Contribution of the C-terminal tri-lysine regions of human immunodeficiency virus type 1 integrase for efficient reverse transcription and viral DNA nuclear import

Affiliation: Zhujun Ao, Éric Cohen & Xiaojian Yao : Département de microbiologie et immunologie, Faculté de Médecine, Université de Montréal

Requirements for the selective degradation of CD4 receptor molecules by the human immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum.

BACKGROUND: HIV-1 Vpu targets newly synthesized CD4 receptor for rapid degradation by a process reminiscent of endoplasmic reticulum (ER)-associated protein degradation (ERAD). Vpu is thought to act as an adaptor protein, connecting CD4 to the ubiquitin (Ub)-proteasome degradative system through an interaction with beta-TrCP, a component of the SCFbeta-TrCP E3 Ub ligase complex. RESULTS: Here, we provide direct evidence indicating that Vpu promotes trans-ubiquitination of CD4 through recruitment of SCFbeta-TrCP in human cells. To examine whether Ub conjugation occurs on the cytosolic tail of CD4, we substituted all four Ub acceptor lysine residues for arginines. Replacement of cytosolic lysine residues reduced but did not prevent Vpu-mediated CD4 degradation and ubiquitination, suggesting that Vpu-mediated CD4 degradation is not entirely dependent on the ubiquitination of cytosolic lysines and as such might also involve ubiquitination of other sites. Cell fractionation studies revealed that Vpu enhanced the levels of ubiquitinated forms of CD4 detected in association with not only the ER membrane but also the cytosol. Interestingly, significant amounts of membrane-associated ubiquitinated CD4 appeared to be fully dislocated since they could be recovered following sodium carbonate salt treatment. Finally, expression of a transdominant negative mutant of the AAA ATPase Cdc48/p97 involved in the extraction of ERAD substrates from the ER membrane inhibited Vpu-mediated CD4 degradation. CONCLUSION: Taken together, these results are consistent with a model whereby HIV-1 Vpu targets CD4 for degradation by an ERAD-like process involving most likely poly-ubiquitination of the CD4 cytosolic tail by SCFbeta-TrCP prior to dislocation of receptor molecules across the ER membrane by a process that depends on the AAA ATPase Cdc48/p97.

Effect of calcium-modulating cyclophilin ligand on human immunodeficiency virus type 1 particle release and cell surface expression of Tetherin

The HIV-1 accessory protein Vpu enhances virus particle release by counteracting a host factor that retains virions at the cell surface of infected cells. It was recently demonstrated that cellular protein BST2/CD317/Tetherin restricts HIV-1 release in a Vpu-dependent manner. CAML was also proposed to be involved in this process. We investigated whether CAML is involved in Tetherin cell-surface expression. Here, we show that CAML over-expression in permissive Cos-7 cells or CAML depletion in restrictive HeLa cells has no effect on HIV-1 release nor on Tetherin surface expression, indicating that CAML is not required for Tetherin-mediated restriction of HIV-1 release.

Sexual sensation-seeking and worry about sexually transmitted diseases (STD) and human immunodeficiency virus (HIV) infection among Spanish adolescents. = B??squeda de sensaciones y preocupaci??n por las Enfermedades de Transmisi??n Sexual y VIH en adolescentes espa??oles

Resumen tomado de la publicaci??n. Resumen tambi??n en ingl??s

Human papillomavirus detection from human immunodeficiency virus-infected colombian women's paired urine and cervical samples

Infection, coinfection and type-specific human papillomavirus (HPV) distribution was evaluated in human immunodeficiency virus (HIV)-positive women from paired cervical and urine samples. Paired cervical and urine samples (n = 204) were taken from HIV-positive women for identifying HPV-DNA presence by using polymerase chain reaction (PCR) with three generic primer sets (GP5+/6+, MY09/11 and pU1M/2R). HPV-positive samples were typed for six high-risk HPV (HR-HPV) (HPV-16, -18, -31, -33, -45 and -58) and two low-risk (LR-HPV) (HPV-6/11) types. Agreement between paired sample results and diagnostic performance was evaluated. HPV infection prevalence was 70.6% in cervical and 63.2% in urine samples. HPV-16 was the most prevalent HPV type in both types of sample (66.7% in cervical samples and 62.0% in urine) followed by HPV-31(47.2%) in cervical samples and HPV-58 (35.7%) in urine samples. There was 55.4% coinfection (infection by more than one type of HPV) in cervical samples and 40.2% in urine samples. Abnormal Papanicolau smears were observed in 25.3% of the women, presenting significant association with HPV-DNA being identified in urine samples. There was poor agreement of cervical and urine sample results in generic and type-specific detection of HPV. Urine samples provided the best diagnosis when taking cytological findings as reference. In conclusion including urine samples could be a good strategy for ensuring adherence to screening programs aimed at reducing the impact of cervical cancer, since this sample is easy to obtain and showed good diagnostic performance.

Mapping the immune response to the outer domain of a human immunodeficiency virus-1 clade C gp120

The outer domain (OD) of human immunodeficiency virus (HIV)-1 gp120 represents an attractive, if difficult, target for a beneficial immune response to HIV infection. Unlike the entire gp120, the OD is structurally stable and contains the surfaces that interact with both the primary and secondary cellular receptors. The primary strain-specific neutralizing target, the V3 loop, lies within the OD, as do epitopes for two cross-reactive neutralizing monoclonal antibodies (mAbs), b12 and 2G12, and the contact sites for a number of inhibitory lectins. The OD is poorly immunogenic, at least in the context of complete gp120, but purposeful OD immunization can lead to a substantial antibody response. Here, we map the antibody generated following immunization with a clade C OD. In contrast to published data for the clade B OD, the majority of the polyclonal response to the complete clade C OD is to the V3 loop; deletion of the loop substantially reduces immunogenicity. When the loop sequence was substituted for the epitope for 2F5, a well-characterized human cross-neutralizing mAb, a polyclonal response to the epitope was generated. A panel of mAbs against the clade C OD identified two mAbs that reacted with the loop and were neutralizing for clade C but not B isolates. Other mAbs recognized both linear and conformational epitopes in the OD. We conclude that, as for complete gp120, V3 immunodominance is a property of OD immunogens, that the responses can be neutralizing and that it could be exploited for the presentation of other epitopes.

Control of human immunodeficiency virus type-1 protease activity in insect cells expressing Gag-Pol rescues assembly of immature but not mature virus-like particles

Expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in insect cells using baculovirus vectors leads to the abundant production of virus-like particles (VLPs) that represent the immature form of the virus. When Gag-Pol is included, however, VLP production is abolished, a result attributed to premature protease activation degrading the intracellular pool of Gag precursor before particle assembly can occur. As large-scale synthesis of mature noninfectious VLPs would be useful, we have sought to control HIV protease activity in insect cells to give a balance of Gag and Gag-Pol that is compatible with mature particle formation. We show here that intermediate levels of protease activity in insect cells can be attained through site-directed mutagenesis of the protease and through antiprotease drug treatment. However, despite Gag cleavage patterns that mimicked those seen in mammalian cells, VLP synthesis exhibited an essentially all-or-none response in which VLP synthesis occurred but was immature or failed completely. Our data are consistent with a requirement for specific cellular factors in addition to the correct ratio of Gag and Gag-Pol for assembly of mature retrovirus particles in heterologous cell types. (C) 2003 Elsevier Science (USA). All rights reserved.

Primary immunodeficiency diseases in Latin America: Proceedings of the Second Latin American Society for Immunodeficiencies (LASID) Advisory Board

Early diagnosis and appropriate therapy are essential for the best prognosis and quality of life in patients with primary immunodeficiency diseases (PIDDs). Experts from several Latin American countries have been meeting on a regular basis as part of an ongoing effort to improve the diagnosis and treatment of PIDD in this region. Three programmes are in development that will expand education and training and improve access to testing facilities throughout Latin America. These programmes are: an educational outreach programme (The L-Project); an immunology fellowship programme; and the establishment of a laboratory network to expand access to testing facilities. This report provides the status of these programmes based on the most recent discussions and describes the next steps toward full implementation of these programmes. (C) 2010 SEICAP. Published by Elsevier Espana, S.L. All rights reserved.

Critical, issues and needs in management of primary immunodeficiency diseases in Latin America

Experts from six Latin American countries met to discuss critical issues and needs in the diagnosis and management of primary immunodeficiency diseases (PIDD). The diagnosis of PIDD is generally made following referral to an immunology centre located in a major city, but many paediatricians and general practitioners are not sufficiently trained to suspect PIDD in the first place. Access to laboratory testing is generally limited, and only some screening tests are typically covered by government health programmes. Specialised diagnostic tests are generally not reimbursed. Access to treatment varies by country reflecting differences in healthcare systems and reimbursement policies. An online PIDD Registry Programme for Latin America has been available since 2009, which will provide information about PIDD epidemiology in the region. Additional collaboration across countries appears feasible in at least two areas: a laboratory network to facilitate the diagnosis of PIDD, and educational programmes to improve PIDD awareness. In total, these collaborations should make it possible to advance the diagnosis and management of PIDD in Latin America. (C) 2010 SEICAP. Published by Elsevier Espana, S.L. All rights reserved.

Autoimmunity in common variable immunodeficiency

immunodeficiency (CVID), the most common symptomatic primary immunodeficiency in adulthood. Different authors report high prevalences of autoimmune diseases in CVID, and several mechanisms have been proposed to explain this apparent paradox. Genetic predisposition, under current surveillance, innate and adaptive immunity deficiencies leading to persistent/recurrent infections, variable degrees of immune dysregulation, and possible failure in central and peripheral mechanisms of tolerance induction or maintenance may all contribute to increased autoimmunity. Conclusions Data on the clinical/immunological profile of affected patients and treatment are available mostly concerning autoimmune cytopenias, the most common autoimmune diseases in CVID. Treatment is based on conventional alternatives, in association with short experience with new agents, including rituximab and infliximab. Benefits of early immunoglobulin substitutive treatment and hypothetical premature predictors of autoimmunity are discussed as potential improvements to CVID patients` follow-up.

Slower rescue of ER homeostasis by the unfolded protein response pathway associated with common variable immunodeficiency

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinemia and recurrent infections. Herein we addressed the role of unfolded protein response (UPR) in the pathogenesis of the disease. Augmented unspliced X-box binding protein 1 (XBP-1) mRNA concurrent with co-localization of IgM and BiP/GRP78 were found in one CVID patient. At confocal microscopy analysis this patient`s cells were enlarged and failed to present the typical surface distribution of IgM, which accumulated within an abnormally expanded endoplasmic reticulum. Sequencing did not reveal any mutation on XBP-1, neither on IRE-1 alpha that could potentially prevent the splicing to occur. Analysis of spliced XBP-1, IRE-1 alpha and BiP messages after LPS or Brefeldin A treatment showed that, unlike healthy controls that respond to these endoplasmic reticulum (ER) stressors by presenting waves of transcription of these three genes, this patient`s cells presented lower rates of transcription, not reaching the same level of response of healthy subjects even after 48 h of ER stress. Treatment with DMSO rescued IgM and IgG secretion as well as the expression of spliced XBP-1. Our findings associate diminished splicing of XBP-1 mRNA with accumulation of IgM within the ER and lower rates of chaperone transcription, therefore providing a mechanism to explain the observed hypogammaglobulinemia. (C) 2008 Elsevier Ltd. All rights reserved.

Reduced frequency of CD4(+)CD25(HIGH)FOXP3(+) cells and diminished FOXP3 expression in patients with Common Variable Immunodeficiency: A link to autoimmunity?

Common Variable Immunodeficiency (CVID) is a primary immunodeficiency disease characterized by defective immunoglobulin production and often associated with autoimmunity. We used flow cytometry to analyze CD4(+)CD25(HIGH)FOXP3(+) T regulatory (Treg) cells and ask whether perturbations in their frequency in peripheral blood could underlie the high incidence of autoimmune disorders in CVID patients. In this study, we report for the first time that CVID patients with autoimmune disease have a significantly reduced frequency of CD4(+)CD25(HIGH)FOXP3(+) cells in their peripheral blood accompanied by a decreased intensity of FOXP3 expression. Notably, although CVID patients in whom autoimmunity was not diagnosed had a reduced frequency of CD4(+)CD25(HIGH)FOXP3(+) cells, FOXP3 expression levels did not differ from those in healthy controls. In conclusion, these data suggest compromised homeostasis of CD4(+)CD25(HIGH)FOXP3(+) cells in a subset of CVID patients with autoimmunity, and may implicate Treg cells in pathological mechanisms of CVID. (C) 2009 Elsevier Inc. All rights reserved.

Comparison of Two Methodologies for CD4+T Lymphocytes Relative Counting on Immune Monitoring of Patients with Human Immunodeficiency Virus

Considering that counting the percentage of CD4 T lymphocytes can add prognostic information regarding patients infected with HIV, the aim of this study was to evaluate the percentage values of CD4+ T lymphocytes from 81 patients determined by flow cytometry and estimated by flow cytometry in conjunction with a hematology counter. Means were compared through the Student's t-test. Pearson's correlation was determined, and the agreement between results was tested by Bland-Altman. The level of significance was P < 0.05. It was found a significantly higher mean difference between the relative values of CD4+ T lymphocytes to the hematologic counter (P < 0.05), for all strata studied. Positive and significant correlations (P < 0.01) were found between the strata CD4 < 200 cells/mL (r = 0.93), between 200 and 500 cells/mL (r = 0.65), and >500 cells/mL (r = 0.81). The limits of agreement were 1.0 +/- 3.8% for the stratum of CD4 < 200 cells/mL, approximately 2.2 +/- 13.5% for the stratum of CD4 between 200 and 500 cells/mL, and approximately 6.2 +/- 20.4% for the stratum > 500 cells/mL. The differences in the percentages of CD4+ T lymphocytes obtained by different methodologies could lead to conflict when used in clinical decisions related to the treatment and care of people infected with HIV.

Acquired immunodeficiency syndrome in Brazil

Aim. To describe the acquired immunodeficiency syndrome (AIDS) epidemic in Brazil and analyze the impact of federal government measures addressing the problem since its onset.Method. Retrospective review of AIDS epidemic data from its onset in 1980 up to the last published data in June 2001.Results. AIDS was first reported in Brazil in 1980. By 1988, all 27 Brazilian states had diagnosed cases, and until June 2000 more than half of Brazilian municipalities had recorded at least one case of the disease. The AIDS incidence reached its peak between 1996 and 1997 (14.7 per 100,000 population), and then declined between 1998 and 2000 to 9 per 100,000 population. In the last two decades, the proportion of deaths has been also significantly reduced. These were not random events, but reflected the efficiency of the program implemented by the Brazilian Health Ministry's Coordination on Sexually Transmitted Diseases and AIDS. The program includes an epidemiological surveillance modeling system, which records cases from several regular epidemiological bulletins; national network of diagnosis and monitoring of HIV-infected individuals (ill or not); highly active antiretroviral therapy available free to all patients; mother-infant protection program; educational programs on condom use; the introduction of the female condom; development of AIDS studies in different areas to provide practical solutions; constant preoccupation about drug costs accounting for the patent breaking; and national production of many drugs currently in use.Conclusion. Well-planned and implemented national program against AIDS can significantly reduce the burden of this disease to the population.