These characteristics of Tat secretion are remarkably overla

These characteristics of Tat secretion are remarkably overlapping with those of cellular FGF-2, which is also sensitive to ouabain and involves binding of the factor to the Na+,K+-ATPase (Dahl et al., 2000; Florkiewicz et al., 1998; Smith et al., 2001; Trudel et al., 2000; Zeitler et al., 2015), while does not require integrity of the enzymatic function of the pump (Zacherl et al., 2015). Work from other laboratories has also shown that unconventional secretion of both FGF-2 (Temmerman et al., 2008) and HIV-1 Tat (Rayne et al., 2010) require interaction of the two proteins with acidic domains in the phospholipid components of the inner membrane leaflet. Thus, the Na+,K+ ATPase α subunit might represent a preferential landing site for the association of Tat to the inner membrane leaflet, which cooperates with PI(4,5)P2 in docking Tat to the plasma membrane to favor its extracellular export, similar to what proposed for FGF-2 (Zacherl et al., 2015). The ouabain binding site is a cavity in the transmembrane domain at the extracellular interface of the protein (Morth et al., 2007; Yatime et al., 2011). It can be speculated that this interaction determines some spatial modification in the α1 structure, such as to abolish interaction of Tat to the C-terminus. Consistent with this possibility, analysis of the protein structure obtained in the presence of ouabain (Yatime et al., 2011) indicates that the drug indeed induces a modification of the surface formed by the C-terminal cytosolic loops. What is molecular function of extracellular Tat release? Past work from several laboratories has indicated that extracellular Tat exerts a number of pleiotropic activities when released outside the producing cells, ranging from stimulation of gene hcv protease inhibitor to inhibition of the immune response (reviewed in: (Fittipaldi and Giacca, 2005)). A non mutually exclusive, but perhaps more intriguing possibility is that a major function of extracellular Tat is to increase virion infectivity. Indeed, our experiments show that HIV-1 viral preparations, either pseudotyped or carrying a natural Env protein, are significantly less infectious when produced in the presence of ouabain; infectivity could be rescued by the addition of recombinant Tat or supernatants containing the protein. The results of these experiments indicate that a major function of secreted Tat is to increase HIV-1 virion infectivity on its primary CD4+ T cell targets. This effect is likely to be mediated by the association of Tat, on the surface of virions, with cell surface-associated heparan sulfate proteoglycans (Rusnati et al., 1998; Tyagi et al., 2001); the observation that infection of the CHO psg A-745 mutant, which lack proteoglycan biosynthesis, by HIV-1 virions produced in the presence of ouabain cannot be rescued by exogenous Tat addition is consistent with this possibility. Of note, cell treatment with peptides corresponding to the three short cytoplasmic sequences in the C-terminal domain of the α1 protein both inhibited non canonical Tat secretion and suppressed HIV-1 infection. While no drugs are currently available in the clinics targeting Tat or its transactivation functions in the nucleus, the efficacy of these peptides suggests that the competitive inhibition of the Tat-Na+,K+-ATPase α1 subunit interaction might represent a novel strategy for the development of alternative anti-HIV-1 compounds. The design of derivatives of these peptides, or of their peptidomimetics, or the identification of small chemical molecules able to interfere with Tat-α1 binding might represent exciting avenues for future pharmacological development.
Funding Sources This work was supported by the Intramural Funding Programme of the ICGEB to the Molecular Medicine Laboratory in Trieste, Italy.
Conflicts of Interest
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Acknowledgments
Introduction The most recent European Association for the Study of the Liver (2016) Guidelines on treatment of hepatitis C, allowed for shortening the course of treatment with Sofosbuvir/Ledipasvir and with Grazoprevir/Elbasvir for subsets of patients with lower virus load based on cutoff values of baseline serum HCV RNA level (EASL, 2017; Kowdley et al., 2014; Curry et al., 2016).