Identifying features of the lentiviral genome crucial for the establishment of latency

Background Latently infected cells represent a major obstacle to the cure of infection with human immunodeficiency virus type 1 (HIV-1). We have observed that the propensity to establish latency varies greatly among primate lentiviruses. The purpose of our study is to identify the features that determine such variation in order to elucidate the fine molecular mechanisms regulating latency. Materials and methods Using GFP–encoding reporter viruses, the level of infection by SIV, HIV-1 and HIV-2 was quantitated by flow cytometry in cells stably expressing HIV-1 Tat, SIV Tat or vector alone. Results We have observed that SIVmac 239 infects human lymphoid cells much less efficiently than HIV-1. However, ectopic expression of HIV-1 Tat rescued SIV infection. As host cell type can influence viral gene expression, we tested different cell lines for their ability to support lentiviral latency. In both T (Jurkat TAg and C8166) and T-B hybrid cell lines (CEMX174) SIV exhibited a greater ability to remain transcriptionally silent within the human genome than HIV-1 or HIV-2. The different behaviour in terms of latency was particularly evident in CEMX174 cells, where HIV Tat activation caused more than 20-fold increase in the number of GFP-positive cells infected with SIV, while it had little effect on cells challenged with HIV-1 or HIV-2. The lower level of productive infection displayed by SIVmac239 was not due to a reduced ability of SIV Tat to trigger viral expression in human cells because SIV Tat overexpression reactivated latent SIV as well as HIV Tat did. Moreover, HIV-1 chimeric viruses harboring the U3 region of SIV behaved like the parental HIV-1 viruses, suggesting that viral determinants of SIV latency reside in a part of the lentiviral genome different from the promoter region. Conclusions In human cell lines transduction with SIVmac239 largely establishes a latent infection which is reactivated by overexpressing HIV-1 or SIVmac239 Tat. The tendency of SIVmac239 to establish latent infection is attributable neither to a defective SIV Tat activity nor to a reduced promoter activity of the U3 region in human lymphoid cells. The CEMX174 cell line offers a good model to explore the specific viral determinants which allow SIV to enter latency

Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration

HIV-2 and SIVMAC are AIDS-causing, zoonotic lentiviruses that jumped to humans and rhesus macaques, respectively, from SIVSM-bearing sooty mangabey monkeys. Cross-species transmission events such as these sometimes necessitate virus adaptation to species-specific, host restriction factors such as TRIM5. Here, a new human restriction activity is described that blocks viruses of the SIVSM/SIVMAC/HIV-2 lineage. Human T, B, and myeloid cell lines, peripheral blood mononuclear cells and dendritic cells were 4 to \u3e 100-fold less transducible by VSV G-pseudotyped SIVMAC, HIV-2, or SIVSM than by HIV-1. In contrast, transduction of six epithelial cell lines was equivalent to that by HIV-1. Substitution of HIV-1 CA with the SIVMAC or HIV-2 CA was sufficient to reduce HIV-1 transduction to the level of the respective vectors. Among such CA chimeras there was a general trend such that CAs from epidemic HIV-2 Group A and B isolates were the most infectious on human T cells, CA from a 1 degrees sooty mangabey isolate was the least infectious, and non-epidemic HIV-2 Group D, E, F, and G CAs were in the middle. The CA-specific decrease in infectivity was observed with either HIV-1, HIV-2, ecotropic MLV, or ALV Env pseudotypes, indicating that it was independent of the virus entry pathway. As2O3, a drug that suppresses TRIM5-mediated restriction, increased human blood cell transduction by SIVMAC but not by HIV-1. Nonetheless, elimination of TRIM5 restriction activity did not rescue SIVMAC transduction. Also, in contrast to TRIM5-mediated restriction, the SIVMAC CA-specific block occurred after completion of reverse transcription and the formation of 2-LTR circles, but before establishment of the provirus. Transduction efficiency in heterokaryons generated by fusing epithelial cells with T cells resembled that in the T cells, indicative of a dominant-acting SIVMAC restriction activity in the latter. These results suggest that the nucleus of human blood cells possesses a restriction factor specific for the CA of HIV-2/SIVMAC/SIVSM and that cross-species transmission of SIVSM to human T cells necessitated adaptation of HIV-2 to this putative restriction factor

Gelsolin activity controls efficient early HIV-1 infection

HIV-1 entry into target lymphocytes requires the activity of actin adaptors that stabilize and reorganize cortical F-actin, like moesin and filamin-A. These alterations are necessary for the redistribution of CD4-CXCR4/CCR5 to one pole of the cell, a process that increases the probability of HIV-1 Envelope (Env)-CD4/co-receptor interactions and that generates the tension at the plasma membrane necessary to potentiate fusion pore formation, thereby favouring early HIV-1 infection. However, it remains unclear whether the dynamic processing of F-actin and the amount of cortical actin available during the initial virus-cell contact are required to such events

Rational design of HIV vaccines and microbicides: report of the EUROPRISE network annual conference 2010

Novel, exciting intervention strategies to prevent infection with HIV have been tested in the past year, and the field is rapidly evolving. EUROPRISE is a network of excellence sponsored by the European Commission and concerned with a wide range of activities including integrated developmental research on HIV vaccines and microbicides from discovery to early clinical trials. A central and timely theme of the network is the development of the unique concept of co-usage of vaccines and microbicides. This review, prepared by the PhD students of the network captures much of the research ongoing between the partners. The network is in its 5th year and involves over 50 institutions from 13 European countries together with 3 industrial partners; GSK, Novartis and Sanofi-Pasteur. EUROPRISE is involved in 31 separate world-wide trials of Vaccines and Microbicides including 6 in African countries (Tanzania, Mozambique, South Africa, Kenya, Malawi, Rwanda), and is directly supporting clinical trials including MABGEL, a gp140-hsp70 conjugate trial and HIVIS, vaccine trials in Europe and Africa

Galanin pathogenic mutations in temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is a common epilepsy syndrome with a complex etiology. Despite evidence for the participation of genetic factors, the genetic basis of TLE remains largely unknown. A role for the galanin neuropeptide in the regulation of epileptic seizures has been established in animal models more than two decades ago. However, until now there was no report of pathogenic mutations in GAL, the galanin-encoding gene, and therefore its role in human epilepsy was not established. Here, we studied a family with a pair of monozygotic twins affected by TLE and two unaffected siblings born to healthy parents. Exome sequencing revealed that both twins carried a novel de novo mutation (p.A39E) in the GAL gene. Functional analysis revealed that the p.A39E mutant showed antagonistic activity against galanin receptor 1 (GalR1)-mediated response, and decreased binding affinity and reduced agonist properties for GalR2. These findings suggest that the p.A39E mutant could impair galanin signaling in the hippocampus, leading to increased glutamatergic excitation and ultimately to TLE. In a cohort of 582 cases, we did not observe any pathogenic mutations indicating that mutations in GAL are a rare cause of TLE. The identification of a novel de novo mutation in a biologically-relevant candidate gene, coupled with functional evidence that the mutant protein disrupts galanin signaling, strongly supports GAL as the causal gene for the TLE in this family. Given the availability of galanin agonists which inhibit seizures, our findings could potentially have direct implications for the development of anti-epileptic treatmen

Influenza vaccination coverage among medical residents: An Italian multicenter survey

Although influenza vaccination is recognized to be safe and effective, recent studies have confirmed that immunization coverage among health care workers remain generally low, especially among medical residents (MRs). Aim of the present multicenter study was to investigate attitudes and determinants associated with acceptance of influenza vaccination among Italian MRs. A survey was performed in 2012 on MRs attending post-graduate schools of 18 Italian Universities. Each participant was interviewed via an anonymous, self-administered, web-based questionnaire including questions on attitudes regarding influenza vaccination. A total of 2506 MRs were recruited in the survey and 299 (11.9%) of these stated they had accepted influenza vaccination in 2011-2012 season. Vaccinated MRs were older (P = 0.006), working in clinical settings (P = 0.048), and vaccinated in the 2 previous seasons (P < 0.001 in both seasons). Moreover, MRs who had recommended influenza vaccination to their patients were significantly more compliant with influenza vaccination uptake in 2011-2012 season (P < 0.001). "To avoid spreading influenza among patients" was recognized as the main reason for accepting vaccination by less than 15% of vaccinated MRs. Italian MRs seem to have a very low compliance with influenza vaccination and they seem to accept influenza vaccination as a habit that is unrelated to professional and ethical responsibility. Otherwise, residents who refuse vaccination in the previous seasons usually maintain their behaviors. Promoting correct attitudes and good practice in order to improve the influenza immunization rates of MRs could represent a decisive goal for increasing immunization coverage among health care workers of the future. © 2014 Landes Bioscience

Influence of cellular proteins incorporated in HIV-1 virions in modulating viral infectivity

Pi\uf9 di 30 anni dopo la scoperta del virus dell'immunodeficienza umana 1 (HIV-1), l'AIDS rappresenta ancora un' emergenza sanitaria. Lo sviluppo della terapia antiretrovirale (ART) ha rappresentato una pietra miliare nel trattamento degli individui infetti da HIV, tuttavia la diffusione di ceppi resistenti ai farmaci e gli effetti collaterali che insorgono nei pazienti soggetti alla terapia da anni, rendono necessaria la caratterizzazione di nuovi bersagli terapeutici. Un gran numero di proteine \u200b\u200bcellulari vengono incorporate nel virus quando questo gemma dalla membrana cellulare e diversi studi dimostrano che queste proteine contribuiscono in maniera importante all' infettivit\ue0 del virus. Lo scopo di questa tesi era quello di esplorare il ruolo delle proteine HLA-C e gelsolina nel ciclo vitale del virus per generare conoscenze sfruttabili in un prossimo futuro per la messa a punto di nuove strategie terapeutiche. In un lavoro precedente, il nostro gruppo ha dimostrato che molecole HLA-C incorporate nell'envelope di HIV-1 sono in grado di legarsi alla glicoproteina gp120 e di aumentare l'infettivit\ue0 virale. In questa tesi abbiamo studiato questa associazione e per la prima volta abbiamo mostrato la distribuzione subcellulare di queste due proteine. Utilizzando un sistema cellulare privo di b2-microglobulina, abbiamo dimostrato che il complesso HLA-C / Env coinvolge molecole di HLA-C prive di b2-microglobulina, e che l'abilit\ue0 di legarsi ad HLA- C \ue8 una capacit\ue0 unica di HIV-1 Env, che non viene mostrata da altre glicoproteine virali n\ue9 da altre proteine \u200b\u200bdi HIV-1. In considerazione di questi risultati e tenendo conto che le molecole MHC hanno gi\ue0 dimostrato di partecipare al trasporto di proteine \u200b\u200bvirali, ipotizziamo che HLA-C possa agire come chaperon di Env durante il processo di gemmazione. Diversi gruppi hanno mostrato che la riorganizzazione dell' actina corticale \ue8 fondamentale per promuovere l'ingresso del virus nella cellula. Questo processo aumenta la probabilit\ue0 di HIV-1 di interagire con i recettori virali che mediano il suo ingresso nella cellula. In questa tesi dimostriamo per la prima volta che, modificando l' actina corticale, gelsolina guida i recettori implicati nell'infezione virale ad un polo della cellula. Alterando l' attivit\ue0 della gelsolina si inibiscono la riorganizzazione dell'actina e la ridistribuzione dei recettori virali che sono necessari per l'ingresso di HIV-1 nella cellula. Pertanto, proponiamo che gelsolina sia un nuovo fattore che pu\uf2 essere sfruttato per combattere HIV-1.More than 30 years after the discovery of HIV-1, AIDS still represents a pandemic emergency. The implementation of antiretroviral ther- apy (ART) has been a milestone in the treatment of HIV-1 infected individuals, nevertheless the spread of drug-resistant strains and the long-term side effects arising in patients on ART, call for the characterization of new therapeutic targets. A number of host proteins are selectively incorporated into the HIV-1 envelope when the virus buds from the cell membrane and several studies attest the contribution of these virally acquired cell proteins in virus infectivity. The aim of this thesis was to explore the role of HLA-C and gelsolin in the viral life cycle in order to generate knowl- edge exploitable for the design of new therapeutic strategies in the near future. In a past work performed by our lab, HLA-C molecules incorpo- rated within the HIV-1 envelopes have been shown to bind to the envelope glycoprotein gp120 and to enhance viral infectivity. In this thesis we looked for further informations about this association and show for the first time the subcellular distribution of these two interact- ing proteins into the cell. By using a particular model system lacking b2-microglobulin, we provide evidence that the HLA-C /Env complex involves free heavy chains of HLA-C, devoid of b2-microglobulin, and we show that the propensity of Env to associate with HLA-C is a unique capacity of HIV-1 Env, which is not displayed by other viral envelopes nor other HIV-1 proteins. In view of these results and taking into account that MHC molecules have been yet shown to participate in the translocation of viral proteins, we speculate that HLA-C may act as a chaperon of Env during the budding process. Several groups have reported that the reorganization of cortical actin is fundamental to promote efficient viral entry. This process increases the probability of HIV-1 to interact with the viral receptors which mediate its entry into the cell. Here, we provide the first evidence that, by severing cortical actin, gelsolin provokes changes in actin reorganization and drives the receptors implicated in viral infection to one pole of the cell. Altering gelsolin activity perturbs this actin reorganization and the redistribution of viral receptors, severely re- stricting early HIV-1 entry. Thus, we propose that gelsolin is a new factor that can limit HIV-1 infection and accordingly, cell-signals that regulate gelsolin expression and/or its actin-severing may be crucial to combat HIV-1 infection

Association Analysis Between HIV-1 Env and HLA-C Using Bimolecular Fluorescence Complementation

Introduction: HLA-C is selectively incorporated into HIV-1 envelope. We reported that viruses produced from HLA-C silenced cells are less infectious and demonstrated a specific association between HLA-C and gp120. Our purpose is to unravel the HLA-C/gp120 association to reveal new targets for the development of neutralizing antibodies and new anti-HIV compounds. Methods: Fluorescent-tagged HLA-C and gp120 were prepared and used for bimolecular fluorescence complementation (BiFC) analysis to study their association into cellular compartments. To identify gp120 domains involved in the association with HLA-C, different gp120 deletion mutants are being constructed and tested. Results: A BiFC complementation signal between HLA-C and gp120 is detectable in the endoplasmic reticulum (ER), Golgi apparatus, late endosomes and on the cell membrane. Confocal microscopy does not show a co-localization signal between gp120 and [beta]2-microglobulin ([beta]2m). Discussion: The co-localization analysis shows that HLA-C, most likely as an open conformer, interacts with gp120 early in the ER and that they remain associated up to the cell membrane, travelling together trough the Golgi apparatus and late endosomes. No gp120 and [beta]2m co-localization is evident, suggesting a competition between them for HLA-C binding