Characterization of LEDGF/p75 genetic variants and association with HIV-1 disease progression.

BACKGROUND: As Lens epithelium-derived growth factor (LEDGF/p75) is an important co-factor involved in HIV-1 integration, the LEDGF/p75-IN interaction is a promising target for the new class of allosteric HIV integrase inhibitors (LEDGINs). Few data are available on the genetic variability of LEDGF/p75 and the influence on HIV disease in vivo. This study evaluated the relation between LEDGF/p75 genetic variation, mRNA expression and HIV-1 disease progression in order to guide future clinical use of LEDGINs. METHODS: Samples were derived from a therapy-naïve cohort at Ghent University Hospital and a Spanish long-term-non-progressor cohort. High-resolution melting curve analysis and Sanger sequencing were used to identify all single nucleotide polymorphisms (SNPs) in the coding region, flanking intronic regions and full 3'UTR of LEDGF/p75. In addition, two intronic tagSNPs were screened based on previous indication of influencing HIV disease. LEDGF/p75 mRNA was quantified in patient peripheral blood mononuclear cells (PBMC) using RT-qPCR. RESULTS: 325 samples were investigated from patients of Caucasian (n = 291) and African (n = 34) origin, including Elite (n = 49) and Viremic controllers (n = 62). 21 SNPs were identified, comprising five in the coding region and 16 in the non-coding regions and 3'UTR. The variants in the coding region were infrequent and had no major impact on protein structure according to SIFT and PolyPhen score. One intronic SNP (rs2737828) was significantly under-represented in Caucasian patients (P<0.0001) compared to healthy controls (HapMap). Two SNPs showed a non-significant trend towards association with slower disease progression but not with LEDGF/p75 expression. The observed variation in LEDGF/p75 expression was not correlated with disease progression. CONCLUSIONS: LEDGF/p75 is a highly conserved protein. Two non-coding polymorphisms were identified indicating a correlation with disease outcome, but further research is needed to clarify phenotypic impact. The conserved coding region and the observed variation in LEDGF/p75 expression are important characteristics for clinical use of LEDGINs.

Identification of a highly conserved valine-glycine-phenylalanine amino acid triplet required for HIV-1 Nef function.

BACKGROUND: The Nef protein of HIV facilitates virus replication and disease progression in infected patients. This role as pathogenesis factor depends on several genetically separable Nef functions that are mediated by interactions of highly conserved protein-protein interaction motifs with different host cell proteins. By studying the functionality of a series of nef alleles from clinical isolates, we identified a dysfunctional HIV group O Nef in which a highly conserved valine-glycine-phenylalanine (VGF) region, which links a preceding acidic cluster with the following proline-rich motif into an amphipathic surface was deleted. In this study, we aimed to study the functional importance of this VGF region. RESULTS: The dysfunctional HIV group O8 nef allele was restored to the consensus sequence, and mutants of canonical (NL4.3, NA-7, SF2) and non-canonical (B2 and C1422) HIV-1 group M nef alleles were generated in which the amino acids of the VGF region were changed into alanines (VGF→AAA) and tested for their capacity to interfere with surface receptor trafficking, signal transduction and enhancement of viral replication and infectivity. We found the VGF motif, and each individual amino acid of this motif, to be critical for downregulation of MHC-I and CXCR4. Moreover, Nef's association with the cellular p21-activated kinase 2 (PAK2), the resulting deregulation of cofilin and inhibition of host cell actin remodeling, and targeting of Lck kinase to the trans-golgi-network (TGN) were affected as well. Of particular interest, VGF integrity was essential for Nef-mediated enhancement of HIV virion infectivity and HIV replication in peripheral blood lymphocytes. For targeting of Lck kinase to the TGN and viral infectivity, especially the phenylalanine of the triplet was essential. At the molecular level, the VGF motif was required for the physical interaction of the adjacent proline-rich motif with Hck. CONCLUSION: Based on these findings, we propose that this highly conserved three amino acid VGF motif together with the acidic cluster and the proline-rich motif form a previously unrecognized amphipathic surface on Nef. This surface appears to be essential for the majority of Nef functions and thus represents a prime target for the pharmacological inhibition of Nef.

One protein to rule them all: modulation of cell surface receptors and molecules by HIV Nef.

The HIV-1, HIV-2 and SIV Nef protein are known to modulate the expression of several cell surface receptors and molecules to escape the immune system, to alter T cell activation, to enhance viral replication, infectivity and transmission and overall to ensure the optimal environment for infection outcome. Consistent and continuous efforts have been made over the years to characterize the modulation of expression of each of these molecules, in the hope that a better understanding of these processes essential for HIV infection and/or pathogenesis will eventually highlight new therapeutic targets. In this article we provide an extensive review of the knowledge gained so far on this important and evolving topic.

Rho GTPase Cdc42 is essential for human T-cell development.

BACKGROUND: Rho GTPases are involved in the regulation of many cell functions, including some related to the actin cytoskeleton. Different Rho GTPases have been shown to be important for T-cell development in mice. However, their role in human T-cell development has not yet been explored. DESIGN AND METHODS: We examined the expression and activation of Rho GTPases along different stages of T-cell development in the human thymus. Early stage human thymocytes were transduced with constitutively active and dominant negative mutants of different Rho GTPases to explore their role in human T-cell development, as analyzed in fetal thymus organ cultures. The use of these mutants as well as Rho GTPase-specific inhibitors allowed us to explore the role of GTPases in thymocyte migration. RESULTS: We found that the expression of several Rho GTPases is differently regulated during successive stages of T-cell development in man, suggesting a specific role in human thymopoiesis. In chimeric fetal thymus organ culture, T-cell development was not or only mildly affected by expression of dominant negative Rac1 and Rac2, but was severely impaired in the presence of dominant negative Cdc42, associated with enhanced apoptosis and reduced proliferation. Kinetic analysis revealed that Cdc42 is necessary in human T-cell development both before and after expression of the pre-T-cell receptor. Using inhibitors and retrovirally transferred mutants of the aforementioned Rho GTPases, we showed that only Rac1 is necessary for migration of different thymocyte subsets, including the early CD34(+) fraction, towards stromal cell-derived factor-1 alpha. Constitutively active mutants of Rac1, Rac2 and Cdc42 all impaired migration towards stromal cell-derived factor-1 alpha and T-cell development to different degrees. CONCLUSIONS: This is the first report on Rho GTPases in human T-cell development, showing the essential role of Cdc42. Our data suggest that enhanced apoptotic death and reduced proliferation rather than disturbed migration explains the decreased thymopoiesis induced by dominant negative Cdc42.