Kevin K. Ariën

Is HIV-1 evolving to a less virulent form in humans?

During the rapid spread of HIV-1 in humans, the main (M) group of HIV-1 has evolved into ten distinct subtypes, undergone countless recombination events and diversified extensively. The impact of this extreme genetic diversity on the phenotype of HIV-1 has only recently become a research focus, but early findings indicate that the dominance of HIV-1 subtype C in the current epidemic might be related to the lower virulence of this subtype compared with other subtypes. Here, we explore whether HIV-1 has reached peak virulence or has already started the slow path to attenuation.

The predominance of Human Immunodeficiency Virus type 1 (HIV-1) circulating recombinant form 02 (CRF02_AG) in West Central Africa may be related to its replicative fitness

BackgroundCRF02_AG is the predominant HIV strain circulating in West and West Central Africa. The aim of this study was to test whether this predominance is associated with a higher in vitro replicative fitness relative to parental subtype A and G viruses. Primary HIV-1 isolates (10 CRF02_AG, 5 subtype A and 5 subtype G) were obtained from a well-described Cameroonian cohort. Growth competition experiments were carried out at equal multiplicity of infection in activated T cells and monocyte-derived dendritic cells (MO-DC) in parallel.ResultsDual infection/competition experiments in activated T cells clearly indicated that CRF02_AG isolates had a significant replication advantage over the subtype A and subtype G viruses. The higher fitness of CRF02_AG was evident for isolates from patients with CD4+ T cell counts >200 cells/μL (non-AIDS) or CD4+ T cell counts <200 cells/μL (AIDS), and was independent of the co-receptor tropism. In MO-DC cultures, CRF02_AG isolates showed a slightly but not significantly higher replication advantage compared to subtype A or G isolates.ConclusionWe observed a higher ex vivo replicative fitness of CRF02_AG isolates compared to subtype A and G viruses from the same geographic region and showed that this was independent of the co-receptor tropism and irrespective of high or low CD4+ T cell count. This advantage in replicative fitness may contribute to the dominant spread of CRF02_AG over A and G subtypes in West and West Central Africa.

HIV Nef: Role in Pathogenesis and Viral Fitness

Conserved in all primate lentivirus genomes, Nef promotes viral replication and infectivity, influences the trafficking of a large number of surface receptors and interferes with TCR signalling, consequently modulating T-cell activation. In vivo observations with Long Term Non-Progressors harbouring a Nef-defective HIV and vaccination studies with Nef-deleted SIV in Rhesus macaques have shown a prominent role for Nef in lentiviral pathogenesis. Here we review the functions of Nef involved in viral replication and infectivity and speculate on a possible role for Nef in HIV fitness.

A combined genotypic and phenotypic human immunodeficiency virus type 1 recombinant virus assay for the reverse transcriptase and integrase genes

With the approval of the first HIV-1 integrase inhibitor raltegravir and a second one in phase III clinical development (elvitegravir), genotypic and phenotypic resistance assays are required to guide antiretroviral therapy and to investigate treatment failure. In this study, a genotypic and phenotypic recombinant virus assay was validated for determining resistance against integrase inhibitors. The assays are based on the amplification of a region encompassing not only HIV-1 integrase, but also reverse transcriptase and RNAseH. The overall amplification success was 85% (433/513) and increased to 93% (120/129) for samples with a viral load above 3 log(10) copies/ml. Both B and non-B HIV-1 subtypes could be genotyped successfully (93%; 52/56 and 100%; 49/49, respectively) and reproducibly. The phenotypic assay showed a high success rate (96.5%; 139/144) for subtype B (100%; 19/19) and non-B subtypes (92%; 45/49), and was found to be accurate and reproducible as assessed using well-characterized integrase mutants. Using both assays, baseline resistance to raltegravir and elvitegravir in subtype B and non-B HIV-1 strains selected at random was not observed, although integrase polymorphisms were present at varying prevalence. Biological cutoff values were found to be 2.1 and 2.0 for raltegravir and elvitegravir, respectively. In summary, a genotypic and phenotypic integrase resistance assay was validated successfully for accuracy, reproducibility, analytical and clinical sensitivity, and dynamic range.

Analysis of infectious virus clones from two HIV-1 superinfection cases suggests that the primary strains have lower fitness

BackgroundTwo HIV-1 positive patients, L and P, participating in the Amsterdam Cohort studies acquired an HIV-1 superinfection within half a year from their primary HIV-1 infection (Jurriaans et al., JAIDS 2008, 47:69-73). The aim of this study was to compare the replicative fitness of the primary and superinfecting HIV-1 strains of both patients. The use of isolate-specific primer sets indicated that the primary and secondary strains co-exist in plasma at all time points after the moment of superinfection.ResultsBiological HIV-1 clones were derived from peripheral blood CD4 + T cells at different time point, and identified as the primary or secondary virus through sequence analysis. Replication competition assays were performed with selected virus pairs in PHA/IL-2 activated peripheral blood mononuclear cells (PBMCs) and analyzed with the Heteroduplex Tracking Assay (HTA) and isolate-specific PCR amplification. In both cases, we found a replicative advantage of the secondary HIV-1 strain over the primary virus. Full-length HIV-1 genomes were sequenced to find possible explanations for the difference in replication capacity. Mutations that could negatively affect viral replication were identified in the primary infecting strains. In patient L, the primary strain has two insertions in the LTR promoter, combined with a mutation in the tat gene that has been associated with decreased replication capacity. The primary HIV-1 strain isolated from patient P has two mutations in the LTR that have been associated with a reduced replication rate. In a luciferase assay, only the LTR from the primary virus of patient P had lower transcriptional activity compared with the superinfecting virus.ConclusionsThese preliminary findings suggest the interesting scenario that superinfection occurs preferentially in patients infected with a relatively attenuated HIV-1 isolate.

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

BackgroundThe 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.ResultsThe 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.ConclusionBased 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.

A catalogue of putative HIV-1 protease host cell substrates.

Abstract Processing of human immunodeficiency virus (HIV) proteins by the HIV-1 protease is essential for HIV infectivity. In addition, several studies have revealed cleavage of human proteins by this viral protease during infection; however, no large-scale HIV-1 protease degradomics study has yet been performed. To identify putative host substrates in an unbiased manner and on a proteome-wide scale, we used positional proteomics to identify peptides reporting protein processing by the HIV-1 protease, and a catalogue of over 120 cellular HIV-1 protease substrates processed in vitro was generated. This catalogue includes previously reported substrates as well as recently described interaction partners of HIV-1 proteins. Cleavage site alignments revealed a specificity profile in good correlation with previous studies, even though the ELLE consensus motif was not cleaved efficiently when incorporated into peptide substrates due to subsite cooperativity. Our results are further discussed in the context of HIV-1 infection and the complex substrate recognition by the viral protease.

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-1α. Constitutively active mutants of Rac1, Rac2 and Cdc42 all impaired migration towards stromal cell-derived factor-1α 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.

Primate lentiviral Nef proteins deregulate T-cell development by multiple mechanisms

BackgroundA nef gene is present in all primate lentiviral genomes and is important for high viral loads and progression to AIDS in human or experimental macaque hosts of HIV or SIV, respectively. In these hosts, infection of the thymus results in a decreased output of naive T cells that may contribute to the development of immunodeficiency. We have previously shown that HIV-1 subtype B Nef proteins can block human T-cell development. However, the underlying mechanism(s) and the conservation of this Nef function between different groups of HIV and SIV remained to be determined.ResultsWe investigated whether reduction of thymic output is a conserved function of highly divergent lentiviral Nef proteins including those from both types of human immunodeficiency viruses (HIV-1 and HIV-2), their direct simian counterparts (SIVcpz, SIVgor and SIVsmm, respectively), and some additional SIV strains. We found that expression of most of these nef alleles in thymocyte progenitors impaired T-cell development and reduced thymic output. For HIV-1 Nef, binding to active p21 protein (Cdc42/Rac)-activated kinase (PAK2) was a major determinant of this function. In contrast, selective disruption of PAK2 binding did not eliminate the effect on T-cell development of SIVmac239 Nef, as was shown by expressing mutants in a newly discovered PAK2 activating structural motif (PASM) constituted by residues I117, H121, T218 and Y221, as well as previously described mutants. Rather, down-modulation of cell surface CD3 was sufficient for reduced thymic output by SIVmac Nef, while other functions of SIV Nefs contributed.ConclusionsOur results indicate that primate lentiviral Nef proteins impair development of thymocyte precursors into T cells in multiple ways. The interaction of HIV-1 Nef with active PAK2 by HIV-1 seem to be most detrimental, and downregulation of CD3 by HIV-2 and most SIV Nef proteins sufficient for reduced thymic output. Since the reduction of thymic output by Nef is a conserved property of divergent lentiviruses, it is likely to be relevant for peripheral T-cell depletion in poorly adapted primate lentiviral infections.

Nef interferes with development of thymic T cell precursors: differential mechanisms in HIV and SIV

Background HIV infection of the thymus results in a decreased output of naive T cells. We previously showed that expression of the Nef protein alone is sufficient to disturb human thymopoiesis. Additional structure-function studies with mutant Nef alleles suggested a role of PAK2 in this process. In this study we evaluated the effect of Nef alleles from different clinical HIV-1, HIV-2 and SIV isolates and Nef mutants which were specifically mutated in the PAK2 interaction surface for their effect on T cell development.