APOBEC3 costructure exists to date despite extensive research. We and others previously generated HIV-1 Vif costructure models with A3G and A3F by mapping specific contact points between both proteins. Here, we applied a similar approach to HIV-1 Vif and A3H and successfully generated a Vif-A3H interaction model. Importantly, we find that the HIV-1 Vif-A3H interface is distinct from the Vif-A3G and Vif-A3F interfaces, with a small Vif region being important for recognition of both A3G and A3H. Our Vif-A3H structure model informs on how both proteins interact and could guide toward approaches to block the Vif-A3H interface to target HIV replication.

Dataset Information

The Role of RNA in HIV-1 Vif-Mediated Degradation of APOBEC3H.

Publications

The Role of RNA in HIV-1 Vif-Mediated Degradation of APOBEC3H.

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