The HIV-1 accessory protein Vpu enhances virus release by counteracting the host restriction factor tetherin. To further understand the role of host cell proteins in Vpu function, we carried out yeast two-hybrid screening and identified a previously reported Vpu-interacting host factor, small glutamine-rich tetratricopeptide repeat-containing protein (SGTA). While RNAi-mediated depletion of SGTA did not significantly affect levels of tetherin or virus release efficiency, we observed that overexpression of SGTA inhibited HIV-1 release in a Vpu- and tetherin-independent manner. Overexpression of SGTA in the presence of Vpu, but not in its absence, resulted in a marked stabilization and cytosolic relocalization of a 23-kDa, non-glycosylated tetherin species. Coimmunoprecipitation studies indicated that non-glycosylated tetherin is stabilized through the formation of a ternary SGTA/Vpu/tetherin complex. This accumulation of non-glycosylated tetherin is due to inhibition of its degradation, independent of the ER-associated degradation (ERAD) pathway. Because the SGTA-stabilized tetherin species is partially localized to the cytosol, we propose that overexpression of SGTA in the presence of Vpu blocks the translocation of tetherin across the ER membrane, resulting in cytosolic accumulation of a non-glycosylated tetherin species. Although our results do not provide support for a physiological function of SGTA in HIV-1 replication, they demonstrate that SGTA overexpression regulates tetherin expression and stability, thus providing insights into the function of SGTA in ER translocation and protein degradation.
Critical bacterial pathogens of public health and biodefense concerns were engineered to constitutively express Escherichia coli enzyme thymidine kinase (TK) that allows for noninvasive nuclear imaging via phosphorylation and entrapment of radiolabeled nucleoside analog 1-(2′deoxy-2′-fluoro-β-D-arabinofuranosyl)-5-iodouracil (FIAU). Expression of functional TK was established using a nucleoside analog Zidovudine that impeded the growth of tk-engineered bacteria. Significantly, no observable growth differences were detected for FIAU. High resolution mass spectrometry with Pseudomonas aeruginosa PAO1 and its tk variant (PAO1TK) confirmed FIAU phosphorylation and retention only in PAO1TK. In vitro gamma counting with wild-type PAO1, Acinetobacter baumannii and Burkholderia pseudomallei Bp82 and their tk derivatives with [18F]FIAU further confirmed that tk variants selectively incorporated the radiotracer, albeit with varying efficiencies. In vitro [18F]FIAU labeling coupled with in vivo Positron Emission Tomography/Computed Tomography (PET/CT) imaging of PAO1 and PAO1TK confirmed that only PAO1TK can be imaged in mice at sensitivities ≥107 bacteria per infection site. This was further verified by administering [18F]FIAU to animals infected with PAO1 and PAO1TK. Utility of tk-engineered P. aeruginosa in noninvasive PET/CT imaging for bacterial therapeutic evaluation in animals was demonstrated employing antibiotic ciprofloxacin, underscoring the immediate use of PAO1TK and potentially other engineered pathogens for evaluating experimental therapeutics.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.