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1.  Nef Neutralizes the Ability of Exosomes from CD4+ T Cells to Act as Decoys during HIV-1 Infection 
PLoS ONE  2014;9(11):e113691.
Nef is an HIV-1 accessory protein that promotes viral replication and pathogenesis. A key function of Nef is to ensure sustained depletion of CD4 and MHC-I molecules in infected cells by inducing targeting of these proteins to multivesicular bodies (MVBs), and ultimately to lysosomes for degradation. Nef also affects cellular secretory routes promoting its own secretion via exosomes. To better understand the effects of Nef on the exocytic pathway, we investigated whether this viral factor modifies the composition of exosomes released by T lymphocytes. We showed that both CD4 and MHC-I molecules are secreted in exosomes from T cells and that the expression of Nef reduces the amount of these proteins in exosomes. To investigate the functional role for this novel activity of Nef, we performed in vitro HIV-1 infection assays in the presence of distinct populations of exosomes. We demonstrated that exosomes released by CD4+ T cells, but not CD4− T cells, efficiently inhibit HIV-1 infection in vitro. Because CD4 is the main receptor for HIV-1 infection, these results suggest that CD4 molecules displayed on the surface of exosomes can bind to envelope proteins of HIV-1 hindering virus interaction with target cells and infection. Importantly, CD4-depleted exosomes released by CD4+ T cells expressing Nef have a reduced capacity to inhibit HIV-1 infection in vitro. These results provide evidence that Nef promotes HIV-1 infection by reducing the expression of CD4 in exosomes from infected cells, besides the original role of Nef in reducing the CD4 levels at the cell surface.
PMCID: PMC4244142  PMID: 25423108
2.  Modulation of α-Enolase Post-Translational Modifications by Dengue Virus: Increased Secretion of the Basic Isoforms in Infected Hepatic Cells 
PLoS ONE  2014;9(8):e88314.
Hepatic cells are major sites of dengue virus (DENV) replication and liver injury constitutes a characteristic of severe forms of dengue. The role of hepatic cells in dengue pathogenesis is not well established, but since hepatocytes are the major source of plasma proteins, changes in protein secretion by these cells during infection might contribute to disease progression. Previously, we showed that DENV infection alters the secretion pattern of hepatic HepG2 cells, with α-enolase appearing as one of the major proteins secreted in higher levels by infected cells. ELISA analysis demonstrated that DENV infection modulates α-enolase secretion in HepG2 cells in a dose-dependent manner, but has no effect on its gene expression and on the intracellular content of the protein as assessed by PCR and western blot analyses, respectively. Two-dimensional western blots showed that both intracellular and secreted forms of α-enolase appear as five spots, revealing α-enolase isoforms with similar molecular weights but distinct isoeletric points. Remarkably, quantification of each spot content revealed that DENV infection shifts the isoform distribution pattern of secreted α-enolase towards the basic isoforms, whereas the intracellular protein remains unaltered, suggesting that post-translational modifications might be involved in α-enolase secretion by infected cells. These findings provide new insights into the mechanisms underlying α-enolase secretion by hepatic cells and its relationship with the role of liver in dengue pathogenesis. In addition, preliminary results obtained with plasma samples from DENV-infected patients suggest an association between plasma levels of α-enolase and disease severity. Since α-enolase binds plasminogen and modulates its activation, it is plausible to speculate the association of the increase in α-enolase secretion by infected hepatic cells with the haemostatic dysfunction observed in dengue patients including the promotion of fibrinolysis and vascular permeability alterations.
PMCID: PMC4149363  PMID: 25171719
3.  Atypical esthesioneuroblastoma invading oral cavity: a case report and review of the literature 
Diagnostic Pathology  2014;9:10.
Esthesioneuroblastoma is an uncommon tumour of neuroectodermal origin. The authors describe a rare presentation of an atypical esthesioneuroblastoma invading oral cavity. The clinical presentation, aetiology, diagnosis, and management of this condition are discussed. The patient developed significant swelling in the right anterosuperior alveolar mucosa and had moderate tooth mobility. Conventional x-rays and computed tomography revealed a large osteolytic lesion, with imprecise limits. Histological findings along with immunohistochemical staining results and clinical features led to the diagnosis of high-grade esthesioneuroblastoma. Local recurrences and neck metastasis were detected. The rare oral findings produced delayed in diagnosis which may lead to a compromise in planning and execution of further radical management and thus a poor prognosis.
Virtual slides
The virtual slides for this article can be found here:
PMCID: PMC3943513  PMID: 24443792
Esthesioneuroblastoma; Olfactory neuroblastoma; Maxillary neoplasms; Diagnosis; Oral cavity
4.  Differential In Vitro Kinetics of Drug Resistance Mutation Acquisition in HIV-1 RT of Subtypes B and C 
PLoS ONE  2012;7(10):e46622.
HIV-1 subtype B is the most prevalent in developed countries and, consequently, it has been extensively studied. On the other hand, subtype C is the most prevalent worldwide and therefore is a reasonable target for future studies. Here we evaluate the acquisition of resistance and the viability of HIV-1 subtype B and C RT clones from different isolates that were subjected to in vitro selection pressure with zidovudine (ZDV) and lamivudine (3TC).
Methods/Principal Findings
MT4 cells were infected with chimeric virus pseudotyped with RT from subtype B and C clones, which were previously subjected to serial passage with increasing concentrations of ZDV and 3TC. The samples collected after each passage were analyzed for the presence of resistance mutations and VL. No differences were found between subtypes B and C in viral load and resistance mutations when these viruses were selected with 3TC. However, the route of mutations and the time to rebound of subtype B and C virus were different when subjected to ZDV treatment. In order to confirm the role of the mutations detected, other clones were generated and subjected to in vitro selection. RT subtype B virus isolates tended to acquire different ZDV resistance mutations (Q151M and D67N or T215Y, D67D/N and F214L) compared to subtype C (D67N, K70R, T215I or T215F).
This study suggests that different subtypes have a tendency to react differently to antiretroviral drug selection in vitro. Consequently, the acquisition of resistance in patients undergoing antiretroviral therapy can be dependent on the subtypes composing the viral population.
PMCID: PMC3463560  PMID: 23056372
5.  Interactions between SIVNef, SIVGagPol and Alix correlate with viral replication and progression to AIDS in rhesus macaques 
Virology  2009;394(1):47-56.
Infection with Simian Immunodeficiency Virus (SIV) leads to high viral loads and progression to Simian AIDS (SAIDS) in rhesus macaques. The viral accessory protein Nef is required for this phenotype in monkeys as well as in HIV-infected humans. Previously, we determined that HIVNef binds HIVGagPol and Alix for optimal viral replication in cells. In this study, we demonstrated that these interactions could correlate with high viral loads leading to SAIDS in the infected host. By infecting rhesus macaques with a mutant SIVmac239, where sequences in the nef gene that are required for these interactions were mutated, we observed robust viral replication and disease in two out of four monkeys, where they reverted to the wild type genotype and phenotype. These two rhesus macaques also died of SAIDS. Two other monkeys did not progress to disease and continued to harbor mutant nef sequences. We conclude that interactions between Nef, GagPol and Alix contribute to optimal viral replication and progression to disease in the infected host.
PMCID: PMC2767429  PMID: 19748111
SIV; HIV; Nef; monkey infection; pathogenesis; disease progression; GagPol; Alix
6.  Development of a New Methodology for Screening of Human Immunodeficiency Virus Type 1 Microbicides Based on Real-Time PCR Quantification▿  
Potential topical retrovirucides or vaginal microbicides against human immunodeficiency virus type 1 (HIV-1) include nonnucleoside reverse transcriptase inhibitors (NNRTIs). To be successful, such agents have to be highly active against cell-free virions. In the present study, we developed a new real-time PCR-based assay to measure the natural endogenous reverse transcription (NERT) activity directly on intact HIV-1 particles in the presence of reverse transcriptase (RT) inhibitors. We further evaluated the permeability to nevirapine (NVP) and efavirenz (EFV) and their retention within nascent viral particles. We also demonstrated the NVP and EFV inhibitory effects on NERT activity and the impact of resistance mutations measured directly by this new strategy. Furthermore, the results showed a clear correlation between NERT activity and classical infectivity assays. The 50% inhibitory concentrations (IC50s) of NVP and EFV were demonstrated to be up to 100-fold higher for cell-free than for cell-associated virions, suggesting that cell-free virions are less permeable to these drugs. Our results suggest that NVP and EFV penetrate both the envelope and the capsid of HIV-1 particles and readily inactivate cell-free virions. However, the characteristics of these NNRTIs, such as lower permeability and lower retention during washing procedures, in cell-free virions reduce their efficacies as microbicides. Here, we demonstrate the usefulness of the NERT real-time PCR as an assay for screening novel antiretroviral compounds with unique mechanisms of action.
PMCID: PMC1797782  PMID: 17116672
7.  Interactions between Nef and AIP1 proliferate multivesicular bodies and facilitate egress of HIV-1 
Retrovirology  2006;3:33.
Nef is an accessory protein of primate lentiviruses, HIV-1, HIV-2 and SIV. Besides removing CD4 and MHC class I from the surface and activating cellular signaling cascades, Nef also binds GagPol during late stages of the viral replicative cycle. In this report, we investigated further the ability of Nef to facilitate the replication of HIV-1.
To this end, first the release of new viral particles was much lower in the absence of Nef in a T cell line. Since the same results were obtained in the absence of the viral envelope using pseudo-typed viruses, this phenomenon was independent of CD4 and enhanced infectivity. Next, we found that Nef not only possesses a consensus motif for but also binds AIP1 in vitro and in vivo. AIP1 is the critical intermediate in the formation of multivesicular bodies (MVBs), which play an important role in the budding and release of viruses from infected cells. Indeed, Nef proliferated MVBs in cells, but only when its AIP1-binding site was intact. Finally, these functions of Nef were reproduced in primary macrophages, where the wild type but not mutant Nef proteins led to increased release of new viral particles from infected cells.
We conclude that by binding GagPol and AIP1, Nef not only proliferates MVBs but also contributes to the egress of viral particles from infected cells.
PMCID: PMC1526754  PMID: 16764724
8.  Impact of Nelfinavir Resistance Mutations on In Vitro Phenotype, Fitness, and Replication Capacity of Human Immunodeficiency Virus Type 1 with Subtype B and C Proteases 
Human immunodeficiency virus type 1 subtype B and C proteases were manipulated to contain 90M, 88D, or 89L, and their in vitro biological properties were studied. We showed that D30N has significantly more impact in subtype C than in subtype B counterparts, accounting for the reported low prevalence of this mutation in patients failing nelfinavir-based regimens.
PMCID: PMC514783  PMID: 15328124

Results 1-8 (8)