Biomedical subjects

Maureen M Goodenow

Publications and source records attributed to Maureen M Goodenow.

16 recordsLinked to original sources

PubMed2006

HIV-1 coreceptor preference is distinct from target cell tropism: a dual-parameter nomenclature to define viral phenotypes.

HIV-1 infection of cells is mediated by engagement between viral envelope glycoproteins (Env) and a receptor complex comprising CD4 and one of two chemokine receptors, CCR5 and CXCR4, expressed on the surface of target cells. Most CD4+-transformed T cell lines express only CXCR4, but primary lymphocytes and macrophages, the main cellular targets for infection in vivo, express both coreceptors. Cell- and viral strain-specific utilization of these coreceptor pathways, rather than coreceptor expression per se, regulates lymphocyte and macrophage entry and tropism. Virus use of coreceptor[s] (R5, X4, or R5 and X4) and its target cell tropism (lymphocytes, macrophages, and/or transformed T cell lines) are related but distinct characteristics of Envs. A comprehensive classification schema of HIV-1 Env phenotypes that addresses both tropism and coreceptor use is proposed. Defining Env phenotype based on both parameters is important in the development of entry inhibitors and vaccines, for understanding changes in Env that evolve over time in vivo, and for discerning differences among viral species that underlie aspects of pathogenesis and transmission. Recognizing how tropism is related to, yet differs from, coreceptor selectivity is critical for understanding the mechanisms by which these viral characteristics impact pathogenesis.

CD4-Positive T-Lymphocytes↗

PubMed2006

Analysis of HIV-1 CRF_01 A/E protease inhibitor resistance: structural determinants for maintaining sensitivity and developing resistance to atazanavir.

A series of HIV-1 protease mutants has been designed in an effort 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 that of the subtype B (B) protease. Kinetic analysis of these variants using chromogenic substrates showed differences in substrate specificity between pretherapy 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 it aquiring a combination of active site and non-active site mutations. Structural analysis of atazanavir bound to a pretherapy B protease showed that the ability of atazanavir to maintain its binding affinity for 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.

Amino Acid Substitution↗

PubMed2005

Transforming growth factor-beta1 increases CXCR4 expression, stromal-derived factor-1alpha-stimulated signalling and human immunodeficiency virus-1 entry in human monocyte-derived macrophages.

Stromal-derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 play crucial roles in leukocyte migration and activation, as well as embryogenesis, angiogenesis, cancer and viral pathogenesis. CXCR4 is one of the major human immunodeficiency virus-1 (HIV-1) coreceptors on macrophages. In many tissues macrophages are one of the predominant cell types infected by HIV-1 and act as a reservoir for persistent infection and viral dissemination. In patients infected by HIV-1, blood and tissue levels of transforming growth factor-beta1 (TGF-beta1) are increased. The purpose of this study was to evaluate the effects of TGF-beta1 on CXCR4 expression and function in primary human monocyte-derived macrophages (MDMs) and rat microglia. TGF-beta1 up-regulated CXCR4 and enhanced SDF-1alpha-stimulated ERK1,2 phosphorylation in these cells. The increased CXCR4 expression in human MDMs resulted in increased susceptibility of the cells to entry by dual-tropic CXCR4-using HIV-1 (D-X4). In contrast, TGF-beta1 failed to increase CCR5 expression or infection by a CCR5-using virus in MDMs. Our data demonstrate that TGF-beta1 enhances macrophage responsiveness to SDF-1alpha stimulation and susceptibility to HIV-1 by selectively increasing expression of CXCR4. The results suggest that increased expression of CXCR4 on macrophages may contribute to the emergence of dual-tropic X4 viral variants at later stages of HIV-1 infection.

Animals↗

PubMed2005

Complex determinants in human immunodeficiency virus type 1 envelope gp120 mediate CXCR4-dependent infection of macrophages.

Host cell range, or tropism, combined with coreceptor usage defines viral phenotypes as macrophage tropic using CCR5 (M-R5), T-cell-line tropic using CXCR4 (T-X4), or dually lymphocyte and macrophage tropic using CXCR4 alone or in combination with CCR5 (D-X4 or D-R5X4). Although envelope gp120 V3 is necessary and sufficient for M-R5 and T-X4 phenotypes, the clarity of V3 as a dominant phenotypic determinant diminishes in the case of dualtropic viruses. We evaluated D-X4 phenotype, pathogenesis, and emergence of D-X4 viruses in vivo and mapped genetic determinants in gp120 that mediate use of CXCR4 on macrophages ex vivo. Viral quasispecies with D-X4 phenotypes were associated significantly with advanced CD4+-T-cell attrition and commingled with M-R5 or T-X4 viruses in postmortem thymic tissue and peripheral blood. A D-X4 phenotype required complex discontinuous genetic determinants in gp120, including charged and uncharged amino acids in V3, the V5 hypervariable domain, and novel V1/V2 regions distinct from prototypic M-R5 or T-X4 viruses. The D-X4 phenotype was associated with efficient use of CXCR4 and CD4 for fusion and entry but unrelated to levels of virion-associated gp120, indicating that gp120 conformation contributes to cell-specific tropism. The D-X4 phenotype describes a complex and heterogeneous class of envelopes that accumulate multiple amino acid changes along an evolutionary continuum. Unique gp120 determinants required for the use of CXCR4 on macrophages, in contrast to cells of lymphocytic lineage, can provide targets for development of novel strategies to block emergence of X4 quasispecies of human immunodeficiency virus type 1.

Adolescent↗

PubMed2004

Two-year clinical and immune outcomes in human immunodeficiency virus-infected children who reconstitute CD4 T cells without control of viral replication after combination antiretroviral therapy.

OBJECTIVE: To evaluate 96-week clinical and immune outcomes to protease inhibitor-containing antiretroviral therapy. METHODS: A prospective study was conducted of 40 human immunodeficiency virus (HIV)-infected children who displayed viral suppression (VS) with successful immune reconstitution (IS), failure to suppress virus (VF) or develop immune reconstitution (IF), or discordant immune and viral responses (VF/IS) at 24 weeks posttherapy. All children enrolled had viral RNA >4.0 log10 copies per mL and were Centers for Disease Control ad Prevention immune stage 2 or 3. Clinical, viral, and immune outcomes were assessed during the subsequent 72 weeks. RESULTS: VS/IS and VF/IS groups displayed similar sustained increases in CD4 T cells, although viral levels rebounded by 48 and 96 weeks posttherapy to pretherapy levels in the discordant group. The VF/IS outcome group had significant increases in height and weight z scores compared with entry and were similar to the VS/IS group. After treatment, antigen-specific responses after tetanus immunization were similar in the VF/IS and VS/IS groups. Prevalence of HIV-associated illnesses decreased in both VS/IS and VF/IS but not in VF/IF response groups. CONCLUSIONS: The findings indicate that viral replication under the selective pressure of protease inhibitors fails to exhibit the same deleterious impact on T-cell immunity as pretherapy viruses. CD4 T-cell counts may be a better predictor of disease progression and improvement in growth than viral burden in HIV-infected children who receive a protease inhibitor as part of a highly active antiretroviral therapy regimen.

AIDS-Related Opportunistic Infections↗

PubMed2004

Comparing the accumulation of active- and nonactive-site mutations in the HIV-1 protease.

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. Crystal-structure 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.

Binding Sites↗

PubMed2004

Effects of human immunodeficiency virus type 1 infection on CCR5 and CXCR4 coreceptor expression on CD4 T lymphocyte subsets in infants and adolescents.

HIV-1 infection alters expression of CCR5 and CXCR4 on CD4 T cells in adults, although an effect by virus on expression of coreceptor genes in pediatric subjects is unknown. We designed an exploratory study to evaluate surface expression of CXCR4 and CCR5 on CD45RA and CD45RO subsets of CD4 T lymphocytes from 17 HIV-1-infected infants and adolescents and 16 healthy age-matched individuals. While age in the absence of HIV-1 infection was unrelated to coreceptor expression, infection affected coreceptor expression differentially in infants and adolescents. Among infected adolescents, CCR5 and CXCR4 expression was significantly increased on CD4 CD45RO T cells, while CXCR4 was diminished in the CD4 CD45RA subset. Although HIV-1 infection in infants was also associated with increased CXCR4 expression on the CD4 CD45RO subset, in contrast to adolescents, infection in infants had no impact on coreceptor expression within the CD45RA CD4 subset. The proportion of CD4 T cells coexpressing CD45RA and CD45RO was increased by infection in both infants and adolescents. The CD45RA CD45RO subset in culture expressed high levels of CD4, CXCR4, and CD69, an early activation marker, and was highly susceptible to HIV-1 infection and replication. Infection of transitional CD4 T cells coexpressing CD45RA and CD45RO could contribute in part to provirus in either CD45RA or CD45RO subsets. Deleterious effects by HIV-1 infection on CD4 T cell homeostasis were greater in infants then adolescents, indicating that adolescence may be an optimal age group for assessing vaccines to prevent or treat HIV-1 infection.

Adolescent↗

PubMed2004

Impact on genetic networks in human macrophages by a CCR5 strain of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) impacts multiple lineages of hematopoietic cells, including lymphocytes and macrophages, either by direct infection or indirectly by perturbations of cell networks, leading to generalized immune deficiency. We designed a study to discover, in primary human macrophages, sentinel genetic targets that are impacted during replication over the course of 7 days by a CCR5-using virus. Expression of mRNA and proteins in virus- or mock-treated macrophages from multiple donors was evaluated. Hierarchical agglomerative cluster analysis grouped into distinct temporal expression patterns >900 known human genes that were induced or repressed at least fourfold by virus. Expression of more than one-third of the genes was induced rapidly by day 2 of infection, while other genes were induced at intermediate (day 4) or late (day 7) time points. More than 200 genes were expressed exclusively in either virus- or mock-treated macrophage cultures, independent of the donor, providing an unequivocal basis to distinguish an effect by virus. HIV-1 altered levels of mRNA and/or protein for diverse cellular programs in macrophages, including multiple genes that can contribute to a transition in the cell cycle from G(1) to G(2)/M, in contrast to expression in mock-treated macrophages of genes that maintain G(0)/G(1). Virus treatment activated mediators of cell cycling, including PP2A, which is impacted by Vpr, as well as GADD45 and BRCA1, potentially novel targets for HIV-1. The results identify interrelated programs conducive to optimal HIV-1 replication and expression of genes that can contribute to macrophage dysfunction.

CDC2 Protein Kinase↗

PubMed2003

Secondary mutations M36I and A71V in the human immunodeficiency virus type 1 protease can provide an advantage for the emergence of the primary mutation D30N.

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.

Alanine↗

PubMed2003

HIV-1 fitness and macrophages.

HIV-1 comprises a collection of closely related, but not identical, viruses or quasispecies. Fitness represents a selective advantage for propagation among populations of organisms competing in a particular environment and is an important characteristic of viruses because of a link between fitness and pathogenesis. Environmental differences based on the type of cell that is targeted for infection or the cell type that produces virus, impact fitness. CD4-expressing cells of lymphocyte or macrophage lineage are the principal host cells for HIV-1, although the milieu in lymphocytes is distinct from the macrophage environment from the perspective of cell half-life and activation, signal transduction and expression of coreceptors, and bioavailability of antiretroviral drugs. Multiple viral determinants, including entry via envelope glycoproteins, replication by reverse transcriptase, and virion maturation by protease activity, contribute to fitness in different cells and provide targets for current antiretroviral therapies. This review focuses on fitness of HIV-1 in macrophages and examines the impact of protease inhibitors on fitness of quasispecies and an unexplained discordance between fitness and pathogenesis.

Acquired Immunodeficiency Syndrome↗

PubMed2003

Combination antiretroviral therapy results in a rapid increase in T cell receptor variable region beta repertoire diversity within CD45RA CD8 T cells in human immunodeficiency virus-infected children.

Human immunodeficiency virus (HIV) type 1 disrupts the T cell receptor (TCR) variable region (V) beta repertoire in CD8 T cells by impairing thymic capacity and skewing postthymic cellular maturation. The TCR repertoire was examined using spectratyping of CDR3 length diversity within CD45RA and CD45RO CD8 T cells in HIV-infected and healthy children. In healthy children, CDR3 lengths displayed Gaussian distribution in both CD45RA and CD45RO subsets. Vbeta families in HIV-infected children displayed a large proportion of perturbations in both subsets. High virus load and advanced immunosuppression correlated with increased perturbations within CD45RA but not CD45RO CD8 T cells. After therapy and virus suppression, there was rapid reestablishment of Gaussian distributions in CD45RA cells. HIV-1-induced disruption of TCR diversity within CD45RA CD8 T cells correlates with disease progression. Suppression of viral replication by treatment results in the rapid correction of TCR diversity in this CD8 subset because of emergence of new T cells from the thymus.

Adolescent↗

PubMed2003

13. HIV-1 infection.

This review is intended to provide a fundamental perspective on the dynamic interplay between HIV-1 and the immune system, an essential aspect in defining the pathogenesis and treatment of AIDS. HIV-1 infection, the cause of AIDS, is a worldwide pandemic with enormous adverse heath and economic implications, particularly in the developing world. This bloodborne and sexually transmitted disease, which evolved from simian immunodeficiency virus, infects and replicates in helper T cells and macrophages and utilizes CD4 and a chemokine coreceptor for entry. Immune deficiency occurs as a result of virally induced attrition of CD4 T cells, resulting in the development of opportunistic infections and malignancy. Prophylaxis against opportunistic infections is required according to the extent of immune deficiency. HIV-specific immunity can control viral replication and delay disease progression but does not clear infection. Antiretroviral treatment consists of inhibitors that target for viral entry, reverse transcriptase, and viral protease. Therapy can control viral replication, restore immunity, and delay disease progression, but it cannot eliminate infection. Thus chronic infection persists even in treated patients. Antiretroviral drugs have been highly effective in preventing mother-to-child transmission and for postexposure prophylaxis. Several novel vaccines in development hold promise for either effective infection prevention or attenuation of disease progression.

HIV↗

PubMed2003

Human immunodeficiency virus type 1 (HIV-1) induces activation of multiple STATs in CD4+ cells of lymphocyte or monocyte/macrophage lineages.

Human immunodeficiency virus type 1 (HIV-1) impacts the activation state of multiple lineages of hematopoietic cells. Chronic HIV-1 infection among individuals with progressive disease can be associated with increased levels of activated signal transducers and activators of transcription (STATs) in peripheral blood mononuclear cells. To investigate interactions between HIV-1 and CD4(+) cells, activated, phosphorylated STAT proteins in nuclear extracts from lymphocytic and promonocytic cell lines as well as primary monocyte-derived macrophages were measured. Levels of activated STATs increased six- to tenfold in HUT78 and U937 cells within 2 h following exposure to virions. The response to virus was dose-dependent, but kinetics of activation was delayed relative to interleukin-2 or interferon-gamma. Activation of STAT1, STAT3, and STAT5 occurred with diverse viral envelope proteins, independent of coreceptor use or viral replication. Envelope-deficient virions had no effect on STAT activation. Monoclonal antibody engagement of CD4 identified a novel role for CD4 as a mediator in the activation of multiple STATs. Results provide a model for HIV-1 pathogenesis in infected and noninfected hematopoietic cells.

CD4 Antigens↗

PubMed2002

Retroviral proteases.

SUMMARY: The proteases of retroviruses, such as leukemia viruses, immunodeficiency viruses (including the human immunodeficiency virus, HIV), infectious anemia viruses, and mammary tumor viruses, form a family with the proteases encoded by several retrotransposons in Drosophila and yeast and endogenous viral sequences in primates. Retroviral proteases are key enzymes in viral propagation and are initially synthesized with other viral proteins as polyprotein precursors that are subsequently cleaved by the viral protease activity at specific sites to produce mature, functional units. Active retroviral proteases are homodimers, with each dimer structurally related to the larger class of single-chain aspartic peptidases. Each monomer has four structural elements: two distinct hairpin loops, a wide loop containing the catalytic aspartic acid and an alpha helix. Retroviral gene sequences can vary between infected individuals, and mutations affecting the binding cleft of the protease or the substrate cleavage sites can alter the response of the virus to therapeutic drugs. The need to develop new drugs against HIV will continue to be, to a large extent, the driving force behind further characterization of retroviral proteases.

Animals↗

PubMed2002

Increased replication of non-syncytium-inducing HIV type 1 isolates in monocyte-derived macrophages is linked to advanced disease in infected children.

Non-syncytium-inducing (NSI) strains of HIV-1 prevail among most infected children, including pediatric patients who develop advanced disease, severe immune suppression, and die. A study was designed to address the hypothesis that genotypic and/or phenotypic markers can distinguish NSI viruses isolated during early infection from NSI viruses found in advanced disease. Primary HIV-1 isolates, which were obtained from 43 children, adolescents, and adults who displayed a cross-section of clinical disease and immune suppression but were untreated by protease inhibitor antiretroviral therapy, were characterized for replication phenotype in different cell types. Most individuals (81%) harbored NSI viruses and almost half had progressed to advanced disease or severe immune deficiency. About 51% of NSI isolates produced low levels of p24 antigen (median, 142 pg/ml) in monocyte-derived macrophages (MDMs), 31% produced medium levels (median, 1584 pg/ml), and 17% produced high levels (median, 81,548 pg/ml) (p < 0.001). Seven of eight syncytium-inducing isolates also replicated in MDMs and displayed a dual-tropic phenotype that was associated with advanced disease. Replication of NSI viruses in MDMs varied as much as 100- to 1000-fold and was independent of replication in peripheral blood mononuclear cells. Replication in MDMs provided a clear biological feature to distinguish among viruses that were otherwise identical by NSI phenotype, V3 genotype, and CCR5 coreceptor usage. Low-level MDM replication was characteristic of viruses isolated from asymptomatic individuals, including long-term survivors. Enhanced MDM replication was related to morbidity and mortality among patients. Replication levels in MDMs provide a novel prognostic indicator of pathogenic potential by NSI viruses.

Acquired Immunodeficiency Syndrome↗

PubMed2002

Naturally occurring amino acid polymorphisms in human immunodeficiency virus type 1 (HIV-1) Gag p7(NC) and the C-cleavage site impact Gag-Pol processing by HIV-1 protease.

Human immunodeficiency virus type 1 (HIV-1) protease activity is targeted at nine cleavage sites comprising different amino acid sequences in the viral Gag-Pol polyprotein. Amino acid polymorphisms in protease and in regions of Gag, particularly p7(NC) and the C-cleavage site between p2 and p7(NC), occur in natural variants of HIV-1 within infected patients. Studies were designed to examine the role of natural polymorphisms in protease and to identify determinants in Gag that modulate protease processing activity. Closely related Gag-Pol regions from an HIV-1-infected mother and two children were evaluated for processing in an inducible expression system, for protease activity on cleavage-site analogues, and for impact on replication by recombinant viruses. Gag-Pol regions displayed one of three processing phenotypes based on the appearance of Gag intermediates and accumulation of mature p24(CA). Gag-Pol regions that were processed rapidly to produce p24(CA) resulted in high-level replication by recombinant viruses, while slow-processing Gag-Pol variants resulted in recombinant viruses that replicated with reduced kinetics in both T cell lines and peripheral blood mononuclear cells. Direct impact by Gag sequences on processing by protease was assessed by construction of chimeric Gag-Pol regions and by site-directed mutagenesis. Optimal protease activity occurred when Gag and Pol regions were derived from the same gag-pol allele. Heterologous Gag regions generally diminished rates and extent of protease processing. Natural polymorphisms in novel positions in p7(NC) and the C-cleavage site have a dominant effect on protease processing activity. Accumulation of Gag products after processing at the C site appears to delay subsequent cleavage and production of mature p24(CA).

Alleles↗