Constitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusion

The CCR5 chemokine receptor is a rhodopsin-like G protein-coupled receptor that mediates the effects of pro-inflammatory β-chemokines. CCR5 is also the major co-receptor for entry of human immunodeficiency virus (HIV) into human cells. G protein-coupled receptors exist in ensembles of active and inactive conformations. Active receptor conformations can be stabilized by mutations. Although binding of the HIV envelope protein to CCR5 stimulates cellular signaling, the CCR5 conformation that induces fusion of the viral membrane with cellular membranes is not known. We mutated conserved amino acids to generate constitutively active CCR5 receptors, which are stabilized in active conformations, and tested the ability of constitutively active CCR5 receptors to mediate HIV envelope-directed membrane fusion. Mutation of the Asp 3.49(125) and Arg 6.32(225) residues of CCR5 did not cause constitutive activity, but Lys or Pro substitutions for Thr 2.56(82) , in the TxP motif, caused high basal inositol phosphate signaling. Signaling did not increase in response to MIP-1β, suggesting that the Thr 2.56(82) mutants were fully stabilized in active conformations. The Thr 2.56(82) Lys mutation severely decreased cell surface CCR5 expression. Combining the Thr 2.56(82) Lys mutation with an Arg 6.32(225) Gln mutation partially reversed the decrease in expression. Mutants with Thr 2.56(82) Lys substitutions were poor mediators of HIV envelope-directed membrane fusion, but mutants with the Thr 2.65(82) Pro substitution exhibited full co-receptor function. Our results suggest that the Thr 2.65(82) Lys and Thr 2.65(82) Pro mutations stabilize distinct constitutively active CCR5 conformations. Lys in position 2.65(82) stabilizes activated receptor conformations that appear to be constitutively internalized and do not induce envelope-dependent membrane fusion, whereas Pro stabilizes activated conformations that are not constitutively internalized and fully mediate envelope-directed membrane fusion

The Applicability of Wireless Communication in CyberTracker

CyberTracker is software application for mobile devices which is used for data collection. It consists of a mobile client and a desktop server. It has been used with significant success in previous years within various contexts. The most noteworthy being the collection of wildlife information in National Parks throughout Southern Africa. The application allows users to create data entry templates which is then used to record sightings on a mobile handheld computer. When the user wishes to view reports of the sightings, the device has to be physically docked and synchronised with the desktop application. With the data already being stored electronically, it is unfortunate that the information is not available sooner. The CyberTracker extension discussed in this paper allows for users to send data that they collect to a remote database. This functionality will eliminate the need for users to return to base thus increasing productivity as the users routine will not be interrupted. The extension also allows for the possibility of having information sent from the remote database to the mobile device. Wireless technologies such as GPRS and WiFi have been utilised in a number of different fields including medicine, field work and education. These technologies enable users to be mobile by allowing them to have access to remote databases as a means of receiving and updating information. The CyberTracker extension implemented builds on the existing CyberTracker application by using various technologies. These include sending and receiving of information via TCP/IP in the form of structured XML packets. The extended system uses store-and-forward as a means to save collected data when a connection to GPRS/WiFi is unavailable. Two case studies presented illustrate the usefulness of the extension. The case studies focus on the applicability of the extension by trackers in wildlife parks and traffic officers working in the UCT Traffic Department. The extended system could increase the productivity of both trackers and traffic officers by allowing them to carry out their tasks without needing to return to the office. Trackers and traffic officers can then carry on working without any disruptions of having to travel to the office. Data collected can also be available to analysts or administrators on a real-time basis thus reducing time delays of when data is available. The extended system can also be useful in terms of the data that can be sent from the database to the user. Trackers could receive information about where they have been in previous days and traffic officers could receive information about cars that have outstanding tickets. The information presented in this paper shows that the extension to CyberTracker could be useful not only the users currently using CyberTracker but also to new users

SARS-CoV-2 Transmission Risk in the School Environment: a pilot case-ascertained prospective study to inform future school-based surveillance.

Background. There is no current active or passive disease surveillance programme focused on schools in South Africa. As such the country is missing an opportunity to rapidly and effectively flag and address pathogen outbreaks, for example SARS-CoV-2, in a key closed setting. Furthermore, the role of school transmission in the spread of the SARS-CoV-2 virus within communities is uncertain.  Objective. This pilot study, conducted during March 2022 in Cape Town, aimed to indicate the feasibility of conducting intense active contact-tracing in a school environment prior to a large national study to compare school versus community SARS-CoV-2 transmission risk.  Methods. We conducted a pilot school-level case-ascertained prospective study with a component of enhanced surveillance. Following study initiation, the first learner at a participating school who tested SARS-CoV-2 positive (via Polymerase Chain Reaction (PCR) or a Rapid Antigen Test (RAT)) was invited to join the study as the index case and all their school-based close contacts were followed up telephonically, monitored for symptoms for 14 days, and tested using a PCR if any symptoms were reported.  Results. On 8th March 2022, a student with RAT laboratory-confirmed COVID-19 was identified and they and their guardian consented to participate as the index case. Of the 11 eligible close contacts, six provided consent/assent and completed symptom monitoring calls until the end of the 14-day study period. The Secondary Attack Rate (SAR) was 2/11 (18.18%) of all close contacts who were at risk of infection, 2/4 (50.0%) of all those close contacts who developed symptoms, and 2/4 (50.0%) of all those close contacts who developed symptoms and were tested for SARS-CoV-2. During the same period, the school reported that nine of the 926 learner body tested COVID-19 positive (0.97%). Total hours spent conducting monitoring for 6 learners was 27 hours, with each learner requiring approximately 4.5 hours of contact time during the study period.  Conclusion. This is the first South African school-based COVID-19 transmission study, the results of which can inform national discussions regarding the role of schools and school-based active and passive surveillance in pathogen prevention and control.

Dopamine Receptor Activation Increases HIV Entry into Primary Human Macrophages

Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers

IP production and expression of wild type and mutant CCR5 receptors.

<p>HEK-Gqi cells were transiently transfected with wild type or mutant CCR5 receptors, labeled with [³H]<i>myo</i>-inositol and incubated without (basal) or with chemokine agonist, MIP-1β (10⁻⁷ M). Specific CPM denotes the CPM determined for receptor expressing-cells minus the CPM for vector-transfected cells. Data are from a representative experiment performed at least three times in duplicate. B, HEK 293 cells transiently transfected with wild type or mutant CCR5 receptors were stained with a PE-2D7 anti-CCR5 antibody and analyzed by FACS. Data are representative of at least three independent experiments performed in duplicate.</p

Venn diagram depicting ensembles of CCR5 receptor conformations stabilized by mutation of Thr^2.56(82).

<p>Triangles represent receptor conformations stabilized by mutation of Thr^2.56(82) to Lys or Pro. Circles represent receptor conformations that mediate G protein activation, receptor internalization or HIV Env-directed membrane fusion. Mutation of Thr^2.56(82) to Lys stabilizes an ensemble of receptor conformations that activate G protein-mediated signaling and conformations with increased susceptibility to internalization, but not conformations that support HIV Env dependent membrane fusion. The Thr^2.56(82)Pro mutation stabilizes an ensemble of receptor conformations that activate the G protein and conformations that support HIV-1 fusion, but it does not appear to increase population of receptor conformations that result in decreased membrane expression of CCR5.</p

A social network typology and sexual risk-taking among men who have sex with men in Cape Town and Port Elizabeth, South Africa

Despite the high prevalence of HIV among men who have sex with men in South Africa, very little is known about their lived realities, including their social and sexual networks. Given the influence of social network structure on sexual risk behaviours, a better understanding of the social contexts of men who have sex with men is essential for informing the design of HIV programming and messaging. This study explored social network connectivity, an understudied network attribute, examining self-reported connectivity between friends, family and sex partners. Data were collected in Cape Town and Port Elizabeth, South Africa, from 78 men who have sex with men who participated in in-depth interviews that included a social network mapping component. Five social network types emerged from the content analysis of these social network maps based on the level of connectivity between family, friends and sex partners, and ranged from disconnected to densely connected networks. The ways in which participants reported sexual risk-taking differed across the five network types, revealing diversity in social network profiles. HIV programming and messaging for this population can greatly benefit from recognising the diversity in lived realities and social connections between men who have sex with men.

IP production, expression and competition binding of CCR5 receptors with mutations of Thr^2.56(82) and Arg^6.32(225).

<p>A, HEK-Gqi cells were transfected with the wild type (▪) or mutant CCR5 receptors Thr^2.56(82)Lys (•), Thr^2.56(82)Pro (▴), Thr^2.56(82)Lys/Arg^6.32(225)Gln (○) or Thr^2.56(82)Pro/Arg^6.32(225)Gln (Δ). Untransfected cells (□) were used as a negative control. Cells pre-labeled with [³H]<i>myo</i>-inositol were incubated with increasing concentrations of MIP-1β. Data are from a single experiment that is representative of at least three independent experiments performed in duplicate. B, HEK cells were transfected with wild type or mutant CCR5 receptors and stained with PE-2D7 for FACS analysis. Results are mean values ± SEM from at least three independent experiments performed in duplicate. C, HEK 293 cells were transiently transfected with wild type (▪) or mutant CCR5 receptors, Thr^2.56(82)Lys (•), Thr^2.56(82)Pro (▴), Thr^2.56(82)Lys/Arg^6.32(225)Gln (○) or Thr^2.56(82)Pro/Arg^6.32(225)Gln (Δ) and incubated with ¹²⁵I-MIP-1β and various concentrations of unlabelled MIP-1β. Cell-bound radioactivity was collected by filtration and counted. Data are from a single experiment, representative of at least three independent experiments performed in triplicate.</p

IP production and surface expression of wild type and mutant CCR5 receptors.

a<p>significantly different from wild type, p<0.05.</p><p>To assess constitutive- and ligand-stimulated IP production, HEK-Gqi cells transiently expressing wild type or mutant CCR5 receptors were labeled with [H³]-<i>myo</i>-inositol and incubated with buffer (Basal) or MIP-1β (10⁻⁷ M, Stimulated). To assess cell surface expression of receptors HEK 293 cells transiently transfected with wild type or mutant CCR5 constructs were incubated with PE-2D7 antibody before FACS analysis. Every experiment included wild type CCR5 and mock transfected cells. Data are means ± SEM calculated from at least three independent experiments performed in duplicate.</p

Fusion activity of wild type and mutant CCR5 receptors.

<p>A, HOS cells stably expressing CD4 and the luciferase reporter gene were transiently transfected with wild type (▪) or mutant CCR5 receptors Thr^2.56(82)Lys (•), Thr^2.56(82)Pro (▴), Thr^2.56(82)Lys/Arg^6.32(225)Gln (○) or Thr^2.56(82)Pro/Arg^6.32(225)Gln (Δ). CCR5-expressing HOS-CD4-Luc cells were co-cultured overnight with HEK cells transiently expressing tat, rev and Env and luciferase activity was assessed. B, CCR5-expressing HOS-CD4-Luc cells were labeled with PE-2D7 and analyzed by FACS analysis. C, To compare fusion efficiency among mutant receptors that were expressed at different levels the fusion coefficient was derived by dividing the luciferase activity by the mean fluorescence of each construct.</p