Objective HIV-1 replication and microbial translocation occur concomitant with systemic immune activation. This study delineates mechanisms of immune activation and CD4 T cell decline in pediatric HIV-1 infection. Design Cross-sectional and longitudinal cellular and soluble plasma markers for inflammation were evaluated in 14 healthy and 33 perinatally HIV-1-infected pediatric subjects prior to and over 96 weeks of protease-inhibitor-containing combination antiretroviral treatment [ART]. All HIV-1-infected subjects reconstituted CD4 T cells either with suppression of viremia or rebound of drug-resistant virus. Methods Systemic immune activation was determined by polychromatic flow cytometry of blood lymphocytes and ELISA for plasma soluble CD27 [sCD27], soluble CD14 [sCD14], and tumor necrosis factor [TNF]. Microbial translocation was evaluated by limulus amebocyte lysate assay to detect bacterial lipopolysaccharide [LPS] and ELISA for anti-endotoxin core antigen IgM antibodies. Immune activation markers were compared to viral load, CD4% and LPS by regression models. Comparisons between healthy and HIV-1 infected or between different viral outcome groups were performed by non-parametric rank sum. Results Microbial translocation was detected in healthy infants but resolved with age (P<0.05). LPS and sCD14 levels were elevated in all HIV-1 infected subjects (P<0.05 and P<0.0001, respectively) and persisted even if CD4 T cells were fully reconstituted, virus optimally suppressed, and lymphocyte activation resolved by ART. Children with CD4 T cell reconstitution but viral rebound following ART continued to display high levels of sCD27. Conclusions Microbial translocation in pediatric HIV-1-infection is associated with persistent monocyte/macrophage activation independent of viral replication or T cell activation.

Protease inhibitor resistance still poses one of the greatest challenges in treating HIV. To better design inhibitors able to target resistant proteases, a deeper understanding is needed of the effects of accumulating mutations and the contributions of active-and nonactive-site mutations to the resistance. We have engineered a series of variants containing the nonactive-site mutations M46I and I54V and the active-site mutation I84V. These mutations were added to a protease clone (V6) isolated from a pediatric patient on ritonavir therapy. This variant possessed the ritonavir-resistance-associated mutations in the active-site (V32I and V82A) and nonactive-site mutations (K20R, L33F, M36I, L63P, A71V, and L90M). The I84V mutation had the greatest effect on decreasing catalytic efficiency, 10-fold when compared to the pretherapy clone LAI. The decrease in catalytic efficiency was partially recovered by the addition of mutations M46I and I54V. The M46I and I54V were just as effective at decreasing inhibitor binding as the I84V mutation when compared to V6 and LAI. The V6 54/84 variant showed over 1000-fold decrease in inhibitor-binding strength to ritonavir, indinavir, and nelfinavir when compared to LAI and V6. Crystalstructure analysis of the V6 54/84 variant bound to ritonavir and indinavir shows structural changes in the 80's loops and active site, which lead to an enlarged binding cavity when compared to pretherapy structures in the Protein Data Bank. Structural changes are also seen in the 10's and 30's loops, which suggest possible changes in the dynamics of flap opening and closing.The development of resistance to protease inhibitors (PI) 1 during treatment of infection by the Human Immunodeficiency Virus (HIV) still poses one of the greatest challenges in the struggle to limit the virus replicative capacity. After initiation of therapy with single or multiple protease inhibitors, resistance mutations in the protease can appear within weeks. Under a constant drug selection pressure, resistant mutations continue to accumulate. It is clear that there is a correlation between the number of resistance mutations and the level of resistance and cross resistance to multiple protease inhibitors. It is of great interest to understand the level of contributions made by active-and nonactive-site mutations to the development of a high level of resistance (1-6). The HIV protease is a symmetric dimer composed of two 99-residue polypeptides (Figure 1). The dimerization region comprises the floor of the active site, which includes two catalytic aspartic acids (Asp25), one provided by each polypeptide. Unlike the human aspartic proteases that have one flap, two flaps completely cap the active site of the HIV protease. A more detailed description of HIV-1 protease structure, inhibitor binding, and resistance can be found in reviews by Wlodawer and Gustchina, Tomasselli and Heinrikson,.In this study, we analyze the effect of adding the nonactive-site mutations M46I and I54V and the active-site mutation I84V to a post...

The crystal structure of a recombinant form of the proteinase encoded by the feline immunodeficiency virus (FIV PR) has been solved at 2 A resolution and refined to an R-factor of 0.148. The refined structure includes a peptidomimetic, statine-based inhibitor, LP-149, which is an even more potent inhibitor of HIV PR. Kinetic parameters were obtained for the cleavage of five substrates by FIV PR, and inhibition constants were measured for four inhibitors. The structure of FIV PR resembles other related retroviral enzymes although few inhibitors of HIV PR are capable of inhibiting FIV PR. The structure of FIV PR will enhance our knowledge of this class of enzymes, and will direct testing of new proteinase inhibitors in a feline animal model.

Development of resistance mutations in enzymatic targets of human immunodeficiency virus 1 (HIV-1) hampers the ability to provide adequate therapy. Of special interest is the effect mutations outside the active site of HIV-1 protease have on inhibitor binding and virus viability. We engineered protease mutants containing the active site mutation D30N alone and with the nonactive site polymorphisms M36I and/or A71V. We determined the K(i) values for the inhibitors nelfinavir, ritonavir, indinavir, KNI272, and AG1776 as well as the catalytic efficiency of the mutants. Single and double mutation combinations exhibited a decrease in catalytic efficiency, while the triple mutant displayed catalytic efficiency greater than that of the wild type. Variants containing M36I or A71V alone did not display a significant change in binding affinities to the inhibitors tested. The variant containing mutation D30N displayed a 2-6-fold increase in K(i) for all inhibitors tested, with nelfinavir showing the greatest increase. The double mutants containing a combination of mutations D30N, M36I, and A71V displayed -0.5-fold to +6-fold changes in the K(i) of all inhibitors tested, with ritonavir and nelfinavir most affected. Only the triple mutant showed a significant increase (>10-fold) in K(i) for inhibitor nelfinavir, ritonavir, or AG-1776 displaying 22-, 19-, or 15-fold increases, respectively. Our study shows that the M36I and A71V mutations provide a greater level of inhibitor cross-resistance combined with active site mutation D30N. M36I and A71V, when present as natural polymorphisms, could aid the virus in developing active site mutations to escape inhibitor binding while maintaining catalytic efficiency.

Design We sought to investigate the evolutionary and historical reasons for the different epidemiological patterns of HIV-1 in the early epidemic. In order to characterize the demographic history of HIV-1 subtypes A and D in east Africa, we examined molecular epidemiology, geographical and historical data. Methodology We employed high-resolution phylodynamics to investigate the introduction of HIV-1A and D into east Africa, the geographic trends of viral spread, and the demographic growth of each subtype. We also used geographic information system data to investigate human migration trends, population growth, and human mobility. Results HIV-1A and D were introduced into east Africa after 1950 and spread exponentially during the 1970s, concurrent with eastward expansion. Spatiotemporal data failed to explain the establishment and spread of HIV based on urban population growth and migration. The low prevalence of the virus in the Democratic Republic of Congo before and after the emergence of the pandemic was, however, consistent with regional accessibility data, highlighting the difficulty in travel between major population centers in central Africa. In contrast, the strong interconnectivity between population centers across the east African region since colonial times has likely fostered the rapid growth of the epidemic in this locale. Conclusion This study illustrates how phylodynamic analysis of pathogens informed by geospatial data can provide a more holistic and evidence-based interpretation of past epidemics. We advocate that this ‘landscape phylodynamics’ approach has the potential to provide a framework both to understand epidemics' spread and to design optimal intervention strategies.

BackgroundMacrophages provide an interface between innate and adaptive immunity and are important long-lived reservoirs for Human Immunodeficiency Virus Type-1 (HIV-1). Multiple genetic networks involved in regulating signal transduction cascades and immune responses in macrophages are coordinately modulated by HIV-1 infection.Methodology/Principal FindingsTo evaluate complex interrelated processes and to assemble an integrated view of activated signaling networks, a systems biology strategy was applied to genomic and proteomic responses by primary human macrophages over the course of HIV-1 infection. Macrophage responses, including cell cycle, calcium, apoptosis, mitogen-activated protein kinases (MAPK), and cytokines/chemokines, to HIV-1 were temporally regulated, in the absence of cell proliferation. In contrast, Toll-like receptor (TLR) pathways remained unaltered by HIV-1, although TLRs 3, 4, 7, and 8 were expressed and responded to ligand stimulation in macrophages. HIV-1 failed to activate phosphorylation of IRAK-1 or IRF-3, modulate intracellular protein levels of Mx1, an interferon-stimulated gene, or stimulate secretion of TNF, IL-1β, or IL-6. Activation of pathways other than TLR was inadequate to stimulate, via cross-talk mechanisms through molecular hubs, the production of proinflammatory cytokines typical of a TLR response. HIV-1 sensitized macrophage responses to TLR ligands, and the magnitude of viral priming was related to virus replication.Conclusions/SignificanceHIV-1 induced a primed, proinflammatory state, M1HIV, which increased the responsiveness of macrophages to TLR ligands. HIV-1 might passively evade pattern recognition, actively inhibit or suppress recognition and signaling, or require dynamic interactions between macrophages and other cells, such as lymphocytes or endothelial cells. HIV-1 evasion of TLR recognition and simultaneous priming of macrophages may represent a strategy for viral survival, contribute to immune pathogenesis, and provide important targets for therapeutic approaches.

Telomere shortening may reflect the total number of divisions experienced by a somatic cell and is associated with replicative senescence. We found that the average rate of telomere shortening in peripheral blood mononuclear cells (PBMCs) obtained longitudinally from nine different infants during the first 3 years of life (270 bp per year) is more than fourfold higher than in adults and does not correlate with telomerase activity. These results show that the rate of telomere loss changes during ontogeny, suggesting the existence of periods of accelerated cell division. Because human immunodeficiency virus (HIV) preferentially infects actively dividing cells, our observation suggesting accelerated cell division in children may provide an explanation for some of the distinctive pathogenic features of the HIV disease in infants, including higher viral loads and more rapid progression to acquired immunodeficiency syndrome (AIDS).

A series of HIV-1 protease mutants have been designed to analyze the contribution to drug resistance provided by natural polymorphisms as well as therapy-selective (active and non-active site) mutations in the HIV-1 CRF_01 A/E (AE) protease when compared to the subtype-B (B) protease. Kinetic analysis of these variants using chromogenic substrates showed differences in substrate specificity between pre-therapy B and AE proteases. Inhibition analysis with ritonavir, indinavir, nelfinavir, amprenavir, saquinavir, lopinavir, and atazanavir revealed that the natural polymorphisms found in A/E can influence inhibitor resistance. It was also apparent that a high level of resistance in the A/E protease, as with B protease, is due to aquiring a combination of active site and non-active site mutations. Structural analysis of atazanavir bound to a pre-therapy B protease showed that the ability of atazanavir to maintain its binding affinity to variants containing some resistance mutations is due to its unique interactions with flap residues. This structure also explains why the I50L and I84V mutations are important in decreasing the binding affinity of atazanavir.