The membrane proximal region (MPR, residues 649–683) and transmembrane domain (TMD, residues 684–705) of the gp41 subunit of HIV-1’s envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662–683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649–705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM).

Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.

Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models.

The combined use of methamphetamine and opioids has been reported to be on the rise throughout the United States (U.S.). However, our knowledge of this phenomenon is largely based upon reported overdoses and overdose-related deaths, law enforcement seizures, and drug treatment records; data that are often slow, restricted, and only track a portion of the population participating in drug consumption activities. As an alternative, wastewater-based epidemiology (WBE) has the capability to track licit and illicit drug trends within an entire community, at a low cost and in near real-time, while providing anonymity to those contributing to the sewer shed. In this study, wastewater was collected from two Midwestern U.S. cities (2017-2019) and analyzed for the prevalence of methamphetamine and the opioids oxycodone, codeine, fentanyl, tramadol, hydrocodone, and hydromorphone. Monthly 24-hour time-weighted composite samples (n = 48) from each city were analyzed using isotope dilution liquid chromatography tandem mass spectrometry. Results showed that methamphetamine and total opioid consumption (milligram morphine equivalents) in City 1 were strongly correlated only in 2017 (Spearman rank order correlation coefficient, ρ = 0.78), the relationship driven by fentanyl, hydrocodone, and hydromorphone. For City 2, methamphetamine and total opioid consumption were strongly positively correlated during the entire study (ρ = 0.54), with the correlations driven by hydrocodone and hydromorphone. In both cities, hydrocodone and hydromorphone mass loads were highly correlated, suggesting a parent and metabolite relationship. WBE provides important insights into licit and illicit drug consumption patterns in near real-time as they evolve; important information for community stakeholders in municipalities across the U.S.