APOBEC3G impairs the multimerization of the HIV-1 Vif protein in living cells.

Journal of Virology. 2013 Jun; 87(11): 6492-506

Julien Batisse1, Santiago Guerrero1, Serena Bernacchi1, Ludovic Richert2, Julien Godet2, Valérie Goldschmidt2, Yves Mély2, Roland Marquet1, Hugues de Rocquigny2 & Jean-Christophe Paillart1

1Architecture et Réactivité de l’ARN, Université de Strasbourg, CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg Cedex, France;

2Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74 Route du Rhin, 67401, Illkirch Cedex, France.

 

ABSTRACT

The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Vif allows productive infection in non-permissive cells, including most natural HIV-1 target cells, by counteracting the cellular cytosine deaminases APOBEC3G (A3G) and A3F. Vif is also associated with the viral assembly complex and packaged into viral particles through interactions with the viral genomic RNA and the nucleocapsid domain of Pr55Gag. Recently, we showed that oligomerization of Vif into high-molecular mass complexes induces Vif folding and influences its binding to high affinity RNA binding sites present in the HIV genomic RNA. To get further insight into the role of Vif multimerization in viral assembly and A3G repression, we used fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer-based assays (FRET/FLIM) to investigate Vif-Vif interactions in living cells. By using two N-terminal tagged Vif proteins, we show that Vif-Vif interactions occur in living cells. This oligomerization is strongly reduced when the putative Vif multimerization domain (161PPLP164) is mutated, indicating that this domain is crucial, but that regions outside this motif also participate to Vif oligomerization. When co-expressed together with Pr55Gag, Vif is largely relocated to the cell membrane, where Vif oligomerization also occurs. Interestingly, wild-type A3G strongly interferes with Vif multimerization, contrary to an A3G mutant that does not bind to Vif. These findings confirm that Vif oligomerization occurs in living cells partly through its C-terminal motif and suggest that A3G may target and perturb the Vif oligomerization state to limit its functions in the cell.

 

SUPPLEMENT

HIV-1 natural target cells have developed a restriction activity to limit HIV-1 spreading. They express A3G/3F restriction factors that cause lethal damages during the next retroviral cycle. HIV-1 counteracts this restriction by encoding Vif that reduces the encapsidation of A3G/3F via two main mechanisms: (i) A3G degradation through the proteasome pathway and (ii) A3G translation inhibition. The mechanisms that regulate the balance between A3G restriction and Vif-mediated A3G repression are still not well understood and the multimerization state of Vif could play an important role, as previously suggested by other studies [1-3]. Indeed, it has been shown that mutations in the putative Vif multimerization domain (PPLP) reduce the virion infectivity, suggesting a loss of function in A3G repression. In our study, we showed the involvement of the PPLP motif in the multimerization of Vif in living cells. Interestingly, contrary to the presence of Pr55Gag, which relocated Vif to plasma membrane without affecting its multimerization state, the presence of A3G strongly interfered with Vif multimerization. Our data showed that the binding of A3G perturbs and dissociates Vif multimers into monomers and/or dimers most likely by a competition mechanism involving the PPLP motif.

Additionally, we showed the co-existence of two populations of Vif (monomeric and multimeric) that could potentially have distinct but related functions: as monomers, Vif could be in charge of the A3G targeting to the proteasome, whereas as multimers, Vif would be required for other functions, most likely linked to its RNA chaperone activity [4, 5], such as A3G translation inhibition [6] and virus assembly (Figure 1). Moreover, the capacity of Vif to form oligomers could also be a mean to increase its binding affinity to A3G protein. In this model, Vif would recognize A3G under its oligomeric state and A3G binding would lead to the disruption of Vif multimers (Figure 1, right panel). Such a disruption of Vif multimers by A3G could constitute an additional cellular regulation pathway to affect the functions of Vif requiring this oligomeric organization, thus leading to a reduction of virus assembly and spreading.

Jean-Christophe Paillart-1

Figure 1: Model of the dynamics of Vif protein in the cell. Vif is in equilibrium in the cell between oligomeric and monomeric states. In presence of A3G (right panel), the equilibrium is disrupted and the binding of A3G to Vif induces the recruitment of an E3-ubiquitin ligase complex and the degradation of A3G through the proteasome. In its oligomeric state (left panel) and thanks to its RNA chaperoning activity, Vif accurately reduces the translation of A3G via mRNA binding, and is able to regulate the virus assembly (RNA dimerization, Gag processing) and reverse transcription complexes.


Dynamically, we can suggest that during the early step of infection (post-entry), only a few Vif molecules are present to limit A3G function most likely through the proteasome pathway. Later on, after integration and proviral expression, Vif proteins are available in larger amount to assemble into oligomers and achieve other Vif functions.

Interestingly, Vif was partly relocated at the plasma membrane in presence of Pr55Gag but multimerization of Vif still occurred. This relocation leads to the hypothesis that Vif follows Pr55Gag trafficking, suggesting that the co-encapsidated partners of Pr55Gag are not packaged only by co-localization at the plasma membrane but are actively recruited at their synthesis site. Finally, since A3G preserved its ability to affect Vif multimerization in presence of Pr55Gag, this could reflect a direct competition between Vif and A3G for their interaction with Pr55Gag, suggesting another way for Vif to exclude A3G from the assembling viral particles.

Further studies will be needed to understand the role of Vif multimerization in A3G regulation and its different functions in the cell.

 

Reference:

1.   Bernacchi, S., et al., Importance of the proline-rich multimerization domain on the oligomerization and nucleic acid binding properties of HIV-1 Vif. Nucleic Acids Res, 2011. 39(6): p. 2404-15.

2.   Donahue, J.P., et al., The HIV-1 Vif PPLP motif is necessary for human APOBEC3G binding and degradation. Virology, 2008. 377(1): p. 49-53.

3.   Yang, B., et al., Potent suppression of viral infectivity by the peptides that inhibit multimerization of human immunodeficiency virus type 1 (HIV-1) Vif proteins. J Biol Chem, 2003. 278(8): p. 6596-602.

4.   Batisse, J., et al., The role of Vif oligomerization and RNA chaperone activity in HIV-1 replication. Virus Res, 2012.

5.   Henriet, S., et al., Vif is a RNA chaperone that could temporally regulate RNA dimerization and the early steps of HIV-1 reverse transcription. Nucleic Acids Res, 2007. 35(15): p. 5141-53.

6.   Mercenne, G., et al., HIV-1 Vif binds to APOBEC3G mRNA and inhibits its translation. Nucleic Acids Res, 2010. 38(2): p. 633-46.

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