Revealing viral and cellular dynamics of HIV-1 at the single-cell level during early treatment periods

While combination therapy completely suppresses HIV-1 replication in blood, functional virus persists in CD4$^{+}$ T cell subsets in non-peripheral compartments that are not easily accessible. To fill this gap, we investigated tissue-homing properties of cells that transiently appear in the circulating blood. Through cell separation and in vitro stimulation, the HIV-1 "Gag and Envelope reactivation co-detection assay" (GERDA) enables sensitive detection of Gag+/Env+ protein-expressing cells down to about one cell per million using flow cytometry. By associating GERDA with proviral DNA and polyA-RNA transcripts, we corroborate the presence and functionality of HIV-1 in critical body compartments utilizing t-distributed stochastic neighbor embedding (tSNE) and density-based spatial clustering of applications with noise (DBSCAN) clustering with low viral activity in circulating cells early after diagnosis. We demonstrate transcriptional HIV-1 reactivation at any time, potentially giving rise to intact, infectious particles. With single-cell level resolution, GERDA attributes virus production to lymph-node-homing cells with central memory T cells (T$_{CM}$s) as main players, critical for HIV-1 reservoir eradication

Inter- and intra-molecular interactions of Arabidopsis thaliana DELLA protein RGL1

The phytohormone gibberellin and the DELLA proteins act together to control key aspects of plant development. Gibberellin induces degradation of DELLA proteins by recruitment of an F-box protein using a molecular switch: a gibberellin-bound nuclear receptor interacts with the N-terminal domain of DELLA proteins, and this event primes the DELLA C-terminal domain for interaction with the F-box protein. However, the mechanism of signalling between the N- and C-terminal domains of DELLA proteins is unresolved. In the present study, we used in vivo and in vitro approaches to characterize di- and tri-partite interactions of the DELLA protein RGL1 (REPRESSOR OF GA1-3-LIKE 1) of Arabidopsis thaliana with the gibberellin receptor GID1A (GIBBERELLIC ACID-INSENSITIVE DWARF-1A) and the F-box protein SLY1 (SLEEPY1). Deuterium-exchange MS unequivocally showed that the entire N-terminal domain of RGL1 is disordered prior to interaction with the GID1A; furthermore, association/dissociation kinetics, determined by surface plasmon resonance, predicts a two-state conformational change of the RGL1 N-terminal domain upon interaction with GID1A. Additionally, competition assays with monoclonal antibodies revealed that contacts mediated by the short helix Asp-Glu-Leu-Leu of the hallmark DELLA motif are not essential for the GID1A–RGL1 N-terminal domain interaction. Finally, yeast two- and three-hybrid experiments determined that unabated communication between N- and C-terminal domains of RGL1 is required for recruitment of the F-box protein SLY1

Defining the gate domain of the filamentous phage secretin pIV

Appendix 5 removed due to copyright restrictions. Spagnuolo, J., Opalka, N., Wen, W. X., Gagic, D., Chabaud, E., Bellini, P.,...Rakonjac, J. (2010). Identification of the gate regions in the primary structure of the secretin pIV. Molecular microbiology, 76(1), 133-150. doi:10.1111/j.1365-2958.2010.07085.xSecretins are a family of large outer membrane proteins with large-diameter lumens (5-10 nm). This allows them to transport bulky substrates, including folded proteins, or assembled macromolecular structures – filamentous phages and type IV pili. Many proteins exported by secretins are essential for virulence of Gram-negative pathogens. Such a large channel would ordinarily sensitize the bacterial cell to noxious agents. However, secretins do not - the presence of a mobile septum, or gate, across the lumen of the channel prevents access by noxious agents. Despite the importance of the gate in secretin function, the sequence identity of the gate residues is unknown. In this study, in vivo random mutagenesis was used to map amino acid residues involved in gating the filamentous phage secretin - pIV. This approach has identified 34 residues that are involved in the gating mechanism. These residues are predominantly located within the secretin homology domain and organised into two clusters; GATE1 (39 residues) and GATE2 (14 residues). A number of isolated point mutants sensitised Escherichia coli to bile salts and antibiotics. These findings allowed the construction of a site-directed deletion mutant of GATE2, confirming the gate function. This thesis mapped, for the first time, a secretin gate. Given the success of the mutagenesis approach used in this thesis, the method here will be applicable to secretins of pathogenic bacteria and other outer membrane channels whose gate regions have not been determined as yet. Knowing the gate regions of “pathogenic” secretins in turn will help design secretin-targeting antimicrobials

Extracytoplasmic stress responses induced by a model secretin : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand

Pathogenic bacteria export large proteins and protein complexes, including virulence factors, using dedicated transenvelope multiprotein machinery, collectively called secretion systems. Four of these protein export machines found in Gram-negative bacteria, type 2/3 secretion systems, filamentous phage assembly-secretion system and the type 4 pilus assembly system contain large homologous gated channels, called secretins, in the outer membrane. Secretins are radially symmetrical homomultimers (luminal diameter 6-8 nm) interrupted by an internal septum or gate. Expression of these channels imposes a fitness cost to bacteria. While stress induced by model secretin pIV has been previously investigated using microarrays, this thesis is the first RNAseq characterisation of secretin stress responses. Furthermore, this is the first comparison of stress imposed by a closed-gate secretin (wildtype pIV), vs. an isogenic leaky-gate variant, the latter serving as a model of an open-gate substrate-secreting channel. The high sensitivity to changes in gene expression and low background noise of the RNA-seq approach have greatly expanded the known secretin stress responses to include the SoxS, CpxR and RcsB/RcsAB regulons, in addition to the known involvement of the Psp response. A synthetic lethality analysis of candidate genes in these pathways suggested that the leaky-gate secretins, besides rendering the Psp response essential for survival, also stimulate the SoxS and RcsB/RcsAB regulons for protection of the cells. Knowledge of the secretin stress expanded by this work helped identify potential targets for development of much-needed antibiotics against toxinsecreting Gram-negative bacteria

Immunophenotypic characterization of TCR γδ T cells and MAIT cells in HIV-infected individuals developing Hodgkin's lymphoma

Background Despite successful combined antiretroviral therapy (cART), the risk of non-AIDS defining cancers (NADCs) remains higher for HIV-infected individuals than the general population. The reason for this increase is highly disputed. Here, we hypothesized that T-cell receptor (TCR) γδ cells and/or mucosal-associated invariant T (MAIT) cells might be associated with the increased risk of NADCs. γδ T cells and MAIT cells both serve as a link between the adaptive and the innate immune system, and also to exert direct anti-viral and anti-tumor activity. Methods We performed a longitudinal phenotypic characterization of TCR γδ cells and MAIT cells in HIV-infected individuals developing Hodgkin’s lymphoma (HL), the most common type of NADCs. Cryopreserved PBMCs of HIV-infected individuals developing HL, matched HIV-infected controls without (w/o) HL and healthy controls were used for immunophenotyping by polychromatic flow cytometry, including markers for activation, exhaustion and chemokine receptors. Results We identified significant differences in the CD4+ T cell count between HIV-infected individuals developing HL and HIV-infected matched controls within 1 year before cancer diagnosis. We observed substantial differences in the cellular phenotype mainly between healthy controls and HIV infection irrespective of HL. A number of markers tended to be different in Vδ1 and MAIT cells in HIV+HL+ patients vs. HIV+ w/o HL patients; notably, we observed significant differences for the expression of CCR5, CCR6 and CD16 between these two groups of HIV+ patients. Conclusion TCR Vδ1 and MAIT cells in HIV-infected individuals developing HL show subtle phenotypical differences as compared to the ones in HIV-infected controls, which may go along with functional impairment and thereby may be less efficient in detecting and eliminating malignant cells. Further, our results support the potential of longitudinal CD4+ T cell count analysis for the identification of patients at higher risk to develop HL

Chronic hepatitis B and C virus infection and risk for non-hodgkin lymphoma in HIV-infected patients: A cohort study

Background: Non-Hodgkin lymphoma (NHL) is the most common AIDS-defining condition in the era of antiretroviral therapy (ART). Whether chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection promote NHL in HIV-infected patients is unclear. Objective: To investigate whether chronic HBV and HCV infection are associated with increased incidence of NHL in HIVinfected patients. Design: Cohort study. Setting: 18 of 33 cohorts from the Collaboration of Observational HIV Epidemiological Research Europe (COHERE). Patients: HIV-infected patients with information on HBV surface antigen measurements and detectable HCV RNA, or a positive HCV antibody test result if HCV RNA measurements were not available. Measurements: Time-dependent Cox models to assess risk for NHL in treatment-naive patients and those initiating ART, with inverse probability weighting to control for informative censoring. Results: A total of 52 479 treatment-naive patients (1339 [2.6%] with chronic HBV infection and 7506 [14.3%] with HCV infection) were included, of whom 40 219 (77%) later started ART. The median follow-up was 13 months for treatment-naive patients and 50 months for those receiving ART. A total of 252 treatmentnaive patients and 310 treated patients developed NHL, with incidence rates of 219 and 168 cases per 100 000 person-years, respectively. The hazard ratios for NHL with HBV and HCV infection were 1.33 (95% CI, 0.69 to 2.56) and 0.67 (CI, 0.40 to 1.12), respectively, in treatment-naive patients and 1.74 (CI, 1.08 to 2.82) and 1.73 (CI, 1.21 to 2.46), respectively, in treated patients. Limitation: Many treatment-naive patients later initiated ART, which limited the study of the associations of chronic HBV and HCV infection with NHL in this patient group. Conclusion: In HIV-infected patients receiving ART, chronic coinfection with HBV and HCV is associated with an increased risk for NHL