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Host-pathogen interactions in viral infections

 

The HIV-1 Vif protein in the reverse transcription process





 

The HIV-1 auxiliary protein Vif plays an essential role in counteracting the cellular restriction factor APOBEC3G, a cytidine deaminase. Vif prevents incorporation of APOBEC3G in virions, thus reducing the APOBEC3G-mediated deamination and uracil accumulation in the viral genome.

We have shown that Vif functionally interacts with the HIV-1 reverse transcriptase, increasing its catalytic efficiency and its ability to bypass abasic sites, which transiently originate from uracil removal during reverse transcription.

HIV-1 Vif possesses four distinct functional activities:

- Interacts with RNA and DNA through its N-terminal domain

- Binds Zn through a Zn-finger located in the central portion of the protein

- Interacts with APOBEC3G and with the ubiquitination machinery through its C-terminal domain

- Interacts with HIV-1 RT

 

We are currently investigating the functional and spatial relationships among these different properties, trying to identify the domains involved in the interaction with HIV-1 RT.

 

The HIV-1 Rev- human DDX3 interaction

The HIV-1 protein Rev is essential for nuclear export of viral unspliced or partially spliced transcripts. Recently, a functional interaction between Rev and the cellular protein DDX3 has been established.





 

DDX3 is a DEAD-box protein, with RNA helicase and ATPase activities.

With the aim of better understanding the role of DDX3 in HIV-1 infection, we have cloned and expressed human DDX3 and characterized its ATPase activity. Our results show that hDDX3 coupes high catalytic efficiency with relaxed substrate specificity, being able to bind several nucleotide analogs. Based on these results, we undertook the rational design of DDX3 inhibitors as antiviral agents. Through the collaboration with the group of medicinal chemistry led by Prof. Maurizio Botta at the University of Siena, a pharmacophore has been created starting from the X-ray structure of DDX3 in complex with AMP.





 

This has been used in a virtual screening approach to identify potential drug candidates.





 

From the first identified hits, drug optimization led us to develop potent anti-DDX3 compounds capable of blocking HIV replication in infected cells





 

Identification of cellular interactors for the avian influenza NS1 protein

 

The protein NS1 of infleunza viruses is expressed only in infected cells and plays important roles in the modulation of the cell metabolic pathways to the virus advantage.

The extreme C-ter of NS1 contains a PDZ-ligand (PL) domain, capable of potential interactions with hundreds of cellular proteins containing the PDZ module. We have undertook a systematic search for PDZ interactors in human cells for the NS1 proteins of avian influenza viruses. We have identified a novel tandem PL domain in a subset of avian influenza viruses, capable of interacting with several cellular proteins. The H7N1 NS1 protein containing this domain (F3) was compared to the mutant H7N1 protein containing only one domain (F4), and to the H5N1 NS1 in PDZ binding assays with human protein arrays. This led to the identification of several cellular partners for these proteins.





 

One of this proteins was RIL, a regulator of the tyrosine kinase Src. We found that Src directly binds to H7N1 NS1 through its SH3-binding domain. In addition, we found that NS1 activates Src during infection, promoting cell proliferation.

 

 

References

 

Radi M, Falchi F, Garbelli A, Samuele A, Bernardo V, Paolucci S, Baldanti F, Schenone S, Manetti F, Maga G, Botta M. Discovery of the first small molecule inhibitor of human DDX3 specifically designed to target the RNA binding site: towards the next generation HIV-1 inhibitors. Bioorg Med Chem Lett. 2012 Mar 1;22(5):2094-8.

 

Maga G, Falchi F, Radi M, Botta L, Casaluce G, Bernardini M, Irannejad H, Manetti F, Garbelli A, Samuele A, Zanoli S, Esté JA, Gonzalez E, Zucca E, Paolucci S, Baldanti F, De Rijck J, Debyser Z, Botta M. Toward the discovery of novel anti-HIV drugs. Second-generation inhibitors of the cellular ATPase DDX3 with improved anti-HIV activity: synthesis, structure-activity relationship analysis, cytotoxicity studies, and target validation. ChemMedChem. 2011 Aug 1;6(8):1371-89.

 

Garbelli A, Radi M, Falchi F, Beermann S, Zanoli S, Manetti F, Dietrich U, Botta M, Maga G. Targeting the human DEAD-box polypeptide 3 (DDX3) RNA helicase as a novel strategy to inhibit viral replication. Curr Med Chem. 2011;18(20):3015-27. Review.

 

Garbelli A, Beermann S, Di Cicco G, Dietrich U, Maga G. A motif unique to the  human DEAD-box protein DDX3 is important for nucleic acid binding, ATP hydrolysis, RNA/DNA unwinding and HIV-1 replication. PLoS One. 2011 May 12;6(5):e19810.

 

Maga G, Falchi F, Garbelli A, Belfiore A, Witvrouw M, Manetti F, Botta M. Pharmacophore modeling and molecular docking led to the discovery of inhibitors of human immunodeficiency virus-1 replication targeting the human cellular aspartic acid-glutamic acid-alanine-aspartic acid box polypeptide 3. J Med Chem.  2008 Nov 13;51(21):6635-8.

 

Franca R, Belfiore A, Spadari S, Maga G. Human DEAD-box ATPase DDX3 shows a relaxed nucleoside substrate specificity. Proteins. 2007 Jun 1;67(4):1128-37.

 

Bavagnoli L, Dundon WG, Garbelli A, Zecchin B, Milani A, Parakkal G, Baldanti  F, Paolucci S, Volmer R, Tu Y, Wu C, Capua I, Maga G. The PDZ-ligand and Src-homology type 3 domains of epidemic avian influenza virus NS1 protein modulate human Src kinase activity during viral infection. PLoS One. 2011;6(11):e27789.

 

Bavagnoli L, Maga G. The 2009 influenza pandemic: promising lessons for antiviral therapy for future outbreaks. Curr Med Chem. 2011;18(35):5466-75. Review.

 

Campanini G, Piralla A, Paolucci S, Rovida F, Percivalle E, Maga G, Baldanti F. Genetic divergence of influenza A NS1 gene in pandemic 2009 H1N1 isolates with respect to H1N1 and H3N2 isolates from previous seasonal epidemics. Virol J. 2010 Sep 1;7:209.


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